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https://openalex.org/W2140874795	http://www.ajnr.org/content/ajnr/28/7/1384.full.pdf	English	null	Packing Performance of Helical Guglielmi Detachable Coil (GDC) 18 in Intracranial Aneurysms: A Comparison with Helical GDC 10 Coils and Complex Trufill/Orbit Coils	American journal of neuroradiology	2,007	cc-by	3,931	ORIGINAL RESEARCH M. Sluzewski W.J. van Rooij BACKGROUND AND PURPOSE: The purpose of this study was to retrospectively compare the packing performance of helical Guglielmi detachable coil (GDC) 18 (thickness, 0.0135–0.015 inch) with the packing performance of both helical GDC 10 (thickness, 0.010 inch) and complex Trufill/Orbit coils (thickness, 0.012 inch). MATERIALS AND METHODS: From our data base, we selected aneurysms that were exclusively coiled with GDC 18 coils. For every aneurysm treated with GDC 18 coils, we tried to identify a volume- matched control aneurysm treated with exclusively GDC 10 coils or exclusively Trufill/Orbit coils. This process resulted in 32 aneurysm pairs treated with either GDC 18 or GDC 10 coils and 35 aneurysm pairs treated with either GDC 18 or Trufill/Orbit coils. RESULTS: The mean packing of 24.2% of aneurysms treated with GDC 18 was significantly higher than the mean packing of 18.3% of aneurysms treated with GDC 10 (P .0001). The mean packing of 23.1% of GDC 18 coils was not different from the mean packing of 25.1% of Trufill/Orbit coils (P .15). CONCLUSION: In aneurysms of 4 mm or larger, packing performance of helical GDC 18 coils is superior to that of helical GDC 10 coils and equal to that of complex Trufill/Orbit coils. A p p A pproximately 25% of intracranial aneurysms treated with platinum coils show reopening of the lumen with time,1-2 A pproximately 25% of intracranial aneurysms treated with platinum coils show reopening of the lumen with time,1-2 compaction of coil mesh being the most important contribut- ing factor. Compaction can be prevented by increasing the packing attenuation, defined as ratio of inserted coil volume and volume of the aneurysm.3 Higher aneurysm packing can be obtained by inserting a higher volume of coils. Volume of inserted coils is dependent on both coil thickness and inserted coil length. In previous studies, we showed that packing is significantly higher in aneurysms treated with complex- shaped coils of 0.012-inch wire thickness (Trufill/Orbit coils; Cordis Neurovascular, Miami Lakes, Fla) than with aneu- rysms treated with helical coils of 0.010-inch wire thickness (Guglielmi detachable coil [GDC] 10; Boston Scientific, Fre- mont, Calif).4,5 In these studies, the increased packing of 0.012-inch coils over 0.010-inch coils was attributed to the increased coil thickness because the inserted coil length per aneurysm volume was equal. Description of Compared Coil Types Description of Compared Coil Types GDC 18 Coils. GDC 18 coils are available in 2 softness grades, Regular and Soft. GDC 18 Regular coils have a thickness of 0.015 inch and are available in helical shapes ranging from 2 to 20 mm and in 3D shapes ranging from 6 to 20 mm. GDC 18 Soft coils have a thickness of 0.0135 inch and are available in helical shape ranging from 2 to 6 mm. GDC 10 Coils. GDC 10 coils are available in 3 softness grades, Regular, Soft, and Ultrasoft. GDC 10 Regular coils have a thickness of 0.010 inch, and GDC 10 Soft and Ultrasoft have a thickness of 0.0095 inch. GDC 10 coils are available in sizes ranging from 2 to 14 mm. GDC 10 coils are also available in 3D shape in sizes ranging from 3 to 10 mm. p f p yp GDC 18 Coils. GDC 18 coils are available in 2 softness grades, Regular and Soft. GDC 18 Regular coils have a thickness of 0.015 inch and are available in helical shapes ranging from 2 to 20 mm and in 3D shapes ranging from 6 to 20 mm. GDC 18 Soft coils have a thickness of 0.0135 inch and are available in helical shape ranging from 2 to 6 mm. GDC 10 Coils. GDC 10 coils are available in 3 softness grades, Regular, Soft, and Ultrasoft. GDC 10 Regular coils have a thickness of 0.010 inch, and GDC 10 Soft and Ultrasoft have a thickness of 0.0095 inch. GDC 10 coils are available in sizes ranging from 2 to 14 mm. GDC 10 coils are also available in 3D shape in sizes ranging from 3 to 10 mm. Trufill/Orbit Coils. Orbit coils are the evolution of the Trufill detachable coil system with a reduced detachment system profile to allow compatibility with 0.014-inch microcatheters. Trufill/Orbit coils are helical or shaped in a complex 3D configuration, have a thickness of 0.012 inch, and are available in sizes ranging from 2 to 20 mm. Besides the 0.010-inch GDC 10 coils and the 0.012-inch Trufill/Orbit coils, GDC 18 coils (Boston Scientific) with 0.0135- to 0.015-inch wire thickness are commonly used for larger aneurysms. In this study, we retrospectively compared the packing performance of helical GDC 18 coils with the Coiling Procedure. 10 Coils and Complex Trufill/Orbit Coils Aneurysms: A Comparison with Helical GDC Detachable Coil (GDC) 18 in Intracranial Packing Performance of Helical Guglielmi M. Sluzewski and W.J. van Rooij http://www.ajnr.org/content/28/7/1384 https://doi.org/10.3174/ajnr.A0557 doi: 2007, 28 (7) 1384-1387 AJNR Am J Neuroradiol M. Sluzewski and W.J. van Rooij of October 23, 2024. This information is current as http://www.ajnr.org/content/28/7/1384 https://doi.org/10.3174/ajnr.A0557 doi: 2007, 28 (7) 1384-1387 AJNR Am J Neuroradiol Received October 19, 2006; accepted after revision December 8. From the Department of Radiology, St. Elisabeth Ziekenhuis, Tilburg, the Netherlands. Please address correspondence to Menno Sluzewski, MD, PhD, Department of Radiology, St. Elisabeth Ziekenhuis, Hilvarenbeekseweg 60, 5022 GC Tilburg, The Netherlands; e-mail: radiol@knmg.nl DOI 10.3174/ajnr.A0557 ORIGINAL RESEARCH Apparently, the complex shape of the 0.012-inch coils allowed better concentric aneurysm filling with less created dead space. Also in an experimental study, it was shown that complex-shaped coils allowed signif- icantly better aneurysm filling than helical coils of the same thickness.6 packing performance of both helical GDC 10 coils and com- plex Trufill/Orbit coils. In addition, we compared procedural complications, procedural ruptures, and additional treat- ments after coiling in aneurysms treated with any 3 types of coils. DOI 10.3174/ajnr.A0557 Initial Occlusion Rates and Occlusion at 6-Month Follow-Up On the basis of these criteria, 32 pairs of aneurysms treated by either GDC 18 coils or Trufill/Orbit coils and 35 pairs of aneurysms treated by either GDC 18 coils or GDC 10 coils were iden- tified. Of aneurysms treated with GDC 18, 24 were assigned in both matched groups. Aneurysms were treated between 1999 and 2005 on the same equipment by the same operators. Aneurysms Treated with GDC 18 Coils Compared with Aneurysms Treated with Trufill/Orbit Coils. For 35 aneu- rysms treated with GDC 18 and matched with aneurysms treated with Trufill/Orbit, initial occlusion was (near) com- plete in 34 aneurysms and incomplete in 1 aneurysm. (The patient with the initial incompletely occluded aneurysm died 3 weeks later of vasospasm). At 6-month follow-up angiography of 27 aneurysms, 22 aneurysms were (near) completely oc- cluded and 5 aneurysms became incompletely occluded and were retreated. Of 35 aneurysms treated with GDC 18, a 3D coil was used as a first coil in 8. INTERVENTIONAL ORIGINAL RESEARCH INTERVENTIONAL ORIGINAL RESEARCH AL ORIGINAL RESEARCH Clinical Data Assessment. For all aneurysms selected for compar- ison, we assessed the following clinical data: proportion of women, patient mean age, proportion of ruptured aneurysms, proportion of aneurysms located in anterior and posterior circulation, procedural complications of coiling leading to permanent morbidity or mortal- ity, number of procedural ruptures, proportion and mean duration of angiographic follow-up, and number of retreatments. For 35 aneurysms treated with Trufill/Orbit and matched with aneurysms treated with GDC 18, initial occlusion was (near) complete in 34 aneurysms and incomplete in 1 aneu- rysm that was not additionally treated. At 6-month follow-up angiography of 29 aneurysms, 25 aneurysms were (near) com- pletely occluded and 4 aneurysms became incompletely oc- cluded and were retreated. In addition, for all aneurysms grouped by matched pairs, we as- sessed the following data: the number of aneurysms treated with GDC coils in which a 3D coil was used as a first coil, initial occlusion rates dichotomized into (near) complete occlusion (90%–100%) and in- complete occlusion (90%), and occlusion rates at 6 months fol- low-up interval. Results of Comparison Between Different Types of Coils The results of comparisons between GDC 18 coils and GDC 10 coils are summarized in Tables 1 and 2, and the results of comparisons between GDC 18 coils and Trufill/Orbit coils are summarized in Tables 3 and 4. Initial Occlusion Rates and Occlusion at 6-Month Follow-Up Initial Occlusion Rates and Occlusion at 6-Month Follow-Up Aneurysms Treated with GDC 18 Coils Compared with Aneurysms Treated with GDC 10 Coils. For 32 aneurysms treated with GDC 18 and matched with aneurysms treated with GDC 10, initial occlusion was (near) complete in 31 an- eurysms and incomplete in 1 aneurysm. (The patient with the initial incompletely occluded aneurysm died 3 weeks later of vasospasm). At 6-month follow-up angiography of 26 aneu- rysms, 24 aneurysms were (near) completely occluded and 2 aneurysms became incompletely occluded and were retreated. Of 32 aneurysms treated with GDC 18, a 3D coil was used as a first coil in 4. Aneurysm Volume and Packing Calculation. Volume of aneu- rysms was either calculated from 3D angiographic datasets by using machine software or with a custom-designed computer program that reconstructed 3D aneurysms from 2D angiographic images.3,7 Both methods were validated with phantoms. Volume of inserted coils was calculated with a spreadsheet provided by the coil manufacturers, containing volume per centimeter of coils for every coil type used. Packing of the aneurysms was calculated by dividing the volume of inserted coils by the volume of the aneurysm times 100%. For 32 aneurysms treated with GDC 10 and matched with aneurysms treated with GDC 18, initial occlusion was (near) completein31aneurysmsandincompletein1aneurysmthatwas additionally coiled before 6-month follow-up interval. At 6-monthsfollow-upangiographyof28aneurysms,25aneurysms were (near) completely occluded and 3 aneurysms became in- completelyoccludedandwereretreated.Of32aneurysmstreated with GDC 10, a 3D coil was used as a first coil in 15. Aneurysm Matching. From our data base containing over 1000 coiled aneurysms, we selected those aneurysms with known aneurysm volume that were exclusively coiled with GDC 18 coils. For every aneurysm treated with GDC 18 coils, we tried to identify a volume- matched control aneurysm, within a 10% volume range, treated with either exclusively GDC 10 coils or exclusively Trufill/Orbit coils. Se- lection of the volume-matched controls was performed as follows: in case of a single exact volume match, this particular aneurysm was chosen. In case of more than 1 exact volume match, the aneurysm with the same location was selected. In case of 2 exact volume matches on the same location, the rupture status of the aneurysm was the decisive criterion. Description of Compared Coil Types Coiling of aneurysms was performed by ei- ther of the authors on a biplane angiographic unit (Integris BN 3000; Philips Medical Systems, Best, the Netherlands) with the use of gen- eral anesthesia and systemic administration of heparin. Intravenously or subcutaneously administered heparin was continued for 48 hours after the procedure, followed by 80-mg aspirin daily for 3 months. The aim of coiling was to pack the aneurysm as densely as possible, until not 1 more coil could be placed or when complete angiographic 1384 Sluzewski AJNR 28 Aug 2007 www.ajnr.org 1384 of a 3D coil as a first coil between aneurysms treated with GDC 18 and GDC 10. P values .05 were considered statistically significant. occlusion was apparent. With the use of GDC 18 and 10 coils, in some aneurysms, a 3D coil was used as a first coil. With the use of Trufill/ Orbit coils, all inserted coils were complex-shaped except for some 2- and 3-mm-diameter helical coils. Coil sizes were chosen as appropri- ate for the aneurysm size and shape. Coils were neither over- nor undersized. Although maximal aneurysm diameter (20 mm) ex- ceeded maximal coil size in some aneurysms treated with GDC 10 coils (14 mm), coil size was always appropriate for irregular aneurysm shape. Initial Occlusion Rates and Occlusion at 6-Month Follow-Up Mean volumes of the aneu- rysms in both comparisons were nearly identical: For GDC 18 coils versus GDC 10 coils, this was 329 mm3 and 333 mm3 (P .96), and for GDC 18 coils versus Trufill/Orbit coils, this was 297 mm3 and 295 mm3 (P .98). Mean packing of 24.2% of aneurysms treated with GDC 18 coils was significantly higher than mean packing of 18.3% of aneurysms treated with GDC 10 coils (absolute difference, 5.9%; relative difference, 32%). Mean packing of 23.1% of aneurysms treated with GDC 18 coils was not different from mean packing of 25.1% of aneurysms treated with Trufill/Orbit coils (P .15). Statistical Analysis. Mean aneurysm volume, mean packing, mean inserted coil length per aneurysm per cubic millimeter, and mean number of coils used per aneurysm in the 32 aneurysms treated with GDC 18 coils and 32 aneurysms treated with GDC 10 coils were compared by using the t test. Mean aneurysm volume, mean packing, mean inserted coil length per aneurysm per cubic millimeter, and mean number of coils used per aneurysm in the 35 aneurysms treated with GDC 18 coils and 35 aneurysms treated with Trufill/Orbit coils were compared by using the t test. Procedural complications, proce- dural ruptures, and retreatments were compared by using the Fisher exact test. Initial Occlusion Rates and Occlusion at 6-Month Follow-Up The chi square test was used to assess differences in the use AJNR Am J Neuroradiol 28:1384–87 Aug 2007 www.ajnr.org 1385 Table 1: Aneurysm volume and relevant packing results of 32 aneurysms treated with GDC 18 coils compared with 32 volume- matched controls treated with GDC 10 coils GDC 18 GDC 10 P value (t test) Number of aneurysms 32 32 Aneurysm volume .96 Mean 329 mm3 333 mm3 Median 297.5 mm3 309 mm3 Range 34–1333 mm3 35–1377 mm3 SD 230 mm3 237 mm3 Largest aneurysm diameter Mean 10.2 mm 9.9 mm Median 10 mm 9.5 mm Range 4–20 mm 4–20 mm Packing Mean 24.2% 18.3% .0001 Median 24.5% 19.0% Range 11%–32% 9–33% SD 4.97% 4.76% Aneurysm volumes 10 542 mm3 10 643 mm3 Inserted coil length 2436 cm 3419 cm Coil length per mm3 aneurysm volume 0.23 cm 0.32 cm Mean coil length per aneurysm per mm3 0.233 cm 0.345 cm .0001 Total number of coils 145 223 Mean number of coils per aneurysm 4.5 7.0 .005 Table 3: Aneurysm volume and relevant packing results of 35 aneurysms treated with helical GDC 18 coils compared with 35 volume-matched controls treated with complex Trufill/Orbit coils GDC 18 Trufill/Orbit P value (t test) Number of aneurysms 35 35 Aneurysm volume .98 Mean 297 mm3 295 mm3 Median 400 mm3 406.5 mm3 Range 26–1450 mm3 27–1504 mm3 SD 327 mm3 383 mm3 Largest aneurysm diameter Mean 10.3 mm 9.6 mm Median 10 mm 10 mm Range 4–22 mm 4–17 mm Packing .15 Mean 23.1% 25.1% Median 24% 25% Range 8%–33% 11%–36% SD 5.98% 5.67% Aneurysm volumes 14 002 mm3 14 229 mm3 Inserted coil length 2794 cm 4446 cm Coil length per mm3 aneurysm volume 0.20 cm 0.31 cm Mean coil length per aneurysm per mm3 0.224 cm 0.339 cm .0001 Total number of coils 157 238 Mean number of coils per aneurysm 4.5 6.8 .0024 Table 1: Aneurysm volume and relevant packing results of 32 aneurysms treated with GDC 18 coils compared with 32 volume- matched controls treated with GDC 10 coils Table 3: Aneurysm volume and relevant packing results of 35 aneurysms treated with helical GDC 18 coils compared with 35 volume-matched controls treated with complex Trufill/Orbit coils Table 4: Clinical and follow-up characteristics of 35 aneurysms treated with GDC 18 coils compared with 35 volume-matched controls treated with Trufill/Orbit coils GDC 18 Trufill/Orbit P value (t test) Number of aneurysms 35 35 Woman/man 27/8 19/16 Median age 58 years 56 years Anterior circulation 32 24 Posterior circulation 3 11 Ruptured/unruptured 25/10 27/8 Procedural morbidity 1 0 1.0 Procedural rupture 2 0 .5 Angiographic follow-up 27 (77%) 29 (83%) Mean angiographic follow-up 6.4 months 7.0 months Additional treatments 5 of 27 (19%) 4 of 29 (14%) .9 Table 2: Clinical and follow-up characteristics of 32 aneurysms treated with GDC 18 coils compared with 32 volume-matched controls treated with GDC 10 coils GDC 18 GDC 10 P value (t test) Number of aneurysms 32 32 Woman/man 27/5 20/12 Median age 59.5 years 54.5 years Anterior circulation 29 21 Posterior circulation 3 11 Ruptured/unruptured 22/10 27/5 Procedural morbidity 1 (3.1%) 1 (3.1%) 1.0 Procedural rupture 1 (3.1%) 0 1.0 Angiographic follow-up 26 (81%) 28 (88%) Mean angiographic follow-up 12.7 months 15.0 months Additional treatments 2 of 26 (8%) 4 of 28 (14%) .74 Table 4: Clinical and follow-up characteristics of 35 aneurysms treated with GDC 18 coils compared with 35 volume-matched controls treated with Trufill/Orbit coils Table 2: Clinical and follow-up characteristics of 32 aneurysms treated with GDC 18 coils compared with 32 volume-matched controls treated with GDC 10 coils Mean inserted coil length per aneurysm per cubic millime- ter in the aneurysms treated with GDC 18 coils was signifi- cantly lower than that for aneurysms treated with both GDC 10 and Trufill/Orbit coils. Initial Occlusion Rates and Occlusion at 6-Month Follow-Up Mean number of coils used per an- eurysm in the aneurysms treated with GDC 18 coils was sig- nificantly lower than that for aneurysms treated with both GDC 10 and Trufill/Orbit coils. There were no statistically significant differences in procedural complications, proce- dural ruptures, initial occlusion rates, occlusion rates at 6-month follow-up angiography, and retreatment rates in any of the groups. In the aneurysms treated with GDC 10, signifi- cantly more often a 3D coil was used as a first coil as in aneu- rysms treated with GDC 18 (P .006). ing of aneurysms treated with GDC 10 coils. Packing of aneu- rysms treated with GDC 18 coils was not different from pack- ing of aneurysms treated with Trufill/Orbit coils. The mean volume of the selected aneurysms treated with GDC 18 and their matched volume controls was approximately 300 mm3. This is relatively large because GDC 18 coils are mainly used in medium- and large-sized aneurysms. In a previous study with 235 consecutively coiled aneurysms,5 mean aneurysm volume with 150 mm3 was much lower. Mean inserted coil length per aneurysm per cubic millime- ter in the aneurysms treated with helical GDC 18 coils was significantly lower than that for aneurysms treated with helical GDC 10 coils. Apparently, increased coil thickness of GDC 18 is associated with less inserted coil length per cubic millimeter. Because inserted coil volume is the product of coil length and coil thickness, though less coil length is inserted, the increased Discussion hi d In this study, we found that packing of intracranial aneurysms treated with GDC 18 coils was significantly higher than pack- 1386 Sluzewski AJNR 28 Aug 2007 www.ajnr.org thickness still leads to higher inserted coil volume. This out- come may not be surprising because coil thickness influences coil volume by the second power. rysm was significantly lower for GDC 18 coils than for both GDC 10 coils and Trufill/Orbit coils. This finding means that in relatively large aneurysms, the use of GDC 18 coils not only results in higher packing but is also more cost-effective, pro- viding the price per coil is equal. Packing of GDC 10 coils, the most widely used coil, is lower than packing of Trufill/Orbit coils for all aneurysm sizes and also lower than packing of GDC 18 coils in aneurysms of 4 mm or larger. What is more interesting, in previous studies,4,5 no differ- ence in inserted coil length per cubic millimeter was found between helical GDC 10 coils of 0.010-inch thickness and complex Trufill/Orbit coils of 0.012-inch thickness. The better packing of Trufill/Orbit coils compared with GDC 10 coils was, therefore, explained by the increased coil thickness only because equal coil lengths could be inserted. Apparently, the complex shape of the Trufill/Orbit coil allows better concen- tric filling and creates less dead space. In an experimental study, Piotin et al6 found a higher packing by higher inserted coil lengths of complex-shaped Trufill/Orbit coils compared with helical Trufill/Orbit coils of the same thickness. This study showed that inserted coil volume per cubic millimeter of aneurysm is dependent on coil shape for these particular types of coils and that a complex shape allows more inserted coil length. This increased packing of complex Trufill/Orbit coils compared with helical Trufill/Orbit coils could not be con- firmed for GDC coils: we found no difference in packing per- formance in aneurysms treated with either helical GDC 10 coils or complex-shaped GDC 360° coils.8 Apparently, other physical properties of the coils such as softness and shape memory play a role in packing performance. Future research should aim at the best possible combination of coil thickness, coil shape, and other physical properties. Conclusion In aneurysms of 4 mm or larger, packing performance of he- lical GDC 18 coils (0.0135–0.015 inch) is superior to that of helical GDC 10 coils (0.010 inch) and equal to that of Trufill/ Orbit complex-shaped coils (0.012 inch). References 1. Sluzewski M, van Rooij WJ, Rinkel GJ, et al. Endovascular treatment of rup- tured intracranial aneurysms with detachable coils: long-term clinical and serial angiographic results. Radiology 2003;227:720–24 1. Sluzewski M, van Rooij WJ, Rinkel GJ, et al. Endovascular treatment of rup- tured intracranial aneurysms with detachable coils: long-term clinical and serial angiographic results. Radiology 2003;227:720–24 2. Raymond J, Guilbert F, Weill A, et al. Long-term angiographic recurrences after selective endovascular treatment of aneurysms with detachable coils. Stroke 2003;34:1398–403 2. Raymond J, Guilbert F, Weill A, et al. Long-term angiographic recurrences after selective endovascular treatment of aneurysms with detachable coils. Stroke 2003;34:1398–403 3. Sluzewski M, van Rooij WJ, Slob MJ, et al. Relation between aneurysm volume, packing and compaction in 145 cerebral aneurysms treated with coils. Radi- ology 2004;231:653–58 4. Slob MJ, van Rooij WJ, Sluzewski M. Coil thickness and packing of cerebral aneurysms: a comparative study of two types of coils. AJNR Am J Neuroradiol 2005;26:901–03 4. Slob MJ, van Rooij WJ, Sluzewski M. Coil thickness and packing of cerebral aneurysms: a comparative study of two types of coils. AJNR Am J Neuroradiol 2005;26:901–03 5. Slob MJ, van Rooij WJ, Sluzewski M. Influence of coil thickness on packing, re-opening and retreatment of intracranial aneurysms: a comparative study between two types of coils. Neurol Res 2005;27(suppl 1):116–19 5. Slob MJ, van Rooij WJ, Sluzewski M. Influence of coil thickness on packing, re-opening and retreatment of intracranial aneurysms: a comparative study between two types of coils. Neurol Res 2005;27(suppl 1):116–19 We found no differences in procedural complications, pro- cedural ruptures, and retreatment rates in aneurysms coiled with any of the 3 types of coils. The incidence of these events is low, and larger numbers of patients are needed to detect pos- sible differences. However, a positive relation between high packing and less reopening with time was established in pre- vious studies with more patients.3,5 6. Piotin M, Iijima A, Wada H, et al. Increasing the packing of small aneurysms with complex-shaped coils: an in vitro study. AJNR Am J Neuroradiol 2003;24:1446–48 7. Bescos JO, Slob MJ, Slump CH, et al. Volume measurement of intracranial aneurysms from 3D rotational angiography: improvement of accuracy by gradient edge detection. AJNR Am J Neuroradiol 2005;26:2569–72 8. van Rooij WJ, Sluzewski M. Packing performance of GDC 360 degree in intra- cranial aneurysms: a comparison with complex Orbit coils and helical GDC 10 coils. References AJNR Am J Neuroradiol 2007;28:368–70 In the present study, the number of coils needed per aneu- In the present study, the number of coils needed per aneu- AJNR Am J Neuroradiol 28:1384–87 Aug 2007 www.ajnr.org 1387 AJNR Am J Neuroradiol 28:1384–87 Aug 2007 www.ajnr.org 1387
https://openalex.org/W4292598994	https://iopscience.iop.org/article/10.1088/2515-7655/ac889e/pdf	English	null	Quantitative evaluation of biaxial compressive strain and its impact on proton conduction and diffusion in yttrium-doped barium zirconate epitaxial thin films	JPhys energy	2,022	cc-by	14,823	You may also like You may also like Ba0.5Gd0.8La0.7Co2O6 Infiltrated BaZr0.8Y0.2O3 Composite Oxygen Electrodes for Protonic Ceramic Cells Qingjie Wang, Sandrine Ricote, Yu Wang et al. - Self-contained Electrochemical Process to Produce Pure Compressed Hydrogen from Hydrocarbons and Steam Without an External Energy Supply Huayang Zhu, W. Grover Coors, Sandrine Ricote et al. - Investigation of proton conductivity in Sc and Yb co-doped barium zirconate ceramics Avishek Satapathy, Ela Sinha and S K Rout - Ba0.5Gd0.8La0.7Co2O6 Infiltrated BaZr0.8Y0.2O3 Composite Oxygen Electrodes for Protonic Ceramic Cells Qingjie Wang, Sandrine Ricote, Yu Wang et al. - PAPER • OPEN ACCESS PAPER • OPEN ACCESS This content was downloaded from IP address 195.242.1.98 on 24/10/2024 at 06:43 Abstract Proton-conducting oxides, including 20 mol% yttrium-doped BaZrO3 (BZY20), have attracted considerable attention as electrolytes for environmentally friendly electrochemical cells, such as proton ceramic fuel cells (PCFCs) and proton-conducting solid oxide cells. These oxides exhibit fast proton conduction due to the complex physicochemical phenomena of hydration, chemical lattice expansion, proton migration, proton trapping, and local distortion. Using a proton- conducting oxide as an electrolyte film in electrochemical devices introduces an interface, which thermally and chemically generates mechanical strain. Here, we briefly review the current state of research into proton-conducting oxides in bulk samples and films used in electrochemical devices. We fabricated 18 and 500 nm thick 20 mol% BZY20 epitaxial films on (001) Nb-doped SrTiO3 single-crystal substrates to form a model interface between proton-conductive and non-proton-conductive materials, using pulsed laser deposition, and quantified the mechanical strain, proton concentration, proton conductivity, and diffusivity using thin-film x-ray diffractometry, thermogravimetry, secondary ion mass spectrometry, and AC impedance spectroscopy. Compressive strains of −2.1% and −0.85% were measured for the 18 and 500 nm thick films, respectively, and these strains reduced both the proton conduction and diffusion by five and one orders of magnitude, respectively, at 375 ◦C. Analysis based on a simple trapping model revealed that the decrease in proton conduction results from the slower diffusion of mobile protons with a negligible change in the proton trapping contribution. The model shows that the high ohmic resistance reported for a high-performance PCFC with a power density of 740 mW cm−2 at 600 ◦C can be solely explained by the estimated compressive strain in the cells. This study shows that minimizing biaxial compressive strain by appropriate choices of the electrolyte–electrode combination and fabrication process is important for maximizing the performance of electrochemical cells. Quantitative evaluation of biaxial compressive strain and its impact on proton conduction and diffusion in yttrium-doped barium zirconate epitaxial thin films 1 INAMORI Frontier Research Center, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan , y y, , , J p 2 Kyushu University Platform of Inter-Transdisciplinary Energy Research, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan ∗Author to whom any correspondence should be addressed. Keywords: proton-conducting oxide, yttrium-doped barium zirconate, epitaxial thin film, biaxial compressive strain, proton diffusivity, proton trapping S l i l f hi i l i il bl li Supplementary material for this article is available online Supplementary material for this article is available online PAPER OPEN ACCESS RECEIVED 13 June 2022 REVISED 5 August 2022 ACCEPTED FOR PUBLICATION 10 August 2022 PUBLISHED 21 September 2022 Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. OPEN ACCESS RECEIVED 13 June 2022 REVISED 5 August 2022 ACCEPTED FOR PUBLICATION 10 August 2022 PUBLISHED 21 September 2022 Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence. Any further distribution PAPER Quantitative evaluation of biaxial compressive strain and its impact on proton conduction and diffusion in yttrium-doped barium zirconate epitaxial thin films Junji Hyodo1,2and Yoshihiro Yamazaki1,2,∗ 1 INAMORI Frontier Research Center, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan 2 Kyushu University Platform of Inter-Transdisciplinary Energy Research, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan ∗Author to whom any correspondence should be addressed. E-mail: yamazaki.yoshihiro.105@m.kyushu-u.ac.jp Keywords: proton-conducting oxide, yttrium-doped barium zirconate, epitaxial thin film, biaxial compressive strain, proton diffusivity, proton trapping Supplementary material for this article is available online Quantitative evaluation of biaxial compressive strain and its impact on proton conduction and diffusion in yttrium-doped barium zirconate epitaxial thin films gy pp y Huayang Zhu, W. Grover Coors, Sandrine Ricote et al. To cite this article: Junji Hyodo and Yoshihiro Yamazaki 2022 J. Phys. Energy 4 044003 Investigation of proton conductivity in Sc and Yb co-doped barium zirconate ceramics Avishek Satapathy, Ela Sinha and S K Rout - View the article online for updates and enhancements. View the article online for updates and enhancements. This content was downloaded from IP address 195.242.1.98 on 24/10/2024 at 06:43 https://doi.org/10.1088/2515-7655/ac889e J. Phys. Energy 4 (2022) 044003 J. Phys. Energy 4 (2022) 044003 Journal of Physics: Energy Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. 1. Introduction Proton-conducting oxides [1–4] have attracted considerable attention as electrolytes for proton ceramic fuel cells (PCFCs) [5–10] and proton-conducting solid oxide cells [11–15] that can be operated at intermediate temperatures in the range of 300 ◦C–600 ◦C. Intermediate-temperature operation could eliminate the need for expensive platinum catalysts and heat-resistant alloys. It could also prolong the lifetime of cells due to the reduced rearrangement of constitutional elements near the triple-phase boundaries between the gas phase, electrode, and electrolyte at the reduced temperature. Reducing the cost of fuel cell systems and the longer lifetime could help accelerate their adoption. The requirements for the electrolytes are fast ionic conduction © 2022 The Author(s). Published by IOP Publishing Ltd J. Phys. Energy 4 (2022) 044003 J Hyodo and Y Yamazaki and chemical stability under the operating conditions for the cells. The specific requirement for ionic conduction in an electrolyte in fuel cells is more than 0.01 S cm−1 at the operating temperature to attain an area-specific ohmic resistance lower than 0.15 Ωcm2 for a 15 µm thick electrolyte film, which is a typical thickness for reliable manufacture [16]. A 20 mol% yttrium-doped barium zirconate (BZY20) [17] and 60 mol% scandium-doped barium zirconate [18] can meet the requirements for proton conductivity at 450 ◦C and 400 ◦C, respectively. These oxides also exhibit high chemical stability under reducing and oxidizing conditions on each side of an electrode in electrochemical cells. Although fast and stable proton conduction is realized in acceptor-doped barium zirconates, the physical chemistry behind it is complex. Proton conduction in oxides requires a hydration reaction, namely, the incorporation of protons into the lattice, and proton diffusion. Hydration occurs in two steps: partial substitution of a host element with acceptors creates oxygen vacancies in the lattice to maintain the electroneutrality of the system [1, 19]. Upon exposure to a moist gas phase containing water molecules, the oxygen vacancies can be filled with hydroxyl groups [4, 20, 21] with an accompanying chemical expansion of the crystal lattice [1, 22–25]. The incorporated protons rotate around an oxygen and hop along the neighboring oxygens [1], overcoming proton trapping near acceptor dopants [26]. Ab initio calculations suggest that the origin of proton trapping is strong hydrogen bonding near acceptor dopants accompanied by local lattice distortion [27–29]. The proton-dopant association (trap) energy is experimentally determined to be −29 kJ mol−1 for yttrium dopants [26]. 1. Introduction Proton conduction is a macroscopic result of microscopic phenomena, including hydration, chemical lattice expansion, proton migration, proton trapping, and local distortion, which correlate with each other in a complex manner. Forming a BZY20 electrolyte film introduces an interface, which adds the further complexity of mechanical strain due to lattice mismatch in the conduction behavior of protons. Although proton conduction in acceptor-doped barium zirconate thin films has been reported by many groups [1, 30–40], only one report has investigated mechanical strain at an interface and its influence on bulk proton conduction [34], for various thicknesses of BaZr0.6Ce0.4O3 buffer layers between the BZY20 film (15–87 nm in thickness) and (001) MgO substrate. The measured mechanical strain ranged from +0.7% (tensile, with a 30 nm thick buffer layer) to −0.3% (compressive, no buffer layer), where the proton conductivities were measured at 195 ◦C–363 ◦C in a humidified argon atmosphere. The in-plane proton conductivity at 363 ◦C decreased slightly from 0.004 S cm−1 at +0.7% tensile strain to 0.002 S cm−1 without strain, accompanied by an increase in activation energy from 0.39 to 0.44 eV [34]. Consistent with the experimental results, molecular dynamic simulations for 1 mol% (1227 ◦C–1727 ◦C [41]) and 50 mol% (427 ◦C–1027 ◦C [34]) yttrium-doped barium zirconates showed that the proton diffusivity in the out-of-plane direction decreased under biaxial compressive strain due to the enlarged jump distances [34, 41]. The complexity of proton-conducting oxides explains the current scarcity of research into the influence of mechanical strain at the interface on the proton conductivity. y In addition to the lattice mismatch, proton-conducting oxides have two other potential sources of mechanical strain induced at elevated temperatures, namely, differences in thermal expansion coefficients and chemical expansion at the interface. While a report by Fluri et al [34] detailed the initial investigations into this topic, mechanical strains arising from thermal expansion and chemical expansion at elevated temperatures, at which proton conduction occurs, have not been considered. The consideration of all the factors that influence interfacial mechanical strain is important for developing a fundamental understanding of proton-conducting oxides. Insight into the mechanical strain at interfaces in proton-conducting oxides is also important for the use of such oxides in electrochemical devices. 1. Introduction PCFCs have interfaces between the electrolyte and the anode and cathode, where ohmic resistances higher than that expected for bulk samples have been reported for many acceptor-doped barium zirconate electrolytes [6, 10, 42–52], which is problematic with respect to improving the performance of PCFCs. As summarized in figure 1 and table 1 [5–10, 42–76], almost all reports show ohmic resistances higher than 0.15 Ωcm2 at 600 ◦C, whereas calculations based on the proton conductivity of bulk samples for different electrolyte thicknesses [6, 7, 17, 18, 77–80] give values lower than 0.15 Ωcm2. Suspected reasons for the higher ohmic resistances are (a) lower proton concentration due to the dissolution of nickel into the electrolyte [81–83] and (b) the resistance at the interface between the electrolyte and the cathode [6, 10]; however, these have not been verified. The lack of information on the interface and its influence on proton conductivity has hindered the development of electrochemical devices such as PCFCs. y In the present study, we quantified the mechanical strain in BZY20 epitaxial films deposited on (001) Nb-doped SrTiO3 single-crystal substrates to investigate a model interface between proton-conductive and non-proton-conductive materials, with a wide compressive strain range of −0.85% to −2.1%. We determined the influence of misfit, thermal, and chemical strain on proton conductivity and diffusivity in the out-of-plane direction at elevated temperatures. We quantified the proton carrier concentration in the films using secondary ion mass spectrometry (SIMS) to determine the influence of compressive strain on the 2 J. Phys. Energy 4 (2022) 044003 J Hyodo and Y Yamazaki J. Phys. Energy 4 (2022) 044003 Figure 1. Summary of proton conductivities in doped barium zirconate and cerate-based electrolyte films in PCFCs. The y-axis shows the ratio of proton conductivity for electrolyte thin film in anode-supported single cells (σfilm) to that for thick and dense pellets. The colors and symbols represent materials used as the electrolyte and anode in the PCFCs. All data are given in table 1. Figure 1. Summary of proton conductivities in doped barium zirconate and cerate-based electrolyte films in PCFCs. The y-axis shows the ratio of proton conductivity for electrolyte thin film in anode-supported single cells (σfilm) to that for thick and dense pellets. The colors and symbols represent materials used as the electrolyte and anode in the PCFCs. All data are given in table 1. proton concentration and diffusivity as well as proton trapping in the films. 1.1. Strain at an interface between proton-conductive and non-proton-conductive materials Interfaces exist where proton-conductive and non-proton-conductive materials meet in PCFCs, Interfaces exist where proton-conductive and non-proton-conductive materials meet in PCFCs, i.e. electrolyte/anode and electrolyte/cathode interfaces, which cause lattice misfit, thermal, and chemical strains. Lattice misfit strain (εin.misfit) is introduced at the heterointerface due to the difference in atomic spacing between the substrate and film materials. Thermal strain (εin.thermal) appears when the film and substrate materials possess different linear thermal expansion coefficients. Chemical strain (εin.hyd) is induced by hydration, a characteristic reaction of proton-conducting oxides, which causes expansion and shrinkage of the crystal lattice. The in-plane strain, εin, at an elevated temperature at which protons conduct in the oxide may be written as the sum of the three components, εin(T,pH2O) = εin.misfit + εin.thermal −εin.hyd =  ∑ i ∑ j fij dj −di dj + A  + (αsub −αfilm) · (T −T0) −bCβ H. (1) O) = εin.misfit + εin.thermal −εin.hyd =  ∑ i ∑ j fij dj −di dj + A  + (αsub −αfilm) · (T −T0) −bCβ H. (1) (1) Here, i = {h k l} and j = {h′ k′ l′} are the lattice planes of the film and substrate, respectively, that form the interface, f ij is the fraction of the junction at the interface of the i–j plane combination, and di and dj are the atomic spacings on the i and j planes at the thermal-strain-free temperature, T0. A in the misfit strain term is a constant related to the relaxation of the lattice misfit caused by the formation of dislocations and grain boundaries, surface roughness, and the distance from the constrained interface to the relaxed surface. αfilm and αsub are the thermal expansion coefficients for the film and substrate, respectively. εhyd is defined as (ahyd −adry)/adry, where ahyd and adry are the lattice constants under hydrated and dehydrated states, respectively, at a temperature T, and is a function of the proton concentration, CH, with coefficients b and β that express the degree of chemical expansion. When the crystal orientations at the interface between the film and substrate are completely random, i.e. the film is polycrystalline, the macroscopic strain caused by lattice mismatch is averaged to zero. Compressive strain, the subject of this study, is expressed by a negative value of εin in equation (1). εin.misfit depends on the combination of film–substrate materials and crystal orientations. εin.thermal depends on the fabrication process and conditions. 1.1. Strain at an interface between proton-conductive and non-proton-conductive materials Interfaces exist where proton-conductive and non-proton-conductive materials meet in PCFCs, εin.hyd is a function of temperature, water partial pressure, and the chemical composition of the materials. Here, i = {h k l} and j = {h′ k′ l′} are the lattice planes of the film and substrate, respectively, that form the interface, f ij is the fraction of the junction at the interface of the i–j plane combination, and di and dj are the atomic spacings on the i and j planes at the thermal-strain-free temperature, T0. A in the misfit strain term is a constant related to the relaxation of the lattice misfit caused by the formation of dislocations and grain boundaries, surface roughness, and the distance from the constrained interface to the relaxed surface. αfilm and αsub are the thermal expansion coefficients for the film and substrate, respectively. εhyd is defined as (ah d −ad )/ad where ah d and ad are the lattice constants under hydrated and dehydrated states 1. Introduction The results provide information on the physicochemical fundamentals of hydration, proton diffusion, and proton trapping under compressive interfacial strain, and show that this strain can explain the ohmic resistance of the high-performance PCFCs reported by Bae et al [66]. 2. Results and discussion BZY20 thin films with 18 and 500 nm thicknesses were prepared on Nb-doped SrTiO3 (001) single-crystal substrates using pulsed laser deposition (PLD). The thicknesses of the two films were determined from the interference fringes of the (002) diffraction peaks (figures 2(a) and S2) and cross-sectional scanning electron 3 J. Phys. Energy 4 (2022) 044003 J Hyodo and Y Yamazaki able 1. Summary of proton conduction in electrolyte film and power densities for PCFCs. Electrolyte materials are doped barium zirconates or cerates. Rohm, Rbulk, Rtotal, σfilm, σbulk, and PPD are the ohmic area-specific resistance, xpected ohmic area-specific resistance from bulk conductivity and film thickness, total resistance, the total conductivity of electrolyte thin film in single cells, the total conductivity of bulk pellet, and peak power density at 600 ◦C, spectively. 2. Results and discussion lectrolyte materials Thickness (µm) Rohm (Ωcm2) Rbulk (Ωcm2) Rohm/Rtotal σfilm/σbulk PPD (mW cm−2) Coating method Sintering temperature (◦C) Year References (PCFC) References (σbulk) aZr0.4Ce0.4Y0.2O3−δ 35 1.24 0.63 0.87 0.51 160 Co-pressing 1450 2009 [42] [91] aZr0.8Y0.2O3−δ 20 1.40 0.09 0.52 0.06 70 Co-pressing 1400 2010 [43] [17] aZr0.1Ce0.7Y0.1Yb0.1O3−δ ∼15 0.55 0.05 0.34 0.10 200 Co-pressing 1400 2010 [44] [78] aZr0.1Ce0.7Y0.2O3−δ ∼30 0.50 0.16 0.65 0.33 393 Co-pressing 1450 2010 [53] [79] aZr0.9Y0.1O3−δ 20 0.88 0.09 0.77 0.10 169 Co-pressing 1450 2011 [55] [17]a aZr0.1Ce0.7Y0.1Yb0.1O3−δ ∼15 0.36 0.05 0.40 0.15 289 Co-pressing 1350 2011 [56] [78] aZr0.1Ce0.7Y0.2O3−δ 20 0.38 0.11 0.52 0.28 350 Slurry coating 1400 2011 [45] [79] aZr0.1Ce0.7Y0.2O3−δ 17.6 0.38 0.10 0.74 0.25 747 Tape casting 1300 2011 [46] [79] aZr0.4Ce0.4Y0.2O3−δ ∼20 0.91 0.35 0.74 0.39 194 Co-pressing 1400 2012 [47] [91] aZr0.7Sn0.1Y0.2O3−δ 12 0.46 0.78 0.53 1.69 214 Slurry coating 1400 2013 [57] [57] aZr0.1Ce0.7Y0.1Yb0.1O3−δ ∼60 1.60 0.21 0.71 0.13 225 Slurry coating 1400 2013 [58] [78] aZr0.1Ce0.7Y0.1Yb0.1O3−δ ∼10 0.20 0.04 0.48 0.18 430 Slurry coating 1400 2013 [59] [78] aZr0.1Ce0.7Y0.2O3−δ ∼20 0.45 0.07 0.58 0.14 320 Co-pressing 1350 2013 [48] [79] aZr0.1Ce0.7Y0.1Yb0.1O3−δ ∼15 0.73 0.05 0.49 0.07 193 Co-pressing 1450 2013 [60] [78] aZr0.8Y0.15In0.05O3−δ 12 0.58 0.73 0.54 1.26 221 Slurry coating 1400 2014 [61] [61] aZr0.3Ce0.5Y0.2O3−δ ∼27 0.44 0.31 0.69 0.72 396 Co-pressing 1400 2014 [62] [62] aZr0.1Ce0.7Y0.1Yb0.1O3−δ 14.7 0.13 0.05 0.31 0.41 690 Slurry coating 1400 2014 [63] [78] aZr0.1Ce0.7Y0.1Yb0.1O3−δ 24 0.40 0.76 0.88 1.9 600 Slurry coating 1400 2015 [5] [78] aZr0.8Y0.2O3−δ 23 0.31 0.10 0.84 0.32 460 Slurry coating 1400–1450 2015 [5] [17] aZr0.4Ce0.4Y0.2O3−δ ∼5 0.77 0.09 0.30 0.12 102 Slurry coating 1450 2015 [49] [91] aZr0.1Ce0.7Y0.1Yb0.1O3−δ 4 0.36 0.01 0.57 0.04 418 Slurry coating 1300 2016 [64] [78] aZr0.1Ce0.7Y0.2O3−δ 25 0.32 0.14 0.43 0.42 362 Co-pressing 1400 2017 [67] [79] (Continued.) (Continued.) 4 4 J Hyodo and Y Yamazaki J. Phys. Energy 4 (2022) 044003 Table 1. 2. Results and discussion (Continued.) lectrolyte materials Thickness (µm) Rohm (Ωcm2) Rbulk (Ωcm2) Rohm/Rtotal σfilm/σbulk PPD (mW cm−2) Coating method Sintering temperature (◦C) Year References (PCFC) References (σbulk) aZr0.1Ce0.7Y0.1Yb0.1O3−δ 11 0.38 0.04 0.41 0.10 258 Slurry coating 1350 2017 [68] [78] aCe0.55Zr0.3Y0.15O3−δ 5 0.09 0.04 0.49 0.45 1302 Slurry coating 1350 2018 [7] [7] aCe0.4Zr0.4Y0.1Yb0.1O3−δ ∼15 0.16 0.02 0.55 0.15 1098 Slurry coating 1500–1550 2018 [6] [6] aZr0.1Ce0.7Y0.2O3−δ ∼12 0.22 0.06 0.70 0.30 841 Co-pressing 1350 2019 [70] [79] aZr0.1Ce0.7Y0.2O3−δ ∼18 0.26 0.10 0.60 0.37 643 Co-pressing 1320 2019 [71] [79] aZr0.1Ce0.7Y0.1Yb0.1O3−δ ∼10 0.76 0.03 0.72 0.05 243 Slurry coating 1350 2019 [72] [78] aZr0.3Ce0.5Y0.1Yb0.1O3−δ ∼30 0.89 0.06 0.37 0.07 74 Tape casting 1450 2020 [73] [80] aZr0.1Ce0.7Y0.1Yb0.1O3−δ ∼10 0.11 0.03 0.38 0.31 1060 Tape casting 1400 2021 [74] [78] aZr0.1Ce0.7Y0.2O3−δ 15 0.38 0.08 0.60 0.21 417 Co-pressing 1350 2021 [75] [79] aZr0.4Ce0.4Y0.1Yb0.1O3−δ 7 0.07 0.01 0.23 0.16 1474 Slurry coating 1500 2021 [9] [6] aZr0.1Ce0.7Y0.1Yb0.1O3−δ 15 0.17 0.05 0.72 0.31 1160 Slurry coating 1450 2021 [8] [78] aZr0.4Ce0.4Y0.1Yb0.1O3−δ 22 0.071 0.069 0.63 0.97 1184 Tape casting 1400 2022 [10] [10] aCe0.8Y0.2O3−δ 0.7 0.08 0.003 0.42 0.04 1400 PLD n/a 2005 [50] [77] aZr0.8Y0.2O3−δ 4 1.90 0.02 0.77 0.009 110 PLD 800 2010 [54] [17] aCe0.55Zr0.3Y0.15O3−δ 1.5 0.15 0.02 0.38 0.12 508 PLD 1200 (post annealing) 2016 [51] [7] aCe0.8Y0.2O3−δ 1 0.26 0.004 0.67 0.02 1050 RF sputtering 700 (post annealing) 2016 [52] [77] aZr0.8Y0.2O3−δ 0.075 0.12 (400 ◦C) 0.001 0.04 (400 ◦C) 0.01 (400 ◦C) 274 (400 ◦C) PLD 800 2016 [65] [17] aZr0.85Y0.15O3−δ 2.5 0.09 0.01 0.36 0.12 740 PLD 1200 (post annealing) 2017 [66] [17] aCe0.55Zr0.3Y0.15O3−δ 1 0.06 0.01 0.22 0.19 1100 PLD 1200 (post annealing) 2018 [69] [7] aZr0.5Sc0.5O3−δ 1 0.08 (500 ◦C) 0.008 0.30 (500 ◦C) 0.10 (500 ◦C) 931 (500 ◦C) PLD 700 2021 [76] [18]b The total conductivity is assumed to be the same as that for BaZr0 8Y0 2O3−δ. 5 5 J. Phys. Energy 4 (2022) 044003 J Hyodo and Y Yamazaki Figure 2. Crystal structure of BZY20 thin films. (a) A 2θ −θ scan. (b)–(d) (110) Pole figures for BZY20 (BZY) films with thicknesses of (b) 18 nm and (c) 500 nm, and (d) Nb-doped SrTiO3 (STO) substrate. Figure 2. Crystal structure of BZY20 thin films. (a) A 2θ −θ scan. (b)–(d) (110) Pole figures for BZY20 (BZY) films with thicknesses of (b) 18 nm and (c) 500 nm, and (d) Nb-doped SrTiO3 (STO) substrate. 2. Results and discussion microscopy (SEM) images (figure S3). The two films are much thicker than 5 nm, the thickness at which dislocations are intensively formed at the interface with the substrate [36, 84]. By performing electrochemical measurements in the out-of-plane direction, the influence of fast ionic conduction along the dislocations on the total conductivity was minimized. y The two films were found to be epitaxially grown on the (001) single-crystal substrates. The 2θ −θ profiles determined using x-ray diffractometry show peaks that correspond to (00l) planes for both the BZY20 films and the Nb-SrTiO3 substrates, as shown in figure 2(a). This shows that the BZY20 films are oriented perpendicular to the (001) substrates. The pole figures for the (110) plane exhibit poles with four-fold symmetry at Ψ = 45◦in figures 2(b) and (c), where the positions are equivalent to those for the (001) Nb-SrTiO3 single-crystal substrate in figure 2(d). These results indicate cube-on-cube epitaxy on the substrate. A quantitative analysis of the in-plane lattice constant revealed that biaxial compressive strain was induced in the as-prepared BZY20 thin films. The asymmetric 103 reflection was used for reciprocal space mapping (RSM) to gain in-plane and out-of-plane information for the films, as shown in figures 3(a) and (b). Here, qx and qz show the reciprocal spaces horizontal and vertical, respectively, to the direction of the x-rays at an incidence angle of ω = 0, defined as qx,z = 1/dhkl, where dhkl is the (hkl) lattice spacing. The large spots at the bottom of figures 3(a) and (b) represent diffraction from the (103) planes in the BZY20 films. The in-plane and out-of-plane lattice parameters, ain and aout, respectively, obtained from qx and qz using the (103) spots with correlations qx = 1/d100 = 1/ain and qz = 1/d003 = 3/aout are plotted against the sample thickness and compared with that of the bulk BZY20 sample in figure 3(c). The in-plane lattice constant (ain) decreases with the decreasing sample thickness, showing that the in-plane compression is stronger close to the substrate. The biaxial strain induced by the in-plane compression is −2.1% and −0.85% for the 18 and 500 nm thick films, respectively. These results suggest that the compressive strain in the films is mechanically introduced by the substrate. The out-of-plane lattice constant (aout) indicates that the mechanical stress in the BZY20 films is almost fully relaxed, consistent with an elastic body with respect to Poisson’s ratio. 2. Results and discussion The values of aout increase slightly with the decreasing film thickness in figure 3(c), consistent with previous reports on yttrium-doped barium zirconate films [31, 36]. The blue line in figure 3(c) shows the degree of expansion in the out-of-plane 6 J. Phys. Energy 4 (2022) 044003 J. Phys. Energy 4 (2022) 044003 J Hyodo and Y Yamazaki Figure 3. Lattice distortion in BZY20 thin films. (a) and (b) RSM for 103 reflection for samples with different thicknesses. (c) Estimated in-plane and out-of-plane lattice constants. (d) Estimated lattice volume as a function of film thickness. The lattice constants for the bulk sample and Nb-SrTiO3 (Nb-STO) substrate are shown as black and blue dashed lines, respectively. Figure 3. Lattice distortion in BZY20 thin films. (a) and (b) RSM for 103 reflection for samples with different thicknesses. (c) Estimated in-plane and out-of-plane lattice constants. (d) Estimated lattice volume as a function of film thickness. The lattice constants for the bulk sample and Nb-SrTiO3 (Nb-STO) substrate are shown as black and blue dashed lines, respectively. Table 2. Chemical composition and material properties for BZY20 bulk and film samples. The relative dielectric constant (εγ) and apparent activation energy (Qapp) were determined from the bulk proton conductivity. Thickness Compositiona (f.u.−1) εγ (unitless) Qapp (kJ mol−1) Temperature range for Q (◦C) Ba Zr Y H 18 nm 0.96 ± 0.19 (n/a) 0.73 ± 0.15 (n/a) 0.20 ± 0.04 (n/a) 0.091 ± 0.018 118 ± 16 111 ± 2 168–399 500 nm 0.99 ± 0.03 (0.97) 0.71 ± 0.15 (0.82) 0.20 ± 0.04 (0.20) 0.12 ± 0.03 28 ± 5 54 ± 1 29–163 0.5 mm (1.0) (0.75) (0.21) 0.15 55 ± 1 50 ± 1 30–196 a The chemical compositions in parentheses are the results from EDS. The chemical compositions without parenthesis were determined using SIMS in the depth regions of 5–15 nm and 100–400 nm for the 18 and 500 nm thick films, respectively. The BZY20 bulk sample was used as a reference for converting the SIMS signals to chemical compositions. n/a: not available. Table 2. Chemical composition and material properties for BZY20 bulk and film samples. The relative dielectric constant (εγ) an apparent activation energy (Qapp) were determined from the bulk proton conductivity. a The chemical compositions in parentheses are the results from EDS. 2. Results and discussion The chemical compositions without parenthesis were determined using SIMS in the depth regions of 5–15 nm and 100–400 nm for the 18 and 500 nm thick films, respectively. The BZY20 bulk sample was used as a reference for converting the SIMS signals to chemical compositions. n/a: not available. direction for an elastic body with a Poisson’s ratio of ν = 0.237 for non-doped barium zirconate [85]. The blue line matches well with the experimentally determined lattice parameters in the out-of-plane direction, which confirms that the two films are elastic bodies. We therefore do not need to consider out-of-plane stress because it is fully relaxed. As expected for a Poisson’s ratio smaller than 0.5, the apparent lattice volume, V = ain2aout, is smaller for thinner samples, as shown in figure 3(d). A SIMS analysis showed that the chemical compositions of the films are very close to BZY20. Using energy-dispersive x-ray spectrometry (EDS), the chemical composition of the PLD target sample was determined to be Ba1.0Zr0.75Y0.21O3−δ (EDS, table 2). This was used as a reference for determining the chemical compositions of the films using SIMS. The chemical compositions determined using SIMS near the surface and heterointerface are highly uncertain due to differences in ionization yields, and these regions, highlighted in gray in figures 4(a) and (b), were excluded from the chemical analyses. The average chemical compositions thus determined in the thinner and thicker films were found to be Ba0.96±0.19Zr0.73±0.15 Y0.20±0.04O3−δ and Ba0.99±0.03Zr0.71±0.15Y0.20±0.04O3−δ, respectively, both of which are almost equivalent to that of the bulk sample, Ba1.0Zr0.75Y0.21O3−δ. The Ba, Zr, and Y cations in the 500 nm thick film were uniformly distributed in the depth direction. As shown in figure 4(b), the depth profiles of the cations were almost constant. Sharp drops in the signals for 7 7 J Hyodo and Y Yamazaki J. Phys. Energy 4 (2022) 044003 J. Phys. Energy 4 (2022) 044003 Figure 4. Chemical compositions of BZY20 thin films determined by SIMS. (a) A 18 and (b) 500 nm thick films. (c) Proton concentration (CH) in films and bulk sample. The left and right axes in (a) and (b) correspond to the chemical compositions of the films and the secondary ion mass signals for elements observed from the Nb-doped SrTiO3 substrate, respectively. 2. Results and discussion The gray vertical bands in (a) and (b) represent uncertain regions in the quantification based on ionization yields at the surface and heterointerface differences from the bulk domain. The error bars in (c) show the range of reproducibility for the two measurements. The raw thermogravimetry data for the bulk sample are given in figure S6. Figure 4. Chemical compositions of BZY20 thin films determined by SIMS. (a) A 18 and (b) 500 nm thick films. (c) Proton concentration (CH) in films and bulk sample. The left and right axes in (a) and (b) correspond to the chemical compositions of the films and the secondary ion mass signals for elements observed from the Nb-doped SrTiO3 substrate, respectively. The gray vertical bands in (a) and (b) represent uncertain regions in the quantification based on ionization yields at the surface and heterointerface differences from the bulk domain. The error bars in (c) show the range of reproducibility for the two measurements. The raw thermogravimetry data for the bulk sample are given in figure S6. Figure 4. Chemical compositions of BZY20 thin films determined by SIMS. (a) A 18 and (b) 500 nm thick films. (c) Proton concentration (CH) in films and bulk sample. The left and right axes in (a) and (b) correspond to the chemical compositions of the films and the secondary ion mass signals for elements observed from the Nb-doped SrTiO3 substrate, respectively. The gray vertical bands in (a) and (b) represent uncertain regions in the quantification based on ionization yields at the surface and heterointerface differences from the bulk domain. The error bars in (c) show the range of reproducibility for the two measurements. The raw thermogravimetry data for the bulk sample are given in figure S6. strontium and titanium near the interface suggest that the elements in the substrate did not significantly diffuse into the BZY20 layer. Although it was difficult to determine the cation concentration for the 18 nm thick film (figure 4(a)) due to the large uncertainties near the surfaces and interfaces, the homogeneity for the 500 nm thick film implies that the 18 nm thick film is likely also homogeneous. The proton concentrations for the thick and thin films determined by SIMS are equivalent to that for bulk BZY20. The films were hydrated at 400 ◦C for 24 h under a flow of humidified air at pH2O ∼0.02 atm. 2. Results and discussion Proton conductivities in BZY20 thin films. (a) Arrhenius plots of bulk proton conductivities, (b) H/D isotope effects, (c) influence of misfit strain on proton conductivities at 375 ◦C, (d) influence of misfit strain on apparent activation energies for proton conductivities. The red line in (a) shows data from [17]. The solid lines in (b) are guides for the eye, while the dashed line in (b) is the theoretical value. The negative values on the x-axis in (c) and (d) represent compressive strain. Data from [34] are shown in (c) and (d). The solid line in (d) is a result of fitting to a parabolic function, suggested by [34], using all the data including [34]. been previously reported [34], and our data show a smooth extension from the literature data with a parabolic relation, as has been suggested [34] (solid line in figure 5(d)), demonstrating good agreement. The isotope effect between H and D supports proton conduction in BZY20 films. An ideal harmonic been previously reported [34], and our data show a smooth extension from the literature data with a parabolic relation, as has been suggested [34] (solid line in figure 5(d)), demonstrating good agreement. The isotope effect between H and D supports proton conduction in BZY20 films. An ideal harmonic oscillator affecting the vibrational frequency [86] would, in theory, give σHσD−1 = 1.41. Such isotope effects are apparent for all bulk and film samples (figures 5(a) and (b)), although the values of σHσD−1 increase from 1.4 to 4 with the decreasing temperature. The reason for this is related to the difference in the ground-state energy for H and D, which increases at lower temperatures [86]. The relative dielectric constants for the 18 and 500 nm thick films of 118 ± 16 and 28 ± 5, respectively, also agree with the value of 55 ± 1 for the bulk sample, and the literature values [17]. In summary, all the results support protons being the major conduction carriers in the BZY20 films. A reduction in proton diffusion significantly reduces the proton conductivity in BZY20 films. 2. Results and discussion Thermogravimetry results for a SIMS pellet standard are given in figure S4 and those for a powder sample are given in figure S5. The proton concentrations determined by averaging the SIMS signals (red lines) for the 18 and 500 nm thick films in figures 4(a) and (b), excluding the near-surface and interface regions, are 0.091 ± 0.018 and 0.12 ± 0.03 per formula unit, respectively, as shown in figure 4(c). The thinner film had a slightly smaller concentration than the bulk BZY20, by ∼40% at most, whereas the thicker film had a concentration that was equivalent to that for the bulk BZY20. The proton conductivity for the hydrated BZY20 films was significantly reduced compared to that for the bulk sample, more significantly for the thinner film, which exhibited more compressive strain. At about 375 ◦C, for example, the proton conductivities for the 500 and 18 nm thick films were, respectively, about one and five orders of magnitude smaller than those for the bulk, as shown in figure 5(a), with representative Nyquist plots and fits given in figure S6. The conductivity data for the BZY20 bulk sample are consistent with the literature [17]. A plot against biaxial strain shows a gradual decrease in the proton conductivity by five orders of magnitude (figure 5(c)), and a gradual increase in apparent activation energy for proton conduction, Qapp, from 50 ± 1 to 111 ± 2 kJ mol−1 (figure 5(d)) with the increasing compressive strain to −2.1%. The influence of biaxial strain on the activation energy for proton conduction in BZY20 films has 8 J. Phys. Energy 4 (2022) 044003 J. Phys. Energy 4 (2022) 044003 J Hyodo and Y Yamazaki Figure 5. Proton conductivities in BZY20 thin films. (a) Arrhenius plots of bulk proton conductivities, (b) H/D isotope effects, (c) influence of misfit strain on proton conductivities at 375 ◦C, (d) influence of misfit strain on apparent activation energies for proton conductivities. The red line in (a) shows data from [17]. The solid lines in (b) are guides for the eye, while the dashed line in (b) is the theoretical value. The negative values on the x-axis in (c) and (d) represent compressive strain. Data from [34] are shown in (c) and (d). The solid line in (d) is a result of fitting to a parabolic function, suggested by [34], using all the data including [34]. Figure 5. 2. Results and discussion The temperature dependence of εin.hyd given in figure S7 for the 500 nm thick film is considered for solid lines in (b) and (c), which increases the compressive strain from −0.85% into green area (details and resulting parameters are given in supporting information and table S1, respectively). The yellow and orange regions in (c) represent the contributions of D0 and Ea, respectively, to the retarded mobile proton diffusivity (DH.m). Figure 6. Influence of in-plain strain, εin, on proton diffusion behavior in BZY20 thin films. (a) Arrhenius plots of apparent proton diffusivities, DH.app, (b) parameters for mobile proton diffusion and proton trapping, and (c) mobile proton diffusivity and mobile proton concentration at 375 ◦C. The green dashed line in (a) shows data from [26]. The negative values on the x-axes of εin in (b) and (c) represent compressive strains. The solid lines in (a) and (b) show the results of fitting the obtained data using equation (4). The temperature dependence of εin.hyd given in figure S7 for the 500 nm thick film is considered for solid lines in (b) and (c), which increases the compressive strain from −0.85% into green area (details and resulting parameters are given in supporting information and table S1, respectively). The yellow and orange regions in (c) represent the contributions of D0 and Ea, respectively, to the retarded mobile proton diffusivity (DH.m). feature of proton trapping [26], and is one order of magnitude smaller than that for the bulk sample, whereas that for the 18 nm thick film shows a linear decrease by up to five orders of magnitude. To investigate slow proton diffusion in the films, a simple proton trapping model is introduced, similar to that for hydrogen trapping in metals [87], where mobile and trapped protons in a solid are locally in equilibrium, OH• O.m Ktrap ←→OH• O.trap. (2) (2) Here, the left and right sides of the equation represent mobile and trapped protons, respectively, and Ktrap is the equilibrium constant for proton trapping. The phenomenological equilibrium model is used without specifying the position of protons because of the lack of information on how the compressive strain changes the position of protons associated with the dopant under the biaxial compressive strain. 2. Results and discussion As expected from the close equivalence of the proton concentration (40% difference at most), the apparent proton diffusivity, DH.app, obtained by inputting the proton concentration for bulk BZY20 (black line in figure 4(c)) into the Nernst–Einstein relationship is one and five orders of magnitude smaller for the 500 and 18 nm thick films, respectively, than those for the bulk samples in this work and the literature [26] for the whole temperature range in figure 6(a), noting that the results for bulk BZY20 in this work and the literature are consistent. The apparent proton diffusivity for the 500 nm thick film shows a downward curvature, a 9 J. Phys. Energy 4 (2022) 044003 J. Phys. Energy 4 (2022) 044003 J Hyodo and Y Yamazaki Figure 6. Influence of in-plain strain, εin, on proton diffusion behavior in BZY20 thin films. (a) Arrhenius plots of apparent proton diffusivities, DH.app, (b) parameters for mobile proton diffusion and proton trapping, and (c) mobile proton diffusivity and mobile proton concentration at 375 ◦C. The green dashed line in (a) shows data from [26]. The negative values on the x-axes of εin in (b) and (c) represent compressive strains. The solid lines in (a) and (b) show the results of fitting the obtained data using equation (4). The temperature dependence of εin.hyd given in figure S7 for the 500 nm thick film is considered for solid lines in (b) and (c), which increases the compressive strain from −0.85% into green area (details and resulting parameters are given in supporting information and table S1, respectively). The yellow and orange regions in (c) represent the contributions of D0 and Ea, respectively, to the retarded mobile proton diffusivity (DH.m). Figure 6. Influence of in-plain strain, εin, on proton diffusion behavior in BZY20 thin films. (a) Arrhenius plots of apparent Figure 6. Influence of in-plain strain, εin, on proton diffusion behavior in BZY20 thin films. (a) Arrhenius plots of apparent proton diffusivities, DH.app, (b) parameters for mobile proton diffusion and proton trapping, and (c) mobile proton diffusivity and mobile proton concentration at 375 ◦C. The green dashed line in (a) shows data from [26]. The negative values on the x-axes of εin in (b) and (c) represent compressive strains. The solid lines in (a) and (b) show the results of fitting the obtained data using equation (4). 2. Results and discussion The concentration of mobile protons is expressed as [OH• O.m] = [OH• O]total 1 + Ktrap , = [OH• O]total 1 + Bexp(−Etrap/RT) (3) (3) where the brackets denote the concentration of the respective species, and R, T, Etrap, and B are the universal gas constant, absolute temperature, proton trapping energy, and pre-exponential factor for proton trapping, respectively (see the full description in the supporting information). The apparent proton diffusivity, DH.app, for macroscopic proton transport is the product of the diffusivity of mobile protons, DH.m, and the fraction of mobile protons relative to the total number of protons, DH.app = DH.m [OH• O.m] [OH• O]total , = D0 exp ( −Ea RT ) [OH• O.m] [OH• O]total (4) (4) where D0 is a pre-exponential factor and Ea is the migration energy for the diffusion of mobile protons. The model independently returns the parameters that describe the impact of mobile proton diffusion and proton trapping on proton diffusivity. The parameters obtained without considering εin.hyd are listed in where D0 is a pre-exponential factor and Ea is the migration energy for the diffusion of mobile protons. The model independently returns the parameters that describe the impact of mobile proton diffusion and proton trapping on proton diffusivity. The parameters obtained without considering εin.hyd are listed in 10 J. Phys. Energy 4 (2022) 044003 J Hyodo and Y Yamazaki J. Phys. Energy 4 (2022) 044003 Table 3. Parameters for proton diffusivity in proton trapping model. These values are obtained for a 500 nm thick film and bulk samples with constant εin. Those dependent on εin are listed in table S1. Thickness D0 (10−5 cm2 s−1) Ea (kJ mol−1) B (unitless) Etrap (kJ mol−1) 500 nm 2.6 ± 0.8 28 ± 1 (1.8 ± 0.9) × 10−3 −28 ± 2 0.5 mm 10 ± 3 23 ± 1 (5.6 ± 2.1) × 10−4 −30 ± 1 3. Parameters for proton diffusivity in proton trapping model. These values are obtained for a 500 nm thick film and bulk samples constant εin. Those dependent on εin are listed in table S1. table 3 and plotted as circles against the in-plane strain in figure 6(b). The compressive strain decreases and increases D0 and Ea, respectively, the parameters for the diffusivity of mobile protons. 2. Results and discussion The negligible influence of compressive strain on proton trapping shows that the hydrogen bond angle around YO6 octahedra, considered to be trapping sites [27, 29], is not altered by the strain. A compressive strain of −1% slightly alters the shape of YO6 octahedra, but trapped protons bonded to the apex of YO6 octahedra have enough freedom to maintain the initial hydrogen bond angle. This could be the reason why compressive strain does not have a large influence on proton trapping. The mechanical strain accumulated in the electrolyte of a PCFC can be estimated using equation (1). A PCFC with a high power density of 740 mW cm−2 at 600 ◦C, composed of a BaZr0.75Y0.15O3−δ and nickel cermet anode, a 2.5 µm thick BaZr0.75Y0.15O3−δ polycrystalline electrolyte, and a La0.6Sr0.4CoO3−δ cathode [66], was selected for the evaluation. The parameters εin.misfit, αBZY20, αNiO, T0, and εin.hyd for the PCFC were 0 (polycrystalline), 1200 ◦C (maximum temperature that the cell experienced in the fabrication process), 9.05 × 10−6 K−1 (median of reported values [1, 22–25]), 13.08 × 10−6 K−1 (median of reported values of 11.23 −14.93 × 10−6 K−1 [90]), and 8.47 × 10−3 · C0.62 H [25], respectively. It is noted that α for nickel, 13 × 10−6 K−1 [24], is equivalent to that for NiO, so there will be a negligible difference between nickel metal and its oxide in the evaluation of strain. By inserting these material parameters for the high-performance PCFC into equation (1), we obtain the temperature dependence of the in-plane thermal and chemical strain shown in figure 7(a). For the estimate of εin.hyd, it is assumed that the proton concentration in the electrolyte in the fuel cell, CH, is identical to that for the bulk sample (black line in figure 4(c)). Lowering the temperature increases both the thermal and chemical strain in the electrolyte of PCFCs to −0.5% and −0.3%, respectively, thus −0.8% in total, at room temperature (figure 7(a)). The estimate of the mechanical strain combined with the quantitative model for proton conduction under compressive strain constructed in this work reveal why proton conductivity in the electrolyte of actual fuel cells is much lower than that for the constituent materials. The estimated mechanical strain gives values for the four diffusion parameters D0, Ea, B, and Etrap in the model, which simulates the proton conductivity in the electrolyte of a high-performance PCFC (red line in figure 7(b)). 2. Results and discussion Both contribute almost equally to a significant decrease in DH.m of more than one order of magnitude at εin = −1.12% compared with εin = 0 at 375 ◦C in figure 6(c). The increase in Ea observed under biaxial compression is in good agreement with the results of molecular dynamics simulations [41]. In contrast, Etrap and B, the parameters for proton trapping (figure 6(b)) and CH.m (figure 6(c)) have a negligible influence from a compressive strain of −1% considering uncertainties in the experiments and fitting. Since Etrap quantitatively correlates with the hydration enthalpy, a parameter describing the proton concentration, CH [88], it is reasonable that both CH in figure 4(c) and Etrap in figure 6(b) have an almost negligible influence from compressive strain. An influence due to compressive strain on mobile proton diffusion and proton trapping is observed over the whole temperature range examined in figure S8. All the results consistently support the idea that biaxial compressive strain mainly suppresses the diffusivity of mobile protons in the film. The larger activation energy for mobile proton diffusion, Ea, and the lower D0 under a −1% compressive strain are related to the shrinkage of the perovskite structure and anisotropic deformation of BO6 octahedra. The activation energy for mobile proton diffusion represents the energy needed to overcome the saddle point from the initial state [89]. The smaller lattice volume in the BYZ20 thin film in figure 3(d) requires more energy than for the bulk sample. Anisotropically deformed BO6 octahedra under compressive strain break the even energy landscape around oxygen sites that are important for fast proton diffusion with lower activation energy in a cubic structure, leading to a lower local crystal symmetry with a larger activation energy. A greater variety of proton sites under compressive strain causes higher entropy in the initial and final states before and after proton diffusion, respectively, which reduces the change in the configurational entropy between the initial and transition states. Similarly, the change in vibrational entropy, related to the vibrational frequencies of protons affected by the local environment in the solid, is lowered for the resulting lower D0. Mobile proton diffusion behavior for larger Ea and lower D0 in a biaxially compressed film can be consistently understood in terms of the physicochemical fundamentals of diffusion in solids. 3. Conclusions Biaxial in-plain strain, proton concentration, proton conductivities, and proton/deuteron isotope effects in high-quality epitaxial BZY20 films, 18 and 500 nm in thickness, grown on Nb-doped SrTiO3 single crystal substrates by PLD, were quantitatively evaluated in the temperature range between 28 ◦C and 604 ◦C (400 ◦C for proton concentration) and compared with those for a compositionally equivalent bulk sample. Thin-film X-ray diffractometry, SIMS, SEM-EDS, thermogravimetry, and AC impedance spectroscopy were used. Both films showed epitaxial growth on Nb-doped SrTiO3 (001) single-crystal substrates. The chemical compositions of the films were almost equivalent to that of the bulk sample. The biaxial compressive misfit strains introduced into the 500 and 18 nm thick films were −0.85% and −2.1%, respectively. The proton concentration for the 500 nm thick film hydrated at 400 ◦C for 24 h at pH2O ∼0.02 atm was almost equivalent to that for the bulk sample, while that for the 18 nm thick film was slightly smaller, by ∼40% at most. The compressive misfit strains of −0.85% and −2.1% in the 500 and 18 nm thick films inhibit both proton conduction and proton diffusion by one and five orders of magnitude, respectively, at 375 ◦C. An analysis based on a simple proton trapping model quantified the influence of biaxial compressive strain on four parameters related to proton diffusion, namely the pre-exponential factor and activation energy for mobile protons, and the pre-exponential factor and energy for proton trapping. The analysis revealed that the retardation of proton diffusion in the film did not result from the contribution of proton trapping but from the slower diffusion of mobile protons. A biaxial compressive misfit strain of −0.85% increased the activation energy for mobile protons from 23 to 28 kJ mol−1 and decreased the pre-exponential factor from 1.0 × 10−4 to 2.6 × 10−5 cm2 s−1, resulting in a more than one order of magnitude retardation at 375 ◦C. The retarded mobile proton diffusion with larger Ea and lower D0 in the biaxially compressed film is understood to be related to the non-uniform energy states of protons due to anisotropic deformation of BO6 octahedra in the compressed perovskite lattice. 2. Results and discussion The simulation quantitatively agrees with the values (red circles) reported for a high-performance PCFC [66] and the degree of suppression of the proton conductivity, σfilm/σbulk, by a factor of 0.22–0.36, in the literature (table 1). Although the contact 11 J. Phys. Energy 4 (2022) 044003 J Hyodo and Y Yamazaki J. Phys. Energy 4 (2022) 044003 Figure 7. Estimates of (a) biaxial compressive strain, εin, and (b) proton conductivity, σH, in a PCFC. Negative values of εin express compressive strain. The red and blue regions in (a) show thermal and chemical strains, respectively. The black line in (b) shows the proton conductivities in bulk samples while the red filled circles show the values obtained from the ohmic resistance of a high-performance PCFC with a power density of 740 mW cm−2 at 600 ◦C [66]. The red line in (b) shows the simulated values for the high-performance PCFC [66] based on the constructed model. The red shaded region in (b) shows the uncertainties due to the scattered values of the thermal expansion coefficients for the NiO anode support and yttrium-doped barium zirconate electrolyte in the literature. Figure 7. Estimates of (a) biaxial compressive strain, εin, and (b) proton conductivity, σH, in a PCFC. Negative values of εin express compressive strain. The red and blue regions in (a) show thermal and chemical strains, respectively. The black line in (b) shows the proton conductivities in bulk samples while the red filled circles show the values obtained from the ohmic resistance of a high-performance PCFC with a power density of 740 mW cm−2 at 600 ◦C [66]. The red line in (b) shows the simulated values for the high-performance PCFC [66] based on the constructed model. The red shaded region in (b) shows the uncertainties due to the scattered values of the thermal expansion coefficients for the NiO anode support and yttrium-doped barium zirconate electrolyte in the literature. resistance [6, 10] and/or interdiffusion of nickel with electrolytes [81, 82] have been considered, the biaxial compressive strain would be a significant contributor to the enlarged ohmic resistance in the reported PCFCs. Our analysis emphasizes that minimizing the biaxial compressive strain in PCFCs by appropriate choices of the electrolyte–electrode combinations and the fabrication process are critical for maximizing the potential of proton-conducting oxides for use in environmentally friendly electrochemical devices. 4.1. Thin-film preparation BaZr0.8Y0.2O3−δ thin films were prepared by the PLD method on Nb-doped SrTiO3 (001) single-crystal substrates (a = 3.905 Å, commercially available from Crystal Base Co., Ltd., Japan) using BaZr0.75Y0.21O3−δ bulk target (lattice constant, a = 4.224 Å). The details of the BZY20 target synthesis are given in the supporting information. The PLD system (PLAD-247, AOV Co. Ltd) was operated with a KrF excimer laser (COMPex Pro 102F, Coherent Inc.) emitting at 248 nm and an energy of 2 J cm−2 with a repetition rate of 1 Hz. During the ablation process, the substrate temperature and oxygen partial pressure were maintained at 700 ◦C and 1 Pa, respectively. The film thickness was controlled by varying the deposition time (10–100 min for the two films, respectively). After deposition, the samples were annealed in the deposition chamber for 5 min to crystallize the deposited films. The chemical composition of the films was measured using EDS in conjunction with SEM (SU3500, Hitachi High-Technologies Co.) and SIMS, as described below. Because the X-ray signal intensity was lower than the detection limit, the chemical composition of the 18 nm thick film could not be determined using EDS. 4.3. Crystal structure analysis The crystal structure of the dehydrated thin films was analyzed using a line focus x-ray source (D8 DISCOVER, Bruker AXS). The 2θ −θ profiles were obtained, and RSM was performed using a monochromatic Cu–Kα line with a one-dimensional detector (LYNXEYE, Bruker AXS). The in-plane and out-of-plane lattice constants were estimated from the RSM results for the 103 peak. For pole figure measurements, a collimated x-ray beam with a diameter of 0.5 mm and a two-dimensional detector (VÅNTEC 500, Bruker AXS) were used. Pole figures for the (110) planes in the deposited BZY20 thin films were recorded with a rotation angle (Φ) step size of 3◦and tilt angles (Ψ) of 35◦and 75◦along the normal direction of the x-rays. 4.2. Chemical composition analysis by SIMS y y The chemical composition of the deposited BZY20 films was measured by SIMS (SC-Ultra, CAMECA) using primary 1 keV Cs+ ions. Positive ion signals corresponding to the mass of the CsM+ matrix (M = H, Ti, Sr, Nb, Zr, Y, and Ba) were recorded. To quantify the Ba, Zr, Y, and H elements in the film, a hydrated BZY20 bulk sample (BaZr0.75Y0.21O2.89H0.15) was used as a reference. The sputtering rate was determined by analyzing the depth of craters for the 500 nm thick sample after a depth profile measurement with a surface profiler (P-16+, KLA-Tencor). We note that it is difficult to distinguish between Nb and Zr–H in our SIMS system equipped with a quadrupole mass spectrometer. 3. Conclusions Energy 4 (2022) 044003 literature. Mechanical compressive strain could be the origin of the large ohmic resistance observed in PCFCs, so minimizing biaxial compressive strain in PCFCs with an appropriate choice of electrolyte–electrode combinations and optimizing the fabrication process for single cells are critical for maximizing the potential applications of proton-conducting oxides as electrolytes in environmentally friendly electrochemical devices. The insights obtained in this study should help in the design of high-performance proton-based electrochemical devices. 3. Conclusions Biaxial in-plain strain, proton concentration, proton conductivities, and proton/deuteron isotope effects in high-quality epitaxial BZY20 films, 18 and 500 nm in thickness, grown on Nb-doped SrTiO3 single crystal substrates by PLD, were quantitatively evaluated in the temperature range between 28 ◦C and 604 ◦C (400 ◦C for proton concentration) and compared with those for a compositionally equivalent bulk sample. Thin-film X-ray diffractometry, SIMS, SEM-EDS, thermogravimetry, and AC impedance spectroscopy were used. Both films showed epitaxial growth on Nb-doped SrTiO3 (001) single-crystal substrates. The chemical compositions of the films were almost equivalent to that of the bulk sample. The biaxial compressive misfit strains introduced into the 500 and 18 nm thick films were −0.85% and −2.1%, respectively. The proton concentration for the 500 nm thick film hydrated at 400 ◦C for 24 h at pH2O ∼0.02 atm was almost equivalent to that for the bulk sample, while that for the 18 nm thick film was slightly smaller, by ∼40% at most. The compressive misfit strains of −0.85% and −2.1% in the 500 and 18 nm thick films inhibit both proton conduction and proton diffusion by one and five orders of magnitude, respectively, at 375 ◦C. An analysis based on a simple proton trapping model quantified the influence of biaxial compressive strain on four parameters related to proton diffusion, namely the pre-exponential factor and activation energy for mobile protons, and the pre-exponential factor and energy for proton trapping. The analysis revealed that the retardation of proton diffusion in the film did not result from the contribution of proton trapping but from the slower diffusion of mobile protons. A biaxial compressive misfit strain of −0.85% increased the activation energy for mobile protons from 23 to 28 kJ mol−1 and decreased the pre-exponential factor from 1.0 × 10−4 to 2.6 × 10−5 cm2 s−1, resulting in a more than one order of magnitude retardation at 375 ◦C. The retarded mobile proton diffusion with larger Ea and lower D0 in the biaxially compressed film is understood to be related to the non-uniform energy states of protons due to anisotropic deformation of BO6 octahedra in the compressed perovskite lattice. The model allows us to estimate the proton conductivity in the electrolyte of PCFC devices considering thermally and chemically introduced biaxial strain, and the results agree well with the reported values in the 12 J. Phys. Energy 4 (2022) 044003 J Hyodo and Y Yamazaki J. Phys. Acknowledgments We acknowledge JST PRESTO and CREST (Grant Nos. JPMJPR10QD and JPMJCR18J3) and JSPS KAKENHI (JP15H02287, JP16H000891, and JP21H01654). The PLD equipment installed in the Next-Generation Fuel Cell Research Center (NEXT-FC) at Kyushu University was used in this study. The chemical composition was analyzed using SEM-EDS at the Center of Advanced Instrumental Analysis, Kyushu University. We would like to thank Dr Hitoshi Morioka at Bruker Japan for his help with the thin-film X-ray diffraction measurement and analysis. Data availability statement All data that support the findings of this study are included within the article (and any supplementary files). k l d All data that support the findings of this study are included within the article (and any supplementary files). 4.4. Conductivity measurements The electrical conductivity of BZY20 thin films in the vertical direction was determined using AC impedance spectroscopy. The configuration of the BZY20 thin-film sample for AC impedance spectroscopy is schematically given in figure S9. A silver layer with a thickness of 650 nm was deposited onto the surfaces of the thin film and Nb-SrTiO3 substrate using DC sputtering (SC-701HMC II, Sanyu Electron Co., Ltd). The areas of silver electrode for the 18 nm and 500 nm film samples were measured to be 0.37 and 0.15 cm2, respectively, using a standard scale. A current collector made of silver mesh (Nilaco Corporation) was pasted onto the silver layer using silver paste (TR-3025, Tanaka Kikinzoku Kogyo), and connected to an AC impedance spectrometer via gold wires. For adhesion, the sample specimens were heated at 750 ◦C for 2 h before the electrochemical measurements. AC impedance spectroscopy was performed in the temperature range of 28 ◦C–604 ◦C in an argon atmosphere saturated with H2O and D2O at 17.5 ◦C and 20 ◦C, respectively (pH2O = pD2O = 0.02atm) with a flow rate of 100 ml min−1. The AC impedance was recorded by sweeping the frequency from 10−1 to 106 Hz with a voltage amplitude of 10–50 mV (Biologic, VSP-300). The Nyquist plots were fitted with results for an equivalent circuit comprising a parallel structure of RC components in the grain interior and electrode regions. Here, R is an ideal resistor with ZR = R and C is a constant-phase element with ZC = (Y(jω)n)−1, where j is √−1, ω is the frequency, and Y and n are constants with 0 ⩽n ⩽1. The results of impedance measurements below 141 ◦C for the 18 nm thick film were not 13 J Hyodo and Y Yamazaki J. Phys. Energy 4 (2022) 044003 used in this work because no obvious semicircles were observed due to an overlap of the response frequencies for the bulk and electrode (figures S6(d) and (h)). used in this work because no obvious semicircles were observed due to an overlap of the response frequencies for the bulk and electrode (figures S6(d) and (h)). ORCID iDs Junji Hyodo https://orcid.org/0000-0001-5700-5791 Yoshihiro Yamazaki https://orcid.org/0000-0002-9375-5239 References [1] Kreuer K D 2003 Proton-conducting oxides Annu. Rev. Mater. 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W2528119460.txt	https://figshare.com/articles/journal_contribution/A_model_providing_long-term_datasets_of_energetic_electron_precipitation_during_geomagnetic_storms/10209353/1/files/18406064.pdf	en		A model providing long‐term data sets of energetic electron precipitation during geomagnetic storms	Journal of geophysical research. Atmospheres	2,016	cc-by	12,158	Journal of Geophysical Research: Atmospheres RESEARCH ARTICLE 10.1002/2015JD024212 Special Section: Energetic Electron Loss and its Impacts on the Atmosphere Key Points: • A model for radiation belt energetic electron precipitation (30-1000 keV) has been developed • It can be used to calculate the energy-ﬂux spectrum of precipitating electron from 1932 to present • The results can be used to produce the ﬁrst >80 year long atmospheric ionization rate data set Correspondence to: A. Seppälä, annika.seppala@fmi.ﬁ Citation: van de Kamp, M., A. Seppälä, M. A. Clilverd, C. J. Rodger, P. T. Verronen, and I. C. Whittaker (2016), A model providing long-term data sets of energetic electron precipitation during geomagnetic storms, J. Geophys. Res. Atmos., 121, doi:10.1002/2015JD024212. A model providing long-term data sets of energetic electron precipitation during geomagnetic storms M. van de Kamp1 , A. Seppälä1 , M. A. Clilverd2 , C. J. Rodger3 , P. T. Verronen1 , and I. C. Whittaker4 1 Finnish Meteorological Institute, Helsinki, Finland, 2 British Antarctic Survey/NERC, Cambridge, UK, 3 Department of Physics, University of Otago, Dunedin, New Zealand, 4 Department of Physics and Astronomy, University of Leicester, Leicester, UK Abstract The inﬂuence of solar variability on the polar atmosphere and climate due to energetic electron precipitation (EEP) has remained an open question largely due to lack of a long-term EEP forcing data set that could be used in chemistry-climate models. Motivated by this, we have developed a model for 30–1000 keV radiation belt driven EEP. The model is based on precipitation data from low Earth orbiting POES satellites in the period 2002–2012 and empirically described plasmasphere structure, which are both scaled to a geomagnetic index. This geomagnetic index is the only input of the model and can be either Dst or Ap. Because of this, the model can be used to calculate the energy-ﬂux spectrum of precipitating electrons from 1957 (Dst) or 1932 (Ap) onward, with a time resolution of 1 day. Results from the model compare well with EEP observations over the period of 2002–2012. Using the model avoids the challenges found in measured data sets concerning proton contamination. As demonstrated, the model results can be used to produce the ﬁrst ever >80 year long atmospheric ionization rate data set for radiation belt EEP. The impact of precipitation in this energy range is mainly seen at altitudes 70–110 km. The ionization rate data set, which is available for the scientiﬁc community, will enable simulations of EEP impacts on the atmosphere and climate with realistic EEP variability. Due to limitations in this ﬁrst version of the model, the results most likely represent an underestimation of the total EEP eﬀect. 1. Introduction Received 14 SEP 2015 Accepted 2 OCT 2016 Accepted article online 6 OCT 2016 After the discovery of the eﬀects that solar proton events (SPEs) have on mesospheric ozone balance in sounding rocket experiments [Weeks et al., 1972] our understanding of the inﬂuence that energetic particle precipitation (EPP) has on the atmosphere has increased signiﬁcantly. We now understand that EPP provides an important source of odd hydrogen (HOx ) and odd nitrogen (NOx ) in the polar middle atmosphere, between the altitudes of about 30 and 90 km. These in turn inﬂuence the polar ozone balance via several chemical reactions and catalytic reaction chains. Much work has been done to include the eﬀect of proton deposition into atmospheric models [Jackman et al., 2008, 2009; Neal et al., 2013; Nesse Tyssøy and Stadsnes, 2015]. Meanwhile, it has also become clear that SPEs are not the sole source of EPP into the atmosphere. Electron precipitation contributes to ionizaton as well [e.g., Spjeldvik and Thorne, 1976] and to the formation of NOx [Callis et al., 1996]. It is now apparent that when long-term (months–years) impacts are considered, the precipitation of energetic electrons may be at least as important as protons [Funke et al., 2014a; Andersson et al., 2014]. Several studies have addressed the so-called EPP indirect eﬀect [Randall et al., 2007], which takes into account the consequences beyond the initial EPP impact region via coupling to atmospheric dynamics and transport of chemically active species, such as NOx . In a recent study, Randall et al. [2015] highlighted the importance of the roles of both atmospheric dynamical events and the missing EPP contribution from energetic electron precipitation (EEP) in order to correctly simulate the polar winter stratosphere-mesosphere region. ©2016. American Geophysical Union. All Rights Reserved. VAN DE KAMP ET AL. Additional interest for inclusion of EPP (SPE + EEP) in atmospheric models arises from results suggesting links coupling the initial polar middle atmosphere chemical changes to dynamical variables in the stratosphere, propagating down to the troposphere and ground level. The impacts of these are similar in magnitude to those arising from variations in solar spectral irradiance [e.g., Rozanov et al., 2012; Seppälä and Clilverd, 2014; Seppälä et al., 2014]. Thus, EPP potentially provides a pathway from the Sun via magnetospheric processes into polar climate variability [Seppälä et al., 2009, 2013]. These linkages are not yet fully understood mainly EEP MODEL FOR GEOMAGNETIC STORMS 1 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 due to the limited capability of chemistry-climate models to include a full description of EPP and its long-term variability [Seppälä et al., 2014]. Currently, most of the “high-top” models (chemistry-climate models with upper boundary in the mesosphere or above), such as ECHAM5/MESSy [Baumgaertner et al., 2011] and WACCM [Jackman et al., 2009], include SPEs as one of the EPP sources producing HOx and NOx . In addition, some, such as WACCM, include lower energy (auroral) electron and proton precipitation, parameterized by geomagnetic activity proxies, as the thermospheric NOx source. However, inclusion of medium- and high-energy electrons has remained a challenge. In the absence of long-term observations of energetic electron ﬂuxes into the atmosphere, proxies that describe the overall impact of energetic particle eﬀects on the atmosphere have been developed. These are mainly limited to estimating the variability of NOx levels in terms of changes in geomagnetic activity as the two are known to be closely linked [Randall et al., 2007]. For model simulations, proxies for EEP-induced NOx production have been developed, most recently by Baumgaertner et al. [2009] and Funke et al. [2014b]. While these proxies can be used in long-term simulations, they are, however, limited in the sense that they only provide an estimate of the EPP-NOx (or NOy ) at the model upper boundary and cannot resolve the in situ vertical production by medium- to high-energy electrons. Information concerning this vertical production is important as it aﬀects how rapidly the EPP-NOx can reach stratospheric altitudes. For HOx , which is also very important for the atmospheric ozone balance especially at mesospheric altitudes [Verronen et al., 2006, 2011; Andersson et al., 2014], this type of proxy driven upper boundary source approach is invalid due to its very short chemical lifetime. The best way of establishing the long-term variability of middle atmosphere EPP-HOx , and its impact on ozone levels and beyond, is to establish an altitude-dependent EEP forcing data set and thus including the in situ HOx production. Several models have been developed which describe EEP patterns as functions of geomagnetic activity, based on statistical analysis of NOAA satellite observations [e.g., Codrescu et al., 1997; Wüest et al., 2005; Wissing and Kallenrode, 2009]. Rather than using one of these models, we chose to develop a new model speciﬁcally for our purpose. The precipitation of medium- and high-energy electrons into the Earth’s atmosphere is concurrent with the level of geomagnetic activity and geomagnetic storms and substorms, which are caused by the same magnetospheric processes. Within the geomagnetic ﬁeld energetic electrons are trapped, transported, and energized in the Van Allen Belts by processes such as radial diﬀusion and very low frequency waves (VLF) [Thorne, 2010]. During periods of high geomagnetic activity the ﬂuxes of energetic electrons in the outer radiation belt can change by orders of magnitude in hours or possibly minutes. Some of the ﬂux variability is caused by the loss of electrons into the atmosphere; the magnetic latitude of the outer radiation belt is such that most of these electrons enter the atmosphere at high latitudes in both the northern and southern magnetic hemispheres. Processes that drive electron precipitation such as VLF whistler mode chorus waves, plasmaspheric hiss waves, and electromagnetic ion-cyclotron (EMIC) waves [Summers et al., 2007], are strongly inﬂuenced by the background density of nonenergetic electrons within the inner magnetosphere—where the dense part is known as the plasmasphere [Carpenter, 1963]. Plasmaspheric hiss processes that dominate within the plasmasphere produce only low levels of precipitation ﬂux. EMIC-driven precipitation processes are focused close to the outer edge of the plasmasphere [Carson et al., 2013]. They produce highly energetic electron precipitation, but are restricted to a region that is small spatially [Clilverd et al., 2015]. Chorus waves are believed to dominate electron precipitation processes outside of the plasmasphere and produce high ﬂuxes of electron precipitation with large spatial extent [Whittaker et al., 2014a]. The level of geomagnetic activity is not only correlated with the magnitude of electron precipitation into the atmosphere but also with the position of the plasmapause, i.e., the boundary between the dense plasmasphere and the less dense regions farther from the Earth [e.g., O’Brien and Moldwin, 2003]. The current study is focused on EEP in the energy range 30–1000 keV. The lower limit of this chosen range is equal to that of the measurement instruments used (see below); the upper limit is applied because above 1 MeV, the ﬂuxes due to chorus waves become very small (see next section) and diﬀerent processes, such as EMIC waves, signiﬁcantly start to contribute, which show a diﬀerent behavior dependent on location and disturbance level, as shown by, e.g., Summers et al. [2007]. VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 2 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Figure 1. Dst and Ap time series for the period for which the Dst index is available. The red lines present the daily mean, while the black lines correspond to 14 day medians, included for clarity. In order to obtain EEP data as input to an atmospheric model dependent on location, time, and geomagnetic activity, direct satellite measurements are useful, such as the NOAA TIROS data of 1984 used by Callis [1997]. However, to undertake long-term simulations of the inﬂuence of geomagnetic activity on the atmosphere it is necessary to describe the variability of the EEP forcing over decadal timescales. The most useful long-term measurement of EEP is currently provided by the NOAA POES constellation, with several satellites at diﬀerent Sun-synchronous polar orbits. The satellites carry electron telescopes capable of measuring the medium energy electron ﬂuxes (30 keV–2.5 MeV) that enter into the atmosphere, in the SEM-2 instrument package [Rodger et al., 2010a, 2010b; Yando et al., 2011]. However, the SEM-2 instrument on board POES, which is a signiﬁcant upgrade from the SEM-1 package, which operated on board earlier NOAA satellites, has been providing measurements only since 1998. Furthermore, during this period initially, only a few satellite observational platforms were in operation, so that a useful globally covering EEP data set is available only from about 2002. Therefore, to provide decadal EEP descriptions an alternative technique must be developed. Whittaker et al. [2014a] used POES electron precipitation observations to show that knowledge of geomagnetic conditions and of the location of the plasmapause can be used to derive simple expressions for the electron precipitation (>30 keV) associated with whistler mode chorus and plasmaspheric hiss. In this study we build on this understanding of the electron precipitation processes, including the importance of the location of the plasmapause, to identify the main electron precipitation characteristics that are required for atmospheric models, i.e., geographical location, temporal variation, and electron energy spectrum. Given the correlation between geomagnetic indices and the strength of electron precipitation [e.g., Whittaker et al., 2014a], the signiﬁcant inﬂuence of the location of the plasmapause on the processes that precipitate electrons into the atmosphere [Hardman et al., 2015], and the ability to describe plasmapause dynamics through geomagnetic indexes, it should be possible to describe EEP using an appropriate long-term geomagnetic index. VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 3 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Fortunately, high-quality geomagnetic indexes were developed as part of the International Geophysical year in 1957, and several are now more than 50 years in length. Ap, Kp, Dst (disturbance storm time index), and AE (auroral electrojet index) are all long-term geomagnetic activity data sets [Mayaud, 1980] that have been used to represent the dynamical behavior of the plasmapause [e.g., Carpenter and Anderson, 1992; O’Brien and Moldwin, 2003]. The globally calculated hourly Dst index also provides a quantitative measure of the severity of magnetic storms, particularly those driven by coronal mass ejections [Borovsky and Denton, 2006]. Dependent on the type of magnetic storms, diﬀerent geomagnetic indices may be the best indicators of the disturbance and hence of the particle precipitation caused by it. An analysis of these goes beyond the scope of this paper, as the purpose of this study is to provide a long-term data set. This makes a practical argument signiﬁcant: both the Dst index and the daily Ap (eﬀective daily values calculated from the 3-hourly Kp index) provide a >50 year uninterrupted data series, which make them useful as inputs in long-term chemistry-climate models. Figure 1 shows the variations of the Dst and Ap indices since 1957, with nondisturbed geomagnetic conditions represented by values close to zero, and large geomagnetic storms indicated by about Dst < −50 nT and Ap >50 (for the daily mean values). An 11 year cycle can be seen in the occurrence of large geomagnetic storms. Summarizing, following the discovery of Andersson et al. [2014], which identiﬁed electron precipitation from the Earth’s radiation belts as a signiﬁcant but previously missing source for important ozone loss at mesospheric altitudes, there is motivation in establishing a long-term EEP data set that could be utilized in chemistry-climate model simulations. This data set would also respond to the issue concerning production of EPP-HOx and potentially help resolve the early timing of the appearance of middle atmosphere dynamical changes arising from EPP, which could not be explained by EPP-NOx transport to stratospheric altitudes [Lu et al., 2008; Seppälä et al., 2013]. The following sections of this paper describe the available data set of satellite observations, the EEP model developed based on the underlying plasmaspheric structure and geomagnetic activity, and the resultant long-term ionization rate data set that can be used as an input to coupled chemistry-climate models. 2. POES/SEM Electron Flux Measurement 2.1. Observations The NOAA Polar Orbiting Environmental Satellites (POES) (http://www.ospo.noaa.gov/Operations/POES/) circle the Earth approximately 14 times a day in Sun-synchronous polar orbits at ∼800–850 km altitudes. On board these satellites is the Space Environment Monitor (SEM-2) [Evans and Greer, 2004; Rodger et al., 2010a], which contains the Medium-Energy Proton and Electron Detector (MEPED). The three electron detectors included in MEPED count detected electrons in three energy pass bands: >30 keV, >100 keV, and >300 keV. The nominal upper energy limit is 2500 keV for all three electron detectors [Evans and Greer, 2004], although higher-energy electrons can also enter the detectors as shown by Yando et al. [2011]. For each energy band, two identical detectors are mounted at right angles of each other: one pointing 9∘ oﬀ from vertically upward (away from Earth), the other 9∘ oﬀ from antiparallel to the spacecraft motion. Both telescopes have a ﬁeld of view that is 30∘ wide and have a temporal resolution of 2 s. At the latitudes of the outer radiation belt and the auroral zone, the near-vertically pointing detector measures some of the electrons that are likely to enter the atmosphere shortly after, i.e., a portion of the bounce loss cone [Rodger et al., 2010b]. The location of each measurement point was converted to McIlwain L shell values (referred henceforth to as L shell, or L) [McIlwain, 1961] using the IGRF model of the magnetic ﬁeld and the National Science Data Center INVAR program [Evans and Greer, 2004; Lam et al., 2010]. The MEPED instruments on board the diﬀerent satellites have all been built at the same time and cross calibrated on the ground. In addition, the instruments undergo a weekly in-ﬂight calibration procedure, which involves stimulating each detector with pulses of known amplitude, to check its response sensitivity. Further, detailed technical description of the SEM-2 measurement instrument is given by Evans and Greer [2004], including calibration and the conversion from detected electron counts to ﬂuxes in units of electrons cm−2 sr−1 s−1 . The electron counts measured by MEPED represent the average ﬂux over the detector aperture, which is equal to the average ﬂux over the entire bounce loss cone if a uniform population of the bounce loss cone can be assumed. During strong disturbances, angle diﬀusion is strong, making this assumption justiﬁed. However, during weaker disturbances, weak diﬀusion keeps the electrons mainly near the edge of the bounce loss cone, and away from the detector, which causes an underestimate of these ﬂuxes. Rodger et al. [2013] VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 4 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 analyzed this eﬀect for substorms, and Simon Wedlund et al. [2014] for radiation belt precipitation. They reached diﬀerent results for the threshold value and the correction factors for this eﬀect, due to the diﬀerent types of scattering/diﬀusion taking place. Nesse Tyssøy et al. [2016] showed in Figure 6 of their paper that the diﬀusion strength is similar between the three energy ranges of the MEPED detectors. A full test of the correction for all POES ﬂuxes at all L shells and at all MLT still needs to be done. At this point, it can only be said as a rough estimate that observed ﬂuxes below about 104 to 105 electrons cm−2 sr−1 s−1 may be underestimating the loss cone ﬂuxes by up to a factor of about 10. The MEPED electron detectors are subject to varying levels of false counts from proton contamination [Rodger et al., 2010a; Yando et al., 2011], either from low-energy ring current protons, or high-energy protons during solar proton events. This situation can be detected using the proton ﬂux telescopes on MEPED. Outside of the high ﬂuxes involved in SPEs, the proton contamination can be removed using the equations given by Lam et al. [2010]. The accuracy of these corrections to the ﬂux have been checked by a multispacecraft comparison between POES and DEMETER [Whittaker et al., 2014b], using the Monte Carlo contamination simulations of Yando et al. [2011]. In the case of very high proton ﬂux levels, such as during SPE, the electron count is dominated by protons and correction would be unreliable. Because of this, all samples concurrent with observations on the MEPED >36 MeV proton detector >3 counts/s have been removed from the POES electron data set. We found that this adequately removes the data dominated by SPE. As a result of this, data measured in and around the South Atlantic Anomaly (SAA) were entirely removed due to the large amount of proton contamination occurring there [Rodger et al., 2013]. This study makes use of the data measured by SEM-2/MEPED on board POES from 2002 to 2012. During this time, the number of measuring satellites increased from two at the start and three from July 2002 to six at the end. All electron precipitation ﬂux measurements were binned as a function of their IGRF L value, covering L = 2 –10 with resolution 0.5, and as a function of UT time, with a 3 h resolution, and for all MLTs together. The L shell range used covers both the outer radiation belt and the dynamical range of the plasmapause (2.5 < L < 6). For each bin, the median electron ﬂux is calculated. Next, in order to obtain daily ﬂuxes, the eight 3 h ﬂux values were linearly averaged over each day, for each energy channel, and each L value. Each satellite traverses the L shells of the outer radiation belt 4 times in each orbit, and therefore, each 1 day sample involves the averaging of roughly 100 to 300 individual passes through the radiation belts. The Sun-synchronous satellites pass through the radiation belts at ﬁxed geographic local times. From July 2002 onward, there are passes at least at 3 × 4 = 12 local times, which are evenly distributed around the clock (only up to June 2002 slightly less so). Furthermore, the globally varying oﬀset between the magnetic and geographic coordinates makes the satellites cover a wide range of magnetic local times (MLT) in 14 passes per day. In cases of low electron ﬂuxes, the detector is limited by the instrument noise ﬂoor. Because of this, all data points where the precipitating electron ﬂux >30 keV was lower than ∼250 electrons cm−2 sr−1 s−1 , were removed and replaced by zeros in all three energy ranges. From the three energy ranges measured by POES SEM-2 it is possible to ﬁt an energy ﬂux spectrum. In a previous measurement campaign, the satellite DEMETER measured the much higher ﬂuxes of precipitating electrons in the drift loss cone at very high spectral resolution [Whittaker et al., 2013]. Diﬀerential spectral ﬂux observations from this campaign showed that a power law relationship is appropriate for precipitating electrons in the medium-energy range in the outer radiation belt [Clilverd et al., 2010]. A simple power law model for the spectral density S of the ﬂux is therefore assumed: S(E) = CE k electrons∕(cm2 sr s keV) (1) where E is the energy of the electrons (keV), C is an oﬀset, and k is the spectral gradient. This spectral density can be integrated to obtain the integrated ﬂux as measured between two energy levels: Eu S(E ′ )dE ′ ∫E C (E k+1 − E k+1 ) = k+1 u = C(ln(Eu ) − ln(E)) F(E) = VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS electrons∕(cm2 sr s) (k ≠ −1) (2) (k = −1). 5 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Figure 2. Typical example of the diﬀerential ﬂux of precipitating electrons measured by DEMETER in the outer radiation belt. In this case the ﬂux has been normalized by the 700 keV value, and a power law (red line) ﬁtted to the <700 keV ﬂuxes. However, this is not accurate for relativistic electrons. At these higher energies (above 700–800 keV) the DEMETER spectrum commonly falls oﬀ much faster with energy than predicted by a power law; a typical example is shown in Figure 2. The energy threshold where this steep decrease occurs varies, and we use 1000 keV as a representative energy for when the power law typically becomes invalid. Therefore, although the upper energy limit of the electron detector sensitivity is at least 2.5 MeV as mentioned before, it is realistic to approximate the upper limit Eu of the spectrum in equation (2) to be 1 MeV. (Although this may be a rough approximation, it has been tested that the results presented in this paper do not signiﬁcantly depend on the exact value of Eu , be it 1 or 2.5 MeV, and therefore also not on the shape of the upper limit, be it a sharp cutoﬀ of a steep slope. This is due to the fact that the contribution of the energies above 1 MeV in equation (2) is in any case very small.) At all data points (i.e., for each time/L value), the function of equation (2) has been ﬁtted to the three measured integrated electron ﬂuxes. Here E is equal to the lower energy threshold of the three detectors (30 keV, 100 keV, and 300 keV), and Eu = 1000 keV. The output of this procedure is expressed in the spectral gradient k and the ﬂux >30 keV (F30 ) resulting from the ﬁt, which is given as a function of C and k by ) C ( 1000k+1 − 30k+1 k+1 = C(ln(1000) − ln(30)) F30 = electrons∕(cm2 sr s) electrons∕(cm2 sr s) (k ≠ −1) (3) (k = −1). F30 and k are available for every point in time and every value of L. Typically, the power law gradient k of the energy spectrum varies from −1 to −4 [Clilverd et al., 2010; Simon Wedlund et al., 2014]. At low ﬂux levels some of the three energy ranges could have ﬂux values that are aﬀected by the instrument noise ﬂoor, and thus produce an unrealistic power law gradient of ∼0; small amounts of noise can produce even more unrealistic positive gradients. However, measurements of precipitating electron ﬂuxes by DEMETER at a much higher spectral resolution [Clilverd et al., 2010] indicated that a reasonable upper limit of the spectral gradient is about −1. Therefore, the maximum value of k in the ﬁtting procedure above has been set to −1. In cases where the three measured ﬂuxes do not ﬁt well to one power law function, the ﬂux >300 keV is assumed to be aﬀected by noise and the ﬁt is performed only on the >30 keV and >100 keV ﬂuxes. This noise inﬂuence was detected as the increased probability of the point at 300 keV to be above the ﬁtted curve, dependent on the mean square error of the curve and the ﬂux >300 keV; this happened roughly whenever the ﬂux >300 keV was below 250 electrons cm−2 sr−1 s−1 . At the end of the process which identiﬁes and compensates for proton contamination, low ﬂux periods, and unrealistic spectral gradients, we are able to produce a 10 year data set with 1 day time resolution, of F30 (ﬂux >30 keV electrons cm−2 sr−1 s−1 ), and k (power law spectral gradient), calculated for each 0.5 L bin. The data VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 6 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 resulting from this process will be referred to as “modiﬁed POES data.” There is no magnetic local time (MLT) information available due to the zonal averaging of the satellite observations. 2.2. Validating the Modiﬁed POES Fluxes A recent long-term comparison has been presented between the energetic particle precipitation measurements from POES SEM-2 and those derived from ground-based subionospheric VLF measurements. Neal et al. [2015] used VLF amplitude observations made by the AARDDVARK network and updated the approach of Clilverd et al. [2010] to extract hourly >30 keV electron precipitation ﬂux values for L = 3 –7. While the data analysis and modeling approaches presented by Neal et al. [2015] are limited to the Northern Hemisphere summer period when the D region is sunlit, the AARDDVARK measurements span 2005–2013, and thus provide a long-term set independent of the POES electron precipitation observations. The calculation was done using a combination of models, among which a propagation model of the lower part of the D region and a model for the electron density proﬁle dependent on EEP ﬂux, which together give the received VLF amplitude as a function of the EEP spectrum characterized by the parameters F30 and k. Inversion of this combined model gave F30 as a function of VLF amplitude, if k is given. More details of the procedure are given by Neal et al. [2015] and Rodger et al. [2012]. Calculation of the AARDDVARK-derived ﬂuxes uses the spectral gradient k provided by the modiﬁed POES electron precipitation data set, in order to determine the ﬂux >30 keV which caused the observed VLF amplitude perturbations. When the POES-derived ﬂuxes were set to zero, a power law spectral gradient of −1 was assumed, as discussed above, and used in the AARDDVARK precipitation ﬂux calculations. Unfortunately, the use of the POES spectra in the AARDDVARK calculations violates the mutual independence of the two data sets. As such, the AARDDVARK ﬂuxes can not be used to completely validate the POES ﬂuxes. However, the greatest uncertainty of the POES ﬂuxes is the scaling from the detector aperture to the bounce loss cone, which might be underestimated due to weak diﬀusion, as described in section 2.1. Nesse Tyssøy et al. [2016] showed in Figure 6 of their paper that the diﬀusion strength is similar between the energy channels considered in our study. However, there are clearly occasions where the diﬀusion strength in the lowest energy MEPED electron channel is approximately a factor of 2 higher compared with the other energy channels. Nevertheless, in most cases it seems a reasonable assumption that the measurements in the integrated ﬂux channels will be proportionally underestimated in the case of weak diﬀusion, and therefore the spectral gradient should not be aﬀected by this missing ﬂux. Using this assumption, we now move to validate the POES ﬂuxes using the AARDDVARK-derived ﬂuxes. Figure 3 shows a comparison between the AARDDVARK-reported >30 keV ﬂuxes and the modiﬁed 10 year data set of POES-derived ﬂux values over the range L = 3 –7. Only the Northern Hemisphere summer period is shown in this ﬁgure, as the Neal et al. [2015] ﬂux extraction approach is limited to daylit ionospheric conditions. Figure 3 (top) shows data from the summer of 2009. This was a particularly quiet period, allowing very clear visual comparisons to be made. As is apparent from this graph, during disturbed conditions with high ﬂux levels, there is very good agreement between the AARDDVARK-extracted >30 keV ﬂux magnitudes and those of the modiﬁed POES ﬂuxes. However, for large periods of time the AARDDVARK-derived precipitating ﬂuxes are smaller than the POES instrumental sensitivity ﬂoor at ∼1 electron count/s (∼100 electrons cm−2 sr−1 s−1 ). In cases where the modiﬁed POES ﬂux values were smaller than 125 cm−2 sr−1 s−1 , these have been set to zero and do not appear on the ﬁgure, while the AARDDVARK observations suggest they should be at least as low as ∼20 electrons cm−2 sr−1 s−1 , which appears to be the AARDDVARK sensitivity limit. Figure 3 (bottom) shows a scatterplot over the full period of the summers of 2005–2013, and a line ﬁtted to all points, giving a close-to-identical relation. In this graph, the AARDDVARK ﬂuxes seem to show more deviations to high values than the POES ﬂuxes. These data points consist of only a few strong events, where due to the calculations to the AARDDVARK data, at very high ﬂux values, small changes in perturbation amplitude correspond to large changes in ﬂux, causing the AARDDVARK-derived ﬂuxes to be less accurate in this area. In the low ﬂux range, the limitation in the POES ﬂuxes due to the noise ﬂoor is clearly seen also in this graph, even though all POES ﬂuxes smaller than 185 cm−2 sr−1 s−1 were removed. All points shown in the graph were taken along in the ﬁtted curve, which does not seem to be inﬂuenced signiﬁcantly by either of these deviations. Excluding solar proton events and data gaps, the cross correlation between the log10 (ﬂuxes) over the Northern Hemisphere summers of 2005–2013 is 0.91. The Neal et al. [2015] study used observations of a VLF transmitter VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 7 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Figure 3. Comparison between the magnitude of the >30 keV precipitating electron ﬂuxes extracted from AARDDVARK observations [Neal et al., 2015] and the modiﬁed POES ﬂuxes described in this study. (top) Summer of 2009 as a function of time; (bottom) scatterplot of 1 day resolution data over summer months (MJJ) 2005–2013, and a linear ﬁtted curve giving y = 1.0518x + 0.0273. on the east coast of North America received in northern Finland. As such, the AARDDVARK ﬂuxes are strictly appropriate for the longitudes of the North Atlantic, while the modiﬁed POES ﬂuxes have been zonally averaged to provide a global precipitation ﬂux indication. Nonetheless, the cross correlation between these data series is very high, providing a validation of the modiﬁed POES ﬂuxes. We will now consider how to apply these ﬂuxes in order to produce multidecade estimates of precipitation into the polar atmosphere. 2.3. Comparison With Geomagnetic Activity A visual representation of the 10 year electron precipitation data set derived from the modiﬁed POES SEM-2 measurements is shown in Figure 4, as a function of Dst and L shell (Figure 4, top) and Ap and L shell (Figure 4, bottom). The Dst index has been averaged to daily resolution in order to match the electron precipitation data set, and the ranges of Dst and Ap have been divided in bins. For each bin the median F30 and median k have been calculated. Dst > 0 nT is not considered further here in order to maintain consistency with the development of the electron precipitation model presented in section 3. Figure 4 (left column) shows the variation of the median ﬂux >30 keV (F30 ). At low geomagnetic activity levels (i.e., at Dst ∼ 0 nT and Ap ∼ 0) the electron precipitation ﬂuxes are low and conﬁned to L > 5. As the geomagnetic disturbance levels increase the ﬂuxes become substantially higher, as well as becoming more constrained to lower L shell values. The movement of the high-precipitation ﬂux region toward lower L shells as geomagnetic activity increases is consistent with the dynamical behavior of the plasmapause. O’Brien and Moldwin [2003] presented several models of the plasmapause expressed in various geomagnetic indices, including Dst and Kp. Since the plasmapause generally responds quickly to a geomagnetic storm, but slowly returns to normal after the storm, they found the best correlation if the geomagnetic index is integrated over a certain period before the time point VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 8 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Figure 4. (left column) The median integrated ﬂux >30 keV, F30 , and (right column) spectral gradient, k, as functions of (top row) L and Dst and (bottom row) L and Ap from the modiﬁed POES ﬂuxes described in section 2. The dashed lines are the approximate plasmapause location Lpp , plotted as a function of mint−1,t Dst and maxt−1,t Ap, respectively. of interest. This period is 24–36 h depending on the parameter. Their best performing models dependent on Dst and Kp have been adapted to our 1 day resolution data sets of Dst and Ap as follows: Lpp (t) = −1.57 log10 \|min...Dst\| + 6.3 (4) Lpp (t) = −0.7430 ln maxt−1,t Ap + 6.5257 (5) where mint−1,t Dst and maxt−1,t Ap indicate the minimum/maximum value of Dst/Ap of the day of interest and the previous day. The coeﬃcients in equation (5) were obtained by ﬁtting to the relation by deﬁnition between Kp and Ap. To show the consistency of the movement of the high-precipitation ﬂux region toward lower L shells with the dynamical behavior of the plasmapause as geomagnetic activity increases, the approximate location of the plasmapause has been included in Figure 4 as a black dashed line. Strong linkages between precipitation location and the plasmapause location have been reported recently [e.g., Lichtenberger et al., 2013; Simon Wedlund et al., 2014; Whittaker et al., 2014a; Clilverd et al., 2015]. The region of high precipitation ﬂux in Figure 4 resides outside of the modeled location of the plasmapause and therefore suggests that VLF whistler mode chorus is the most dominant source of electron precipitation in the medium energy range (30–1000 keV) [Summers et al., 2007; Li et al., 2013; Ni et al., 2014]. Figure 4 (right column) shows the median power law spectral gradient k in the electron precipitation data set. The regions of high median ﬂux largely coincide with the regions where the median spectral gradient is steep (∼−3 to −4), and those with low ﬂuxes coincide with shallow gradients (limited to −1). The response of the spectral gradient to increased geomagnetic activity is also consistent with the behavior of the plasmapause. VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 9 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 3. Development of a Flux and Spectrum Model Based on Magnetic Index and L Shell 3.1. The Model A model for the integrated electron ﬂux was developed by ﬁtting analytic expressions to the dependencies, on Dst or Ap and L, of the median integrated ﬂux F30 and the spectral gradient k from the modiﬁed POES ﬂuxes described in the previous section and shown in Figure 4. To compensate for the movement of the plasmapause position we expressed the dependence on L in terms of the distance from the plasmapause, Spp , deﬁned as Spp = L − Lpp . The location of the plasmapause, Lpp , was calculated according to equations (4) and (5). The optimal expressions for F30 and k were found manually by experimental ﬁtting to the median observed values. In this process, most attention was paid to a good ﬁt to the highest ﬂuxes, because these are the main interest of the database. The coeﬃcients of the expressions were found by least squares error regression. The resulting models for F30 and k, as functions of Dst and Spp are F30 = AebSpp cosh(c(Spp − s)) (6) where A = 597.23(−Dst)1.0878 b = 0.90109(−Dst)0.16200 c = 1.0061(−Dst)0.19921 s = 1∕(−3.5264 × 10−3 Dst + 0.65650) And for the spectral gradient k k= −1 −1 Ee−b Spp + 0.29458 cosh(0.19750(Spp − 5.7000)) (7) where E = 0.40850(−Dst)−0.22247 b = 1.8375(−Dst)0.20602 For positive Dst, we ﬁnd that the electron ﬂuxes are typically low and modeling these is not of prime interest in this study. Therefore, an extra clause in this model is that for any value of Dst ≥ 0 nT, F30 = 0 electrons cm−2 s−1 sr−1 , and k = −1. The models for F30 and k, as functions of Ap and Spp , are F30 = e −b(Spp −s) eA + ec(Spp −s) + d (8) where A = 8.2091Ap0.16255 b = 1.3754Ap0.33042 c = 0.13334Ap0.42616 s = 2.2833Ap−0.22990 d = 2.7563 × 10−4 Ap2.6116 VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 10 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Figure 5. The modeled integrated ﬂux >30 keV F30 and spectral gradient k, (top) as functions of Spp and Dst and (bottom) as functions of Spp and Ap. And for the spectral gradient k k= −1 −1 Ee−bSpp + 0.30450 cosh(0.20098 (Spp − s)) (9) where E = 3.3777Ap−1.7038 + 0.15000 b = 3.7632Ap−0.16034 s = 12.184Ap−0.30111 The results of the above expressions as functions of Dst, Ap, and Spp are shown in Figure 5. The model presented above can be used to estimate the >30 keV ﬂux and spectral gradient k for 2 < L < 10 and at any point in time for which the magnetic index is available. To save space, in the following, this paper will focus mostly on the model using Ap. In order to compare the model results with the modiﬁed POES data, we calculate the F30 and k from Ap over the same time period and the same L values as the POES database. The results were binned as functions of Ap, and median values were calculated for every bin for comparison with Figure 4. The result is shown in Figure 6. Comparison with Figure 4 shows a good agreement with the medians of the values in each Ap∕L bin of the modiﬁed POES data—a more quantitative and more complete comparison follows. VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 11 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Figure 6. (left) The median modeled integrated ﬂux >30 keV, F30 , and (right) spectral gradient, k, calculated over the time period of the modiﬁed POES ﬂux observations, as functions of L and Ap. With F30 and k now known, the integrated ﬂuxes above other energy thresholds E (in keV) can now be calculated (derived from equation (2)): F(E) = F30 = F30 Euk+1 − E k+1 Euk+1 − 30k+1 lnEu − lnE lnEu − ln30 (k ≠ −1) (10) (k = −1), where Eu is equal to the upper energy limit of 1000 keV. 3.2. Comparison With Observations 3.2.1. Time Series Examples Figure 7 shows a comparison of the observed (POES modiﬁed) and modeled integrated ﬂux F30 over the entire POES measurement period, 2002–2012. The comparison is shown for three L values: 4.25, 5.25, and 7.75. The ﬁrst two are located in the heart of the radiation belts, while L = 7.75 corresponds to a region in the outermost parts of the outer radiation belt. Figure 4 shows that during moderate geomagnetic disturbances (Ap between about 50 and 100), these three locations correspond roughly to the equatorward edge of the bulge in precipitating ﬂuxes, the peak of the bulge, and its poleward slope, respectively. For the sake of clarity we have reduced the time resolution for Figure 7, presenting the 2 week median values of both POES and modeled ﬂuxes. 3.2.2. Error Assessment The accuracy of the model has been assessed quantitatively, and more generally in the following analysis. The error of the model in the >30 keV precipitating electron ﬂux has been calculated as follows: 𝜖F30 (t, L) = log10 F30model (t, L) − log10 F30POES (t, L). The database was divided into bins according to Ap, varying almost linearly from 0 to 100 (not the same bins as used in Figures 4–6). For each of these bins, the probability density function (PDF) of 𝜖F30 was derived for all daily data points in the respective Ap bin across all 16 L values. Points where the modiﬁed POES F30 was equal to zero (i.e., removed because the values were so small they were aﬀected by the noise ﬂoor) were excluded from this determination, as these would give 𝜖F30 = ∞, while in reality the size of their error is unknown in these cases. After this, all the PDFs for the diﬀerent Ap values were averaged, to obtain one overall PDF of the error 𝜖F30 . The purpose of this averaging of separate Ap-dependent distributions is normalization, to increase the visibility of the disturbed time results. If the PDF of all data were calculated at once, the result would be dominated by the quiet-time results, as they are the most common situation in the data. Using the averaging, the behavior for diﬀerent levels of disturbance are equally represented in the end result. VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 12 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Figure 7. The observed (modiﬁed POES, blue line) and modeled (Ap model, red line) integrated precipitating >30 keV ﬂux over the entire POES SEM-2 measurement period, at three IGRF L shells: 4.25, 5.25, and 7.75. For clarity, all values shown are medians over 14 days. Figure 8 (top) shows the overall PDF for F30 (blue line). This ﬁgure indicates the PDF is roughly Gaussian, distributed around ∼−0.01, with a standard deviation of 0.69. In order to extend the error analysis to involve the spectral gradient, the integrated ﬂuxes >100 keV and >300 keV (F100 and F300 ), were additionally analyzed. The modeled values were calculated from the modeled F30 and k using equation (10) with E = 100 keV/300 keV and Eu = 1000 keV. The same approach was applied to the F30 and k values obtained from the modiﬁed POES data set, resulting in virtual measured values of F100 and F300 . Similarly as for 𝜖F30 , these were used to calculate the errors 𝜖F100 and 𝜖F300 , but these errors are now inﬂuenced by a combination of the modeling accuracies of both F30 and k. The green and red lines in the upper graph of Figure 8 show the PDFs of 𝜖F100 and 𝜖F300 . They are also roughly Gaussian, with means, respectively, −0.01 and 0.02 and standard deviations 0.63 and 0.71. The errors 𝜖F30 and 𝜖F300 of the Dst-based model are also included (dashed lines), calculated equivalently as that of the Ap model, averaging PDFs for diﬀerent Dst bins. Their means are, respectively, −0.05 and 0.07, and the standard deviations are 0.90 and 0.65. The graph shows that errors of up to about a factor 10 (both ways) are fairly common in all ﬂuxes. This is equal to the general spread of measured values within each bin of Dst/Ap and L, and therefore indicates the limitation of the predicting ability of these parameters. Partly, this variation can be due to the zonal averaging of the data, and a better performance still can be expected when MLT dependence is included in later versions of the models. In order to show the performance of both models dependent on geomagnetic activity, the means and standard deviations of the PDFs for the diﬀerent Ap and Dst bins are shown in Figure 8 (middle). Only results for bins containing more than one time sample (=16 values) are shown. Here it is seen that the standard deviations of VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 13 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Figure 8. (top) The PDF of the error of the Ap model, for three energy channels, and for the Dst model for two energy channels; see text for details. (middle) The mean error (red and blue solid and dashed lines) and standard deviation of the error, 𝜎 , (same as the mean but darker red and blue coloring) of both models, from the distributions for the diﬀerent Dst/Ap bins. (bottom) The means and standard deviations as functions of time, with 3 month resolution. In Figures 8 (top) and 8 bottom, all distributions have been averaged from the PDFs for diﬀerent Dst/Ap bins. VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 14 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 F30 are fairly constant over the range, and the means ﬂuctuate around zero, indicating a stable performance of the models over most of the range. For 𝜖F300 , the mean errors are found to be relatively large in quiet conditions (low Ap and \|Dst\|). This overestimation of ﬂuxes at high energies is due to overestimations of the spectral gradient in quiet conditions. As shown before, quiet conditions are cases where the ﬂuxes tend to be low (see, e.g., Figure 4) and therefore of less interest in this study. In addition, it was shown that the measurement of low ﬂuxes in the POES campaign are relatively inaccurate due to noise. For both reasons, the ﬁtting procedure of equations (7) and (9) was aimed more at the higher ﬂuxes than the lower, which is the reason for the poorer prediction of the spectral index in quiet conditions. In order to show the performance of both models as functions of solar cycle phase, the error distributions were also calculated for 3 month periods over the time period of the database. Also, these were derived by averaging the error distributions for the diﬀerent Ap/Dst bins. Note, however, that any data for high disturbance levels were only measured around solar maximum, causing other results to represent only data for lower disturbance (e.g., during 2006–2011 only Ap < 50 and in 2009 only Ap < 20). The resulting means and standard deviations of these are shown in Figure 8 (bottom). Here it is seen that for the Ap model, the error for F30 is stable over the solar cycle period. The Dst model tends to underestimate F30 just after the solar maximum around 2003, while it performs better during other periods. This underestimation is systematic, as it was seen at all disturbance levels during that period (not shown). The already observed overestimations of F300 for both models seem to increase at solar minimum; however, this is caused by biasing toward lower disturbance levels due to the lack of other data, as mentioned above. The standard deviations are all fairly independent of solar cycle phase. 4. Atmospheric Ionization Rates From the Precipitation Model Using the precipitation model described in section 3 and the spectrum estimate described in section 2, it is now possible to calculate an electron energy-ﬂux spectrum for any Dst/Ap index and L shell combination. Further, these spectra can be used in calculation of altitude-dependent atmospheric ionization rates. To demonstrate this, we have calculated a set of ionization rates for years 2002–2012 from both precipitation models presented above using a parameterization of electron impact ionization by Fang et al. [2010]. For comparison, the ionization rates were also calculated from the ﬂux spectra resulting from the modiﬁed POES data set over the same period. The energy range of the spectrum was set at 30–1000 keV, with 168 logarithmically spaced grid points. A representation of the atmosphere, as needed for the ionization rate calculation, was created using the NRLMSISE-00 model [Picone et al., 2002]. Note that the rates were calculated on a ﬁxed atmospheric pressure grid, and the altitude grid shown in the following ﬁgures is approximated. Although the rates were calculated with 1 day temporal resolution, for clarity we present 14 day median values in the ﬁgures. Figure 9 shows the altitude-dependent ionization rates at L shell 5.25 according to both models and to the modiﬁed POES data. Zero values are shown as blanks in these graphs, while values between 0 and 0.01 cm−3 s−1 are dark blue. This ﬁgure shows that the main part of the ionization due to the energy range considered in this paper is between 70 and 110 km altitude, while the rates decrease rapidly at altitudes below and above. The peak of the ionization, at about 90 km, is caused partly by the 30 keV lower limit of electron spectrum energy. The lower altitude limit of the ionization of this energy range is seen at about 55 km (Figure 9), because the electrons with highest spectrum energy (1000 keV) can not penetrate to altitudes below this height [e.g., Turunen et al., 2009, Figure 3]. It should be noted that the ionization proﬁle shown here is only that caused by electrons in the energy range 30–1000 keV. The proﬁles due to electrons of lower and higher energies will overlap this proﬁle, and show maximum ionization at higher and lower altitudes, respectively. The altitude range which is dominated by ionization from electrons in the energy range considered in this paper, and where the proﬁle of Figure 9 can therefore be assumed to be close to the total ionization proﬁle, is between about 60 and 95 km. Comparing the graphs in Figure 9, the ionization rates according to both models generally agree well with those according to the modiﬁed POES data. Also, here, it can be seen that in the strong disturbance period of 2003, the Dst model slightly underestimates the ionization. Otherwise, the most obvious diﬀerences between the three results are in the low ionization area and the zero values indicated as blanks in the graphs. The POES VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 15 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Figure 9. Ionization rates due to 30–1000 keV radiation belt electron precipitation from 2002 to 2012 at L shell 5.25, according to (top) the Dst model, (middle) the Ap model, and (bottom) POES observations. ionizations are occasionally zero, in case of low or removed ﬂuxes (see section 2.1). The Dst model gives zero even more often, in cases of positive Dst values. The Ap model remains consistently positive in all these cases. Figure 10 presents the L shell distribution of the ionization rates at 89 km altitude, near the peak of ionization of this energy range. Large variability is seen with respect to the L shell, with the highest ionization rates reaching 2 × 103 cm−3 s−1 . The main region with large ionization values lie between L shells 5 and 10, i.e., including the heart of the radiation belts at the latitudes most likely to be signiﬁcantly impacted by substorms VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 16 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Figure 10. Ionization rates due to 30–1000 keV radiation belt electron precipitation at 89 km altitude in 2002–2012, according to (top) the Dst model, (middle) the Ap model, and (bottom) POES observations. [Cresswell-Moorcock et al., 2013]. At the lower L shells (L <5) the ionization rates are substantially smaller and indicate a more infrequent, event-like behavior compared to that seen at higher L shells. Comparing the graphs in Figure 10, again the Dst model is seen to slightly underestimate the ionization in 2003. Zero values occur in the POES ionizations often for low L values (inside the plasmaspause footprint), while neither model gives zero ionization there. In both Figures 9 and 10, the ionization rates vary in time by more than 4 orders of magnitude. Following the variations in the solar and geomagnetic activity, 2003 has the highest ionization levels across the year while VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 17 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 2009 has the lowest. In May–June 2004, in the midst of an otherwise generally high ionization period, the rates are substantially smaller; this variation is consistent with observations of radiation belt variability over this time [Rodger et al., 2016]. Note that using the model proposed here the ionization rates due to EEP can now also be calculated during the SPE periods (e.g., October–November 2003, December 2006, and early 2012), when any calculations based on POES data would suﬀer from serious proton contamination (see the discussion in Rodger et al. [2013, section 2.3] and Neal et al. [2015, section 6]). The ionization predicted by the model during SPEs would be equal to that during the same levels of disturbance outside of SPEs, assuming that the SPE would not aﬀect this precipitation. Obviously, the additional electron precipitation due to the SPE itself is not predicted by the models. A more detailed analysis of the variability of the ionization in this data set is outside the scope of this paper. A discussion on the longer-term variability of the ionization rates described by the Ap model and examples of resulting atmospheric response are given by Matthes et al. [2016]. 5. Evaluation 5.1. Intercomparison of the Two Models In case the user intends to apply the EEP models presented in this paper to their own atmospheric model, or to any other purpose, a choice between the two models presented in section 3.1 (Dst- and Ap-dependent model) can be made. Both models perform almost equally well, so that the choice can simply depend on preference or suitability of either of the magnetic indices for the intended application. However, each of the two models has small advantages and disadvantages over the other. The Ap model (equations (6) and (7)) has the advantage over the Dst model that its performance is a bit better, see the standard deviation of the modeled ﬂux in Figure 8 (top). This performance is independent of solar cycle phase, as seen in Figure 8 (bottom). Furthermore, if a long-term database is needed, the index Ap has the advantage of being available further back into the past than Dst (from 1932). The Dst model (equations (8) and (9)), even though its overall error standard deviation is slightly less good, has the potential advantage of a higher time resolution. The index Dst is available at a time resolution of 1 h. Since Dst is, more than Ap, linked to dynamics of the inner magnetospheric electric ﬁeld which causes erosion of the plasmasphere [O’Brien and Moldwin, 2003], it can be expected that this index responds quickly to dynamics of the plasmapause. It has been tested using POES observations (not shown in this paper) that the Dst model performs still almost as well at a time resolution of 3 h as it does at 1 day: the mean error stays close to zero and the standard deviation only slightly increases. A disadvantage of the Dst-dependent model is the underestimation of the ﬂux just after solar maximum. Another disadvantage may be the zero ﬂux values which the Dst model can give during quiet conditions when Dst ≥ 0 nT, and which may not be desired in some applications. 5.2. Limitations of the Models The precipitation models presented above and the ionization rates based on them have certain limitations. The presented ionization rates are zonally averaged and have no information on longitudinal variability of the precipitation. This variability may explain some of the spread of the error shown in Figure 8. We have applied this approach in the ﬁrst version of the electron precipitation model because of the large size of the POES data set. In future it may be possible to retain the information on the magnetic local time coverage of the observations, which could be used to obtain some information on the longitudinal variability of the precipitating ﬂuxes. This, however, is a signiﬁcant task and therefore the ionization rates from the model currently represent the average longitudinal precipitation. Still, they show good agreement with the AARDDVARK measurements which have much less longitudinal averaging. Furthermore, the measurements are relatively inaccurate for low ﬂux levels. First, weak diﬀusion may cause the electrons to be located near the edge of the bounce loss cone and missed by the detector, causing underestimations (although the AARDDVARK validation shows that this error is not very large). Second, the low ﬂux measurements are subject to noise, causing overestimations (even after the precautions described in section 2.1). Third, the ﬁtting of the models to the observations was mainly aimed at the higher ﬂux levels. Because of all these reasons, the models will not be very accurate for low ﬂux levels, as was indeed seen in Figure 8. However, since the purpose of the models is mainly predicting the larger ﬂux levels, this is not a cause of great concern. VAN DE KAMP ET AL. EEP MODEL FOR GEOMAGNETIC STORMS 18 Journal of Geophysical Research: Atmospheres 10.1002/2015JD024212 Finally, we have focused these models on medium energy EEP in the energy range 30–1000 keV, which will likely be dominated by precipitation due to chorus waves. We plan for mechanisms working at higher energies to be included in an updated version of the geomagnetic index driven electron precipitation model in the future. As not all precipitation mechanisms can be presently included, it should be noted that the ionization rates based on the precipitation model remain an underestimation of the total ionization from energetic electron precipitation into the atmosphere. 6. Conclusions We have developed a precipitation model for radiation belt energetic electron precipitation in the energy range 30–1000 keV, which can be used to calculate the ﬁrst ever >80 year long atmospheric ionization rate data set. The EEP model is based on available observational data from satellites, the plasmasphere structure and geomagnetic activity. The satellite precipitation measurements have been partly validated through comparison with electron precipitation measurements from the AARDDVARK network. Results from the EEP model, which uses Dst or Ap index to determine the varying geomagnetic activity levels, compare well with processed observational data over the period of 2002–2012. The main impact from the ionization from EEP is focused on the mesosphere-lower thermosphere altitudes (70–110 km), with the lower limit of the ionization located at about 55 km altitude. This lower limit is a result of the limitation of the energy range in this ﬁrst version of the EEP model presented here. Future work will focus on adding subsequent precipitation mechanisms, which will extend the range of impact altitudes, and bring us closer to being able to estimate the total EEP impact into the atmosphere. While the current ionization rates will enable us to make the ﬁrst long-term simulations of EEP impacts on the atmosphere and climate, we note that these remain an underestimation of the total EEP ionization. Acknowledgments We thank the Academy of Finland for supporting this research: M. van de Kamp and A. Seppälä were supported by projects 258165, 265005, and 292806 (CLASP: Climate and Solar Particle Forcing), P. T. Verronen was supported by the project 276926 (SECTIC: Sun-Earth Connection Through Ion Chemistry). M. A. Clilverd was supported by the Natural Environmental Research Council grant NE/J008125/1. A. Seppälä, M. A. Clilverd, C. J. Rodger, and P. T. Verronen would like to thank the International Space Science Institute (ISSI), Bern, Switzerland for supporting the “Quantifying Hemispheric Diﬀerences in Particle Forcing Eﬀects on Stratospheric Ozone” team (Leader: D. R. Marsh). We would like to thank the geomagnetic data suppliers and the World Data Center for Geomagnetism, Kyoto, for making the Dst index data available via http://wdc.kugi.kyoto-u.ac.jp. The NOAA/POES data used in this study were made available by the National Oceanic and Atmospheric Administration. AARDDVARK data are available from the AARDDVARK Konsortia (see http://www.physics.otago.ac.nz/ space/AARDDVARK_homepage.htm). Other data presented in the paper are available from the corresponding author (annika.seppala@fmi.ﬁ). VAN DE KAMP ET AL. To enable long-term studies of EEP impact on the atmosphere, the ionization rates calculated from the EEP model results are available for the scientiﬁc community for use with chemistry-climate models, at http://solarisheppa.geomar.de/cmip6. A discussion on the ionization rates and their variability over the data set duration has been published separately [Matthes et al., 2016]. References Andersson, M. E., P. T. Verronen, C. J. Rodger, M. A. Clilverd, and A. Seppälä (2014), Missing driver in the Sun-Earth connection from energetic electron precipitation impacts mesospheric ozone, Nat. Commun., 5, 5197, doi:10.1038/ncomms6197. Baumgaertner, A. J. G., P. Jöckel, and C. 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https://openalex.org/W2315230561	https://zenodo.org/record/1217235/files/5116ijbiss01.pdf	English	null	ASSESSING THE IMPACT OF RELATIONSHIP QUALITY ON ONLINE ADOPTION	Deleted Journal	2,016	cc-by	6,334	ternational Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 ternational Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 Abstract Relationships are strongly embedded in Indian culture due to its high-context nature. Because of this, the role of relationship marketing has been advanced in a variety of services viz., banking, retailing, telecom, etc and hence the competition amongst various service providers has increased. Traditionally, relationship management used to be personal interaction based phenomenon but with IT as an enabler for online channels, the need for creating and growing relationships have transpired in a big way. With the advent of technological changes in the business environment, customers are seeking better alternatives to reduce the frequency of visiting the bank branch physically and are relying more on the virtual mode for transacting. Hence, banks and other service organizations are strongly focussing on the digital (online) channels for facilitating monetary transactions and hence gaining competitive advantage. In the virtual mode, the concept of relationships appears to be unrealistic due to the absence of human interface. However, significant studies have been conducted in this regard which focus on the influence of relationship quality factors viz., trust, commitment, satisfaction, etc. towards adopting the online channel for carrying out financial transactions. Relationship quality focuses on evaluating the strength of relationships, which affects customer loyalty. There exists a dearth of such significant studies in the Indian context. The present research will focus on bridging this gap in the literature. This paper will follow a causal research design for empirically analysing the impact of RQ factors towards adopting online mode for banking transactions. It will also investigate the future intentions of the customers toward using the online channels for carrying out financial transactions. Dr. Renu Aggarwal YMCA, Faridabad and Monica Bhardwaj Fortune Institute of International Business, Plot no 5, Rao Tula Ram Marg,,VasantVihar, Delhi, Delhi, India- 110057 Dr. Renu Aggarwal YMCA, Faridabad and Monica Bhardwaj Fortune Institute of International Business, Plot no 5, Rao Tula Ram Marg,,VasantVihar, Delhi, Delhi, India- 110057 Keywords Relationship Quality, Online, Adoption, India 1.INTRODUCTION Relationship marketing in the context of banking aims at establishing and maintaining long-term relationships with the customers (Ritter, 1993). The task of relationship becomes more challenging for online transactions (Mukherjee and Nath, 2003). In order to understand this challenge in a more sophisticated manner an assessment of the relationship strength is required. This can be judged using Relationship Quality (RQ) measures. DOI : 10.14810/ijbiss.2016.5101 1 Banking industry has been strongly enabled by the internet, which makes it convenient for the consumers to transact without physically visiting the bank.Commonly used banking operations by the customers via online medium include access to account information, funds transfer and bill payment. E-banking involves using internet technologies viz., internet, ATMs, mobile phones,etc. 1 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 for carrying out banking transactions (Abbasnejad, 2006 cited by Esmaeili et al, 2013). E-banking systems communicate customers about the banks’ efforts at three levels viz., notification, communication, and transaction (Ghane et al., 2010). Consumers are using internet banking for online shopping, mobile recharges, travel booking, movie ticket booking, etc.Consumers are benefitted in terms of easy access to their account, which can be controlled by them without any intervention/help from the bank employees and hence the banking consumers can make prompt financial decisions. In other words, online banking system is a self-service technology interface Meuteret al (2000). An important and challenging issue for the banks is the perceived security, which is perhaps due to the absence of bank employees and hence the banks are required to look at initiatives for developing online banking relationships and hence shall look at strengthening the online relationship quality (Warrington et al, 2000). Banks are expected to inculcate trust in the relationships such that the online banking is promoted which further looks at building customer loyalty. The importance of trust is at the paramount level due to the complexities arising from the virtual environment (Mukherjee and Nath,2003). Interestingly, trust is understood as the key factor, which is one of the components of RQ. The other important factors include commitment and satisfaction. These are discussed in detail under the review of literature section. The popularity of online banking has propelled since mid 1990s across the globe especially among the developed countries (Pikkarainenet al., 2004). The rate of penetration for online banking has risen immensely in the last few years. 1.INTRODUCTION In 2012, the total number of internet users were 137 million which is expected to rise to somewhere in the range of 330 million to 370 million (Internet World Stats, 2010; McKinsey Digital Consumer Research, 2013). In addition, the digital maturity index suggests that banking industry in India is at the starting stage of digital maturity (Mckinsey Analysis, 2013) while industries like personal computing and travel have reached the digital acceleration levels in India. According to the McKinsey Digital Consumer Survey 2012, the penetration for digital high-value consumers is 24% and online banking is used 0.19 times per week, while for overall consumers it is 7%, who use online banking 0.02 times per week. It is worth noting that the purchases made by the digital high-value consumers is just 1% while researching for the same and making final decision for purchase score 64% and 42% respectively thereby suggesting the lack of trust in the virtual medium. According to Mckinsey’s (2012) article on the impact of internet on India, the online payment system needs to be strengthened for which the Indian banking consumers exhibit low levels of trust and confidence. However, on the positive note, India still has a good scope for improvement in online payment systems and internet readiness, which is also mentioned in the same report. Therefore, it is important to evaluate the factors, which significantly affect the consumer’s intention to adopt the online medium for banking and payments and their plan to continue with the online banking system in future.In addition to this, the online technology interface provided by the bank has been evolving over the years and hence suggest the need for research towards the adoption of online banking which is aimed at enhancing the customer intentions (Loureiro et al., 2014). This study attempts to integrate RQ and online banking adoption thereby extending the current state of knowledge. 2 ternational Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 To address this, following research questions are put forward- To address this, following research questions are put forward- RQ1: Which relationship quality factors are the significant influencers of online banking adoption? RQ2: How do relationship quality factors influence the consumer’s adoption process towards online banking? 2.REVIEW OF LITERATURE It is well understood in the literature that much of the research pertaining to online adoption refers to the Technology Acceptance Model (TAM) and Theory of Reasoned Action (TRA). However, there have been many variations in the conceptualization and application of the model, which has resulted into various modified versions. In addition, the elusive nature of the online environment calls for more focus on the human factors, which ponder on relationships and hence the conceptualization of RQ towards its influence on adoption intentions holds importance. The consideration of RQ has been different across various studies in a very creative manner thereby carving out different dimensions. Trust and Satisfaction have been widely accepted however, several researches also consider Commitment as the key factor comprising RQ (Johnson et al., 2004). Initially, the research by Crosby et al (1990) discussed trust and satisfaction as the antecedents of RQ while Wong and Sohal(2002) contributed commitment as the other dimensions but still as the antecedent to RQ. However, there are several research works, which consider these factors as dimensions of RQ and not as antecedents. These include the works of Storbacka et al (1994), Henning-Thurau and Klee (1997), Dorsch et al (1998), Smith(1998), Baker et al (1999), Garbarino and Johnson (1999) , Lang and Colgate (2003), Walter et al (2003), Van Bruggen et al (2005), Ulaga and Eggert ( 2006), Leonidou et al (2006), Carr (2006). However, only the work of Lang and Colgate (2003) focus on banks and more specifically the online banking. The need for a fresh research perspective is expected which considers these three dimensions, which have been among the more popular RQ dimensions in the extant literature. These three main RQ factors are discussed in the following paragraphs along with a focus towards the online adoption. 1.INTRODUCTION The next section of the paper discusses the review of literature in which the factors pertaining to RQ viz., trust, commitment and satisfaction are reviewed. This is followed by the methodology section wherein the data collection method and the survey instrument along with scale development are discussed. Next, analysis of data is presented along with the structural model. This is followed by the discussion and managerial implications. 2.1.Trust Trust is the confidence exhibited by the buyer in the seller’s future performance. Trust is a key construct of RQ and a favourably perceived future performance results in high level of RQ and hence positively influences customer loyalty (Zhang et al., 2011).Trust holds greater importance in the online environment in relation to the offline-banking environment (Ratnasingham, 1998). Trust is related with sub-factors viz., benevolence, competence, integrity, predictability, etc. which is based on customer’s beliefs about the quality of services provided by the service 3 3 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 provider in online contexts (Gefen et al.,2003). Yee and Faziharudean (2010) discussed, through review of literature, how trust influences customer loyalty. Cyr et al (2007) found a direct and positive association between trust and loyalty in the context of e-services while the same was supported by Lin and Wang (2006) in the m-commerce context. In addition, Floh and Treiblmaier (2006) discussed trust and satisfaction apart from website quality as antecedents to customer loyalty in the online banking context.Yee and Faziharudean (2010) empirically investigated the impact of trust along with other factors on customer loyalty and found trust significantly influencing the customer loyalty in Malaysian online banking environment. Kim et al (2013) studied the mediating role of trust on the usage intentions in online shopping context. Yousafzai et al (2009)found that trust is an antecedent of behavioural intention and holds a multidimensional position in adoption to internet banking. Moreover, online banking is conditional to evoking of trust in the consumer’s mind, which depends on the ability of the bank. Trust also had significant and positive effect on internet banking adoption in the Vietnamese context (Chong et al, 2010). Some research works, which focus on TAM, also included trust in the structural model while evaluating the adoption intentions. Trust was considered as an antecedent to PU and PEOU dimensions of TAM and found that the trust has positive influence on both these dimensions (Erikssonet al, 2005). Also, PU and PEOU in turn influence the attitude towards online banking (Chiou and Shen, 2012). Suh and Han (2002) found that trust significantly influences customer’s attitude towards adoption to online banking. Suh and Han (2002) found trust as a key factor in elucidating customer attitude towards the adoption of online banking (Suh and Han, 2002). 2.1.Trust It is the attitude, which along with trust and PU influences online banking usage intentions. It is worth noting that attitude towards adoption is also understood as acceptance which is an antecedent to adoption intentions which in-turn might influence continuance usage intentions. Contrary to this,Kesharwani and Bisht (2012) found trust as an impediment towards online banking transactions in the Indian context when used as an extra dimension to TAM. 2.4.Attitude Situation of risk and uncertainty can be reduced by the involvement of trust (Mayer et al., 1995). Such challenging situations are common in the context of online banking. An increase in trust would result into positive attitude towards the online banking and hence would influence the intention to adopt the online mode for carrying out banking transactions. This point has also been reiterated by Jarvenpaa et al (1999) in the context of an internet store and also in the context of online banking (Liu et al, 2005). In the present study, the authors also contemplate that satisfaction and commitment also influence the attitude in a similar manner. 2.3.Satisfaction Satisfaction holds a great deal of importance in the online context (Nusair and Kandampully, 2008).An evaluation of seller’sperformance by the buyer results into the level of satisfaction. In addition, the confirmation or disconfirmation from the expectation of the consumers is another conceptualization of satisfaction(Hennig-Thurau and Hansen, 2000) which arises from expectancy-disconfirmation (Oliver, 1981). However, this theory has limitations due to the lack of understanding by the customers about their expectations especially owing to the complexities of the online environment (Allagui and Temessek, 2004). This calls for measuring satisfaction through customer opinions which is accumulated together to provide a measure of overall satisfaction based on a holistic experience (Garbarino and Johnson, 1999). For online banking environments it is the online contact with the customers over the period of time, which defines satisfaction (Liang et al,2008) which comes from the interactive online interfaces provided for the customers (Allagui and Temessek, 2004). A high level of RQ is a function of a high level of satisfaction. Satisfaction being a construct of RQ is highly likely in increasing consumer loyalty (Zhang et al, 2011). According to Mols (1998), online banking environment provides more satisfaction and satisfaction with the existing offering would provide more scope for adapting to the online banking channels. Therefore, past performance acts as an assurance of future performance and hence adoption becomes imminent as it is an additional benefit and more convenient due to the deployment of the technology (Rexha, 2003). In addition, in the context of banking, loyalty is key issue and hence customer satisfaction requires monitoring (Aurier and N’Goala, 2010) in order to achieve a high level of RQ. 2.2.Commitment In the context of consumer behaviour,commitment is a long-term wish for consumer to maintain the relationships (Moorman et al., 1993). Commitment can take three forms viz, attitudinal, instrumental and temporal (Gundlach et al., 1995) and also commitment is revealed in adoption of online banking (Rexha et al., 2003). It is also affects behaviour and attitude in a positive manner (Johnson et al., 2006). In fact, behaviour and attitude are the pointers to loyalty (Chiu et al., 2009). Behaviour can be elaborated as intention to purchase and intention to adopt in the online context. Attitude has been however understood differently in the literature where Chiu et al(2009) discuss it as willingness to recommend while in TAM it is an antecedent to intention to adopt. Looking into the RQ literature it is seen that commitment has found place as a dimension of RQ in different service industry settings. These include electronic products retailing (Heenig-Thurau, 2000), apparel retailing (De Wulf et al, 2003; De Cannie`re et al. 2009), travel agency (Moliner et al, 2007; Beatson et al, 2008). Studies pertaining to the banking industry involving the conceptualization of commitment as a dimension of RQ include the works of Lang and Colgate (2003) and Ndubisi (2007) and both these studies focus more on the intangible aspects with the former focussing on the online activities. 4 4 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 2.5.Continuance Usage Intention Continuance usage intention is related with the loyal behaviour of the customers towards the online banking. This is connected with continuing the relationship in future and not dropping it. Sometimes this phenomenon is also referred as relational continuity. Satisfaction is understood to be a key predictor of relational continuity (Leuthesser and Kohli, 1995). It is highly expected that relationships between the bank and the customer should develop with time which is contingent to high satisfaction levels due to which the customers are loyal and don’t switch their service providers (Mols, 1999). A variety of researches exist on online adoption related to banking and other e-commerce environments, which also include influence of RQ factors on intention to adopt online medium for aforementioned situations.A holistic perspective on the key RQ factors exhibiting their 5 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 influence on online adoption needs profound exploration and analysis. Trust, commitment and satisfaction have been discussed under various research works sometimes together but in most cases the three dimensions did not appear together for the purpose of evaluation of their consequences towards online adoption. For the present study, Trust Commitment and Satisfaction are the three factors considered which constitute RQ. In other words, the concept of RQ in the online context has not been widely discussed in a holistic manner. Brun et al (2014) work is perhaps the first significant study, which discusses online RQ comprising of trust, commitment and satisfaction. In addition, Rajaobelina et al (2013) have also included the very same three dimensions of RQ for classifying the online banking customers. 3.1.Sample and Data Collection Data was collected from the youth using mall-intercept method from various malls of Delhi-NCR. In addition, students from selected colleges in the region were also targeted for data collection. The students selected were either registered for undergraduate or postgraduate courses and both were mainly from the management streams. Other respondents who were contacted through the mall intercept method were mostly the working professionals. Mall intercept method has been found useful in various studies (O'Cass andGrace, 2008) and also in Indian context (Prasad and Aryasri, 2011, Tripathi and Dave, 2013). A qualifier question was included in the survey instrument to ascertain that the respondents were either casual or frequent users of online banking while at the same time non-users were excluded from the analysis. About 1000 respondents were contact, which resulted into 466 usable questionnaires for further analysis thereby giving a response rate of about 46%. The low response rate is quite usual in survey responses in Indian context especially among the youth who are at times casual users of the service. 3.2.Instruments and Measures The scale items for the three RQ factors along with attitude, adoption intention and continuance usage intentions were extracted from the literature. Attitude towards adoption and behavioural intention were culled from the study by Taylor and Todd (1995) with some modifications. The items for trust, commitment, satisfaction and continuance usage intention were extracted and adapted from the work of Liang et al (2012) 3.3.Hypothesis Based on the review of literature the following hypothesis are proposed – Based on the review of literature the following hypothesis are proposed H1: Trust directly and positively influences Attitude towards Adoption of online banking H2: Commitment directly and positively influences Attitude towards Adoption of online banking H3: Satisfaction directly and positively influences Attitude towards Adoption of online banking H4: Attitude towards Adoption directly and positively influences Intention to Adopt online banking 6 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 H5: Intention to Adopt online banking directly and positively influences Continuance Usage Intention Figure 1: Structural model Trust Behavioural Intentions Continuance Usage Intentions Commitment Satisfaction Attitude H3 H2 H1 H4 H5 Figure 1: Structural model 4.ANALYSIS AND DISCUSSION First, an Exploratory Factor Analysis was conducted using SPSS for all the variables. The KMO measure was 0.901 and was found significant. The communalities score were all above 0.50 and the total variance extracted was 74% resulting into six factors. The reliability scores were measured through Cronbach’s alpha. Then the measurement model was evaluated using CFA through AMOS software first for the RQ factors only and then for the whole model together. For the RQ factors only the model fit was found within the acceptable levels with CMIN/DF=2.314, RMR= 0.061, GFI= 0.951, CFI=0.981, RMSEA= 0.075. All items loaded significantly on their respective constructs. Next, CFA was subjected on RQ factors along with attitude towards adoption, intention to adopt and continuance intention. In this case, all the items loaded significantly on their respective dimensions. The model fit was acceptable with CMIN/DF=2.529, RMR= 0.053, GFI= 0.908, CFI=0.948, RMSEA 0 051 Trust Behavioural Intentions Continuance Usage Intentions Commitment Satisfaction Attitude H3 H2 H1 H4 H5 Trust Satisfaction Commitment H3 H1 Attitude Behavioural Intentions Figure 1: Structural model 4.2.Construct Validity It measures the level to which an observed variable measures the unobserved factors for which they are created. It includes face validity, convergent validity and discriminant validity. Face validity is established by extracting the scale items from the extant literature and the same have been adjusted to fit with the present study. Convergent validity was established by investigating the factor loadings and AVE. All the factor loadings (or standardized estimates) were significant at p < 0.001. Reliability scores were pre- established and were all greater than 0.7. All these estimates were higher than 0.7. AVE figures were all greater than 0.50. In addition, AVE was greater than square of the inter-construct correlation. Hence, discriminant validity was established. 4.ANALYSIS AND DISCUSSION First, an Exploratory Factor Analysis was conducted using SPSS for all the variables. The KMO measure was 0.901 and was found significant. The communalities score were all above 0.50 and the total variance extracted was 74% resulting into six factors. The reliability scores were measured through Cronbach’s alpha. Then the measurement model was evaluated using CFA through AMOS software first for the RQ factors only and then for the whole model together. For the RQ factors only the model fit was found within the acceptable levels with CMIN/DF=2.314, RMR= 0.061, GFI= 0.951, CFI=0.981, RMSEA= 0.075. All items loaded significantly on their respective constructs. Next, CFA was subjected on RQ factors along with attitude towards adoption, intention to adopt and continuance intention. In this case, all the items loaded significantly on their respective dimensions. The model fit was acceptable with CMIN/DF=2.529, RMR= 0.053, GFI= 0.908, CFI=0.948, RMSEA= 0.051. 7 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 4.1.Scale Reliability All the latent constructs show signs of high reliability with Cronbach’s Alpha > 0.70 (Hair et al., 2006) and hence the scale is reliable. In addition, composite reliability and average variance extracted scores are taken for all the RQ factors and for the dependent constructs. These are provided in the Table # 1.Composite reliability obtained for all the latent variables is greater than 0.70 and hence it crosses the minimum acceptable values (Carmines and Zeller, 1988). AVE for each factor is greater than 0.5 and hence it is above the minimum acceptable values (Fornell and Larcker, 1981). 4.3.Testing the Structural model The structural model provides an adequate fit with CMIN/DF= 3.279, RMR= 0.057, GFI= 0.905, CFI= 0.918, RMSEA= 0.059. The hypotheses results are provided in Table #2. Except for H2, which is aimed at testing the influence of commitment onattitude towards adopting online banking, all other hypotheses are significant at p<0.001. Results indicate that trust and satisfaction directly and positively influence attitude towards adoption of online banking, which in turn directly and positively influence Intention towards online adoption, which further directly and positively influences intention to continue the online banking usage in future. Table# 1: Measurement model statistics Constructs Measurement Items Estimate AVE CR Trust Trust1 0.745 0.730 0.889 Trust2 0.918 Trust3 0.890 Commitment Commitment1 0.829 0.697 0.889 Commitment2 0.885 Table# 1: Measurement model statistics International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 Commitment3 0.787 Satisfaction Satisfaction1 0.948 0.839 0.940 Satisfaction2 0.879 Satisfaction3 0.919 Attitude Adoption3 0.918 0.813 0.929 Adoption2 0.910 Adoption1 0.877 Behavioural Intentions B_Int1 0.763 0.759 0.903 B_Int2 0.941 B_Int3 0.899 Continuance Usage CU_Int1 0.873 0.692 0.871 CU_Int2 0.834 CU_Int3 0.786 Table #2: Hypothesis testing Hypotheses Hypothesised Paths Standardised Path Co- efficients p- value Results H1 Adoption <--- Trust 0.417 * Hypothesis Supported H2 Adoption <--- Commitment -0.036 0.671 Hypothesis Not Supported H3 Adoption <--- Satisfaction 0.393 * Hypothesis Supported H4 BI <--- Adoption 0.749 * Hypothesis Supported H5 CUI <--- BI 0.764 * Hypothesis Supported Table #2: Hypothesis testing 5.DISCUSSION AND MANAGERIAL IMPLICATIONS 9 The present study is aimed at testing how relationship quality can influence the adoption of online banking by the customers. Insights are gained into the key relationship quality factors, which are identified and validated using the CFA approach. These dimensions can be used by the managers 9 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 International Journal of Business Information Systems Strategies (IJBISS) Vol.5, No.1,February 2016 to understand the consumer behaviour related to online adoption by the bank customers in the Indian context where the penetration levels of online banking are limited despite a relatively large number of internet users. These factors also bring forward a very important aspect of decision- making process, which comes from attitude towards adoption, which is influenced, by trust and satisfaction. Attitude acts an influencer to behavioural intention to adopt online mode for banking. The difference between attitude and intention to adopt is critical but the managerial understanding about the online adoption process is important at one-step further. The intention to adopt might be casual but if the experience werepositive, it would help in adopting online mode for banking more regularly. This focus is necessary for the banks, as online mode is more cost effective than other modes for transactions. 6. LIMITATIONS AND SCOPE FOR FURTHER RESEARCH The current study is limited in its geographical scope of Delhi-NCR, which shall not be considered as a reference for understanding the consumer behaviour for the whole country. In addition, more RQ factors can be derived from the literature, which might help in expanding the academic horizon in the context of online banking adoption. There might be a possibility of a second order constructs particularly comprising of trust and satisfaction especially in the case of online environments (Zhang et al., 2011). 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https://openalex.org/W4389815380	https://journal.lembagakita.org/index.php/jemsi/article/download/1099/790	Indonesian	null	Pengaruh Fasilitas, Pelayanan, Harga Dan Pemberian Diskon Terhadap Kepuasan Pelanggan (Studi Kasus Pada Warnet @N-Kom Net)	JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi)	2,023	cc-by-sa	4,435	Pengaruh Fasilitas, Pelayanan, Harga Dan Pemberian Diskon Terhadap Kepuasan Pelanggan (Studi Kasus Pada Warnet @N-Kom Net) aruh Fasilitas, Pelayanan, Harga Dan Pemberian Diskon Terhadap Kepuasan Pelanggan (Studi Kasus Pada Warnet @N-Kom Net) Tri Utama Tambunan Prodi Manajemen, Fakultas Ekonomi dan Bisnis, Universitas Labuhanbatu, Jalan Sisingamangaraja No.126 A Kec. Rantau Selatan, Kab. Labuhanbatu, Sumatera Utara, Indonesia tamatambunan19@gmail.com Yuniman Zebua Prodi Manajemen, Fakultas Ekonomi dan Bisnis, Universitas Labuhanbatu, Jalan Sisingamangaraja No.126 A Kec. Rantau Selatan, Kab. Labuhanbatu, Sumatera Utara, Indonesia oinitehezeb@gmail.com Junita Lubis Prodi Manajemen, Fakultas Ekonomi dan Bisnis, Universitas Labuhanbatu, Jalan Sisingamangaraja No.126 A Kec. Rantau Selatan, Kab. Labuhanbatu, Sumatera Utara, Indonesia junitayuri@yahoo.com Abstrak Penelitian ini bertujuan untuk mengetahui bagaimana pengaruh fasilitas, pelayanan, pemberian diskon dan harga terhadap kepuasan pelanggan pada warnet @N-Kom Net. Penelitian ini menggunakan metode pengumpulaan data dengan menyebarkan kuesioner. Populasi dan sample yang digunakan dalam penelitian ini adalah konsumen warnet @N-Kom Net sebanyak 100 responden. Pengujian hipotesis dalam penelitian ini menggunakan alat analisis uji validitas, uji realibilitas, uji asumsi klasik regresi linier berganda, (uji t), (uji F) dan koefesien determinasi (R2). Hasil penelitian ini menunjukkkan bahwa secara Parsial Fasilitas berpengaruh Positif dan Signifikan terhadap Kepuasan Pelanggan pada Warnet @N-Kom Net. Secara Parsial Pelayanan tidak berpengaruh terhadap Kepuasan Pelanggan pada Warnet @N-Kom Net karena kurangnya jumlah pekerja pada warnet @N-Kom Net membuat kinerja pegawai menjadi lambat. Secara Parsial Harga tidak berpengaruh terhadap Kepuasan Pelanggan pada Warnet @N-Kom Net karena harga yang diberikan oleh Warnet @N-Kom Net cenderung sama dengan harga yang terdapat pada warnet lainnya. Secara Parsial Pemberian Diskon tidak berpengaruh terhadap Kepuasan Pelanggan pada Warnet @N-Kom Net karena pemberian diskon hanya diberikan kepada orang-orang tertentu dan warnet @N-Kom Net tidak pernah memberikan diskon secara terang-terangan. Secara Simultan Variabel Fasilitas, Pelayanan, Harga dan Pemberian Diskon secara bersama tidak memiliki pengaruh signifikan terhadap Kepuasan Pelanggan pada Warnet @N-Kom Net. Kata Kunci : Fasilitas, Pelayanan, Harga, Pemberian Diskon, Kepuasan Pelanggan Kata Kunci : Fasilitas, Pelayanan, Harga, Pemberian Diskon, Kepuasan Pelanggan Article’s History: Received 14 April 2023; Received in revised form 18 April 2023; Accepted 14 Mei 2023; Published 1 Juni 2023. All rights reserved to the Lembaga Otonom Lembaga Informasi dan Riset Indonesia (KITA INFO dan RISET). Suggested Citation: Tambunan, T, U., Zebua, Y., & Lubis, J. (2023). Pengaruh Fasilitas, Pelayanan, Harga Dan Pemberian Diskon Terhadap Kepuasan Pelanggan (Studi Kasus Pada Warnet @N-Kom Net). JEMSI (Jurnal Ekonomi, Manajemen, Dan Akuntansi), 9 (3). 642 – 649. https://doi.org/10.35870/jemsi.v9i3.1099 Suggested Citation: JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 9 (3) Juni Tahun 2023, Hal 642-649. JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 9 (3) Juni Tahun 2023, Hal 642-649. Pengaruh Fasilitas, Pelayanan, Harga Dan Pemberian Diskon Terhadap Kepuasan Pelanggan (Studi Kasus Pada Warnet @N-Kom Net) Pendahuluan Pada zaman yang serba modern seperti saat ini peran usaha seperti warung internet atau yang biasa lebih dikenal dengan istilah warnet sangat dibutuhkan, terutama dikalangan masyarakat yang sedang duduk di bangku pendidikan dan para pekerja. Karena mereka bisa dengan mudah mendapatkan kebutuhan yang mereka butuhkan seperti pembilan alat tulis kantor ataupun melakukan aktivitas print dan fotocopy. Dewasa ini warnet sangat banyak kita temukan di daerah atau lingkungan sekitar kita namun kebanyakan warnet yang tersedia saat ini tidak memiliki fasilitas yang lengkap, kebanyakan dari warnet yang dijumpai saat ini cenderung hanya menyediakan fasilitas untuk pengguna atau pemain game online saja. Namun ada beberapa warnet yang 642 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 9 (3) Juni Tahun 2023, Hal 642-649. menyediakan seperti yang dibutuhkan untuk orang-orang yang membutuhkan jasa ataupun pembelian namun tak jarang juga masih banyak terjadi hal-hal yang masih belum memuaskan pengguna atau pembelinya baik seperti fasilitas yang tidak lengkap, pelayanan yang kurang baik, pemberian diskon yang tidak merata, harga yang cenderung berubah-ubah sehingga membuat pembeli atau pengguna tidak merasa terpuaskan. menyediakan seperti yang dibutuhkan untuk orang-orang yang membutuhkan jasa ataupun pembelian namun tak jarang juga masih banyak terjadi hal-hal yang masih belum memuaskan pengguna atau pembelinya baik seperti fasilitas yang tidak lengkap, pelayanan yang kurang baik, pemberian diskon yang tidak merata, harga yang cenderung berubah-ubah sehingga membuat pembeli atau pengguna tidak merasa terpuaskan. Warnet @N-Komnet adalah sebuah tempat atau usaha yang termasuk dalam kategori layanan jasa dan penjualan, terletak di Jalan Perisai Rantauprapat dengan lokasi yang cukup strategis karena berada di tengah- tengah kota Rantauprapat. Dalam penelitian ini peneliti mendapatkan beberapa permasalahan yang terjadi pada warnet @N-Kom Net mulai dari fasilitas yang kurang lengkap pada warnet tersebut dan sering terjadi permasalahan lainnya seperti terdapat banyaknya komputer yang tidak beroperasi lagi, printer yang sering eror, komputer yang pengoperasiannya lambat dan banyak lagi. Selain itu Pelayanan pada warnet @N-Kom Net juga kurang cukup memuaskan karena hanya terdapat 1 orang jumlah pekerja yang membuat pelayanan pada warnet tersebut kurang efisien atau lambat. Warnet @N-Kom Net juga sering memberikan diskon kepada pelanggannya namun pemberian diskon tersebut terkesan tidak adil atau tidak merata karena mereka hanya memberikan diskon kepada orang-orang yang dikenal atau pelanggan tetap mereka dan juga kepada orang-orang yang berbelanja banyak saja. Harga-harga yang mereka berikan untuk setiap penjualan atau jumlah print kepada pelanggannya cukup standar yaitu sama dengan kebanyakan warnet di kota ini. Pendahuluan Karena beberapa permasalahan tersebut pelanggan cukup berpikir untuk menggunakan atau datang untuk melakukan pembelian pada warnet tersebut karena beberapa alasan seperti fasilitas, pelayanan, pemberian diskon dan harga yang kurang sesuai dengan keinginan konsumen menyebabkan konsumen kurang puas melakukan pembelian atau penggunaan jasa pada warnet @N-Kom Net. Fasilitas Tidak bisa dipungkiri jika kita ingin terpuaskan dalam mengerjakan suatu pekerjaan maka fasilitas yang digunakan atau disediakan hendaknya lengkap atau bagus. Fasilitas merupakan suatu objek yang penting guna meningkatkan suatu kepuasan, kepusan yang dimaksud bisa berbentuk kenyamanan pelanggan, dapat memenuhi kebutuhan pengguna dan kenyamanan pengguna layanan (Oetama & Sari, 2017). Sedangkan fasilitas menurut (Kotler & Keller, 2016) adalah peralatan-peralatan yang disediakan oleh pihak penjual jasa untuk mendukung kenyamanan konsumen. g y Menurut (Fandy & Gregorius, 2016) dalam mewujudkan fasilitas terdapat beberapa indikator untuk mengevaluasi hal tersebut, yaitu : 1. Perencanaan Spasial Berkaitan dengan unsure jarak, lokasi, bentuk, dan ukuran. Hal ini sangat berkaitan dengan pemanfaatan waktu. 1. Perencanaan Spasial Berkaitan dengan unsure jarak, lokasi, bentuk, dan ukuran. Hal ini sangat berkaitan dengan pemanfaatan waktu. 2 Perencanaan Ruang 2. Perencanaan Ruang Faktor yang meliputi perencanaan interior dan arsitektur, seperti penempatan perabotan, dan perlengkapan didalam ruangan. g tor yang meliputi perencanaan interior dan arsitektur, seperti penempatan perabotan, dan perlengkapan lam ruangan. k 3. Perlengkapan Sebagai instrumen pelengkap yang dapat mempersembahkan kenyamanan, sebagai dekorasi atau sebagai infrastruktur pendukung didalam penggunaan barang para pelanggan. 3. Perlengkapan Sebagai instrumen pelengkap yang dapat mempersembahkan kenyamanan, sebagai dekorasi atau sebagai infrastruktur pendukung didalam penggunaan barang para pelanggan. 4 Tata Cahaya dan Warna 4. Tata Cahaya dan Warna Pengaturan pada cahaya dan warna ruangan yang sesuai aktivitas yang dilakukan serta suasana yang ingin dibangun dalam ruangan tersebut. y Pengaturan pada cahaya dan warna ruangan yang sesuai aktivitas yang dilakukan serta suasana yang ingin dibangun dalam ruangan tersebut. 5 Instruksi yang disampaikan secara grafis 5. Instruksi yang disampaikan secara grafis Penampilan visual, penempatan, penentuan bentuk fisik, pemilihan warna, pencahayaan, dan pemilihan bentuk perwajahan lambing yang ingin digunakan untuk maksud tertentu. y g p g Penampilan visual, penempatan, penentuan bentuk fisik, pemilihan warna, pencahayaan, dan pemilihan bentuk perwajahan lambing yang ingin digunakan untuk maksud tertentu. Harga Saat melakukan pembelian atau saat menggunakan jasa maka hal yang pertama ditanyakan atau yang menjadi pertimbangan adalah harga, apakah sesuai dengan yang kita harapkan atau tidak. Harga adalah beberapa uang yang ditukarkan untuk sebuah produk atau jasa, harga merupakan sejumlah nilai yang ditukarkan konsumen untuk mendapatkan sejumlah manfaat atau suatu barang dan jasa (Armstrong et al., 2014). Menurut (Ramli, 2013) harga merupakan nilai relatif oleh suatu produk namun bukanlah indikator tetap yang menunjukkan besarnya sumber daya yang dibutuhkan untuk membu Indikator harga menurut (Rangkuti, 2011) adalah sebagai berikut : g ( g ) g 1. Penilaian mengenai harga secara keseluruhan Harga yang telah ditetapkan oleh perusahaan dapat dianalisa dengan melihat tanggapan yang diberikan konsumen terhadap harga tersebut, apakah konsumen telah menerima harga yang ditetapkan dengan manfaat yang diterima. 1. Penilaian mengenai harga secara keseluruhan Harga yang telah ditetapkan oleh perusahaan dapat dianalisa dengan melihat tanggapan yang diberikan konsumen terhadap harga tersebut, apakah konsumen telah menerima harga yang ditetapkan dengan manfaat yang diterima. 2. Respons terhadap kenaikan harga Jika terjadi kenaikan harga dari suatu produk, sebaiknya dilihat bagaimana respon konsumen terhadap kenaikan harga tersebut, apakah akan mempengaruhi keputusan dalam membeli produk tersebut ataukah sebaliknya. 2. Respons terhadap kenaikan harga Jika terjadi kenaikan harga dari suatu produk, sebaiknya dilihat bagaimana respon konsumen terhadap kenaikan harga tersebut, apakah akan mempengaruhi keputusan dalam membeli produk tersebut ataukah sebaliknya. p y 3. Harga produk tertentu dibandingkan produk yang sama apabila ditempat lain. Konsumen dalam menentukan produk yang akan dibeli, akan membandingkan harga tersebut harga produk di tempat lain kebanyakan perusahaan dalam menawarkan produknya menetapkan harga berdasarkan suatu kombinasi barang secara fisik ditambah beberapa jasa lain serta keuntungan yang memuaskan. Pelayanan Jika kita pergi untuk melakukan suatu pembelian atau penggunaan jasa, kita akan berharap akan mendapatkan pelayanan yang sebaik mungkin demi kenyamanan agar pembelian atau penggunaan jasa yang kita inginkan sesuai dengan yang kita harapkan. Kualitas pelayanan adalah sebuah pengukur seberapa tinggi suatu pelayanan diberikan untuk memenuhi ataupun melebihi harapan pelanggan (Siboro & Suhardi, 2020). 643 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 9 (3) Juni Tahun 2023, Hal 642-649. Sedangkan menurut (Firdaus & Kasmir, 2021) pelayanan adalah suatu tindakan untuk memberikan kepuasan terhadap konsumen. Kualitas pelayanan merupakan salah satu faktor penilaian yang konsumen dapatkan, persepsi konsumen terhadap 5 (lima) indikator spesifik dari kinerja pelayanan. Adapun kelima indikator kualitas pelayanan tersebut yang dijelaskan oleh (Fandy & Gregorius, 2016) sebagai berikut : 1. Berwujud (Tangibles), berkenaan dengan daya tarik fasilitas fisik, perlengkapan, dan material yang digunanakn perusahaan, serta penampilan karyawan. 1. Berwujud (Tangibles), berkenaan dengan daya tarik fasilitas fisik, perlengkapan, dan material yang digunanakn perusahaan, serta penampilan karyawan. g p p p y 2. Keandalan (Reliability), berkaitan dengan kemampuan perusahaan untuk memberikan pelayanan yang akurat sejak pertama kali tanpa membuat kesalahan apapun dan menyampaiakan jasanya sesuai dengan waktu yang disepakati. 2. Keandalan (Reliability), berkaitan dengan kemampuan perusahaan untuk memberikan pelayanan yang akurat sejak pertama kali tanpa membuat kesalahan apapun dan menyampaiakan jasanya sesuai dengan waktu yang disepakati. 3. Daya Tanggap (Responsiveness), berkenaan dengan kesediaan dan kemampuan untuk membantu para pelanggan dan memproses permintaan mereka, serta menginformasikan kapan jasa akan diberikan dan kemudian memberikan jasa secara cepat. 4. Jaminan (Assurance), berkenaan dengan perilaku para karyawan mampu menumbuhkan kepercayaan pelanggan terhadap perusahaan dan perusahaan bisa menciptakan rasa aman bagi para pelanggan. 5. Empati (Emphaty), berarti bahwa perusahaan memahami masalah para pelanggan dan bertindak demi kepentingan pelanggan, serta memberikan perhatian personal kepada para pelanggan dan memiliki jam operasi yang nyaman. Kepuasan Pelanggan Saat melakukan suatu pembelian atau penggunaan jasa maka kita akan mencari yang sesuai dengan keinginan kita agar kita merasa nyaman dan puas telah melakukan aktifitas pembelian atau penggunaan jasa pada tempat tersebut. Kepuasan pelanggan adalah sebuah perbandingan mengenai kualitas pelayanan yang dirasakan oleh pelanggan, jika kualitas yang didapatkan pelanggan tidak sesuai dengan yang diharapkan maka terjadilah ketidakpuasan, jika kualitas yang didapatkan pelanggan sesuai yang diharapkan maka pelanggan akan puas, dan jika kualitas yang didapatkan pelanggan lebih dari apa yang diharapkan maka pelanggan akan merasa sangat puas (Riyanto, 2018). Jika perusahaan mampu memberikan pelayanan yang baik maka semakin tinggi pula kepuasan pelanggan (Bhuwana et al., 2013). gg p p p gg ( ) Menurut (Tjiptono, 2014) mengemukakan bahwa terdapat beberapa indikator yang dapa kepuasan pelanggan. indikator tersebut yaitu : 1. Kesesuaian Harapan Merupakan tingkat kesesuaian antara kinerja produk yang diharapkan oleh konsumen dengan yang dirasakan oleh konsumen. Seperti pelayanan oleh karyawan yang diperoleh sesuai atau melebihi dengan yang diharapkan. y g p 2. Minat berkunjung kembali Merupakan kesediaan pelanggan untuk berkunjung kembali atau melakukan pembelian ulang terhadap produk terkait. Seperti berminat untuk berkunjung kembali karena pelayanan yang diberikan oleh karyawan memuaskan dan berminat untuk berkunjung kembali karena fasilitas penunjang yang diediakan memadai. 2. Minat berkunjung kembali Pemberian Diskon Saat melakukan pembelian atau penggunaan jasa kita pasti mengharapkan diskon atau potongan harga, apalagi jika kita melakukan aktifitas pembelian atau penggunaan jasa yang cukup banyak atau melakukan pembelian dengan jumlah banyak maka wajar jika kita menginginkan atau mendapat potongan harga. Potongan harga atau yang bisa disebut dengan diskon adalah suatu langkah perusahaan untuk menarik minat pelanggan atau pembeli dengan cara yang cepat (Kusnawan et al., 2019). Selain itu diskon juga merupakan pengurangan atau penurunan harga normal suatu produk dalam kurun waktu tertentu untuk menarik minat beli pelanggan (Sonata, 2019). ( , ) Adapun beberapa indikator diskon menurut (Baskara, 2015) yaitu : 1. Besarnya potongan harga 2. Masa potongan harga 3. Jenis produk yang mendapatkan discount 644 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 9 (3) Juni Tahun 2023, Hal 642-649. Metodologi Penelitian Jenis penelitian ini adalah kuantitatif, penelitian dilaksanakan di warnet @N-Kom Net. Teknik pengumpulan data yang digunakan dalam penelitian ini adalah teknik menggunakan instrument survey kuesioner dengan metode skala Likert. Penetapan sampel pada penelitian ini menggunakan rumus Wibisono, hal ini disebabkan karena jumlah populasi belum diketahui (Wibisono, 2003: 52). Jumlah sampel yang ditentukan berdasarkan rumus Wibisono adalah sebagai berikut : = =96 g = =96 Keterangan: n : Jumlah sampel Z : Tingkat kepercayaan sebesar 95% = 1,96 σ : Standar deviasi populasi = 0,25 e : Tingkat kesalahan sebesar 5% = 0,05 = =96 Keterangan: n : Jumlah sampel Z : Tingkat kepercayaan sebesar 95% = 1,96 σ : Standar deviasi populasi = 0,25 e : Tingkat kesalahan sebesar 5% = 0,05 Z : Tingkat kepercayaan sebesar 95% = 1,96 e : Tingkat kesalahan sebesar 5% = 0,05 Berdasarkan hasil perhitungan dengan menggunakan rumus Wibisono, ditetapkan jumlah sampel pada penelitian ini sebesar 96 namun digenapkan menjadi 100 responden. Hasil dan Pembahasan Uji Validitas • Jika rhitung > rtabel maka butir pernyataan tersebut adalah valid • Jika rhitung < rtabel maka butir pernyataan tersebut tidak valid Tabel 1 Hasil Uji Validitas Variabel Item Pernyataan r hitung r tabel Keterangan FASILITAS (X1) X1.1 0,196 0,195 Valid X1.2 0,253 0,195 Valid X1.3 0,358 0,195 Valid X1.4 0,220 0,195 Valid X1.5 0,303 0,195 Valid PELAYANAN (X2) X2.1 0,259 0,195 Valid 645 X2.2 0,249 0,195 Valid X2.3 0,289 0,195 Valid X2.4 0,211 0,195 Valid X2.5 0,255 0,195 Valid HARGA (X3) X3.1 0,325 0,195 Valid X3.2 0,242 0,195 Valid X3.3 0,222 0,195 Valid PEMBERIAN DISKON (X4) X4.1 0,318 0,195 Valid KEPUASAN PELANGGAN (Y) Y.1 0,268 0,195 Valid Y.2 0,229 0,195 Valid Y.3 0,232 0,195 Valid Y.4 0,219 0,195 Valid Sumber: data olah spss Uji Reliabilitas a. Apabila hasil koefisien Alpha > taraf sig. 60% atau 0,6 maka kuesioner tersebut reliable. b A bil h il k fi i Al h t f i 60% t 0 6 k k i t b t tid k li bl eliabilitas a. Apabila hasil koefisien Alpha > taraf sig. 60% atau 0,6 maka kuesioner tersebut reliable. p p g b. Apabila hasil koefisien Alpha < taraf sig. 60% atau 0,6 maka kuesioner tersebut tidak reliable. Tabel 2 Hasil Uji Reliabilitas Tabel 2 Hasil Uji Reliabilitas Tabel 2 Hasil Uji Reliabilitas Variabel Koefisien Alpha Taraf Sig. Keterangan Fasilitas (X1) 0,669 0,60 Reliable Pelayanan (X2) 0,655 0,60 Reliable Harga (X3) 0,677 0,60 Reliable Pemberian Diskon (X4) 0,692 0,60 Reliable Kepuasan Pelanggan (Y) 0,678 0,60 Reliable Keseluruhan variabel 0,700 0,60 Seluruh item reliable Sumber: data olah spss Berdasarkan Tabel 2 hasil uji reliabilitas dapat disimpulkan bahwa seluruh item reliable, dikarenakan nilai cronbach alpa 0,700 lebih besar dari taraf sig. 60%. Uji Normalitas Tabel 3 Hasil Uji One-Sample Kolmogorov-Smirnov Test Unstandardized Residual N 100 Normal Parametersa,b Mean .0000000 Std. Deviation 2.36533969 Most Extreme Differences Absolute .038 Positive .038 Negative -.038 Test Statistic .038 Asymp. Sig. (2-tailed) .200c,d Sumber: data olah spss Uji Normalitas Tabel 3 Hasil Uji One-Sample Kolmogorov-Smirnov Test Unstandardized Residual N 100 Normal Parametersa,b Mean .0000000 Std. Deviation 2.36533969 Most Extreme Differences Absolute .038 Positive .038 Negative -.038 Test Statistic .038 Asymp. Sig. (2-tailed) .200c,d Sumber: data olah spss Berdasarkan Tabel 3 hasil uji Asymp. Sig. (2-tailed) sebesar 0,200 yang berarti lebih besar dari 0,05. Maka disimpulkan bahwa data dalam penelitian ini berdistribusi normal. Uji Normalitas Berdasarkan Tabel 3 hasil uji Asymp. Hasil dan Pembahasan Sig. (2-tailed) sebesar 0,200 yang berarti lebih besar dari 0,05. Maka disimpulkan bahwa data dalam penelitian ini berdistribusi normal. Berdasarkan Tabel 3 hasil uji Asymp. Sig. (2-tailed) sebesar 0,200 yang berarti lebih besar dari 0,05. Maka disimpulkan bahwa data dalam penelitian ini berdistribusi normal. Berdasarkan Tabel 3 hasil uji Asymp. Sig. (2-tailed) sebesar 0,200 yang berarti lebih besar dari 0,05. Maka disimpulkan bahwa data dalam penelitian ini berdistribusi normal. 646 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 9 (3) Juni Tahun 2023, Hal 642-649. Uji Multikolinearitas Tabel 4 Hasil Uji Multikolinearitas Variabel Tolerance VIF Keterangan Fasilitas 0,868 1,152 Tidak terjadi multikolinearitas Pelayanan 0,813 1,230 Tidak terjadi multikolinearitas Harga 0,900 1,112 Tidak terjadi multikolinearitas Pemberian Diskon 0,948 1,055 Tidak terjadi multikolinearitas Sumber: data olah spss JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 9 (3) Juni Tahun 2023, Hal 642-649. Uji Multikolinearitas Tabel 4 Hasil Uji Multikolinearitas Variabel Tolerance VIF Keterangan Fasilitas 0,868 1,152 Tidak terjadi multikolinearitas Pelayanan 0,813 1,230 Tidak terjadi multikolinearitas Harga 0,900 1,112 Tidak terjadi multikolinearitas Pemberian Diskon 0,948 1,055 Tidak terjadi multikolinearitas Sumber: data olah spss Uji Multikolinearitas Berdasarkan Tabel 4 menunjukkan bahwa variabel seluruh variabel dengan nilai Tolerance > 0,10 dan nilai VIF < 10. Maka dapat disimpulkan bahwa semua variabel bebas tidak terjadi multikolinearitas. Uji Hipotesis Parsial (Uji T) Uji Hipotesis Parsial (Uji T) Uji Hipotesis Parsial (Uji T) Uji t digunakan untuk mengetahui apakah variabel bebas (x) dapat memengaruhi variabel terikat (y). • Jika Nilai sig < 0,05 dan t hitung > t tabel (berpengaruh positif dan signifikan) • Jika Nilai sig > 0,05 dan t hitung < t tabel (tidak berpengaruh positif dan signifikan) Tabel 5 Hasil Uji t Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 7.634 3.118 2.448 .016 FASILITAS .283 .140 .217 2.028 .045 PELAYANAN -.082 .120 -.076 -.684 .495 HARGA .197 .146 .142 1.346 .181 PEMBERIAN DISKON -.020 .238 -.009 -.085 .933 Sumber: data olah spss Uji Hipotesis Parsial (Uji T) Uji t digunakan untuk mengetahui apakah variabel bebas (x) dapat memengaruhi variabel terikat (y). • Jika Nilai sig < 0,05 dan t hitung > t tabel (berpengaruh positif dan signifikan) • Jika Nilai sig > 0,05 dan t hitung < t tabel (tidak berpengaruh positif dan signifikan) Tabel 5 Hasil Uji t Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Uji Simultan (Uji f) Uji f digunakan untuk mengetahui apakah dari semua variabel independen (x) yang digunakan dalam model memberikan pengaruh secara bersama-sama atau simultan terhadap variabel dependen (y). memberikan pengaruh secara bersama-sama atau simultan terhadap variabel dependen (y). • Jika Nilai sig < 0,05 dan F hitung > F tabel (secara simultan berpengaruh positif dan signifikan) • Jika Nilai sig >0,05 dan F hitung < F tabel (secara simultan tidak berpengaruh positif dan signifikan) • Jika Nilai sig < 0,05 dan F hitung > F tabel (secara simultan berpengaruh positif dan signifikan) • Jika Nilai sig >0,05 dan F hitung < F tabel (secara simultan tidak berpengaruh positif dan signifikan) Tabel 6 Hasil Uji f ANOVAa Model Sum of Squares df Mean Square F Sig. 1 Regression 31.822 4 7.955 1.364 .252b Residual 553.888 95 5.830 Total 585.710 99 Sumber: data olah spss Tabel 6 Hasil Uji f ANOVAa Model Sum of Squares df Mean Square F Sig. 1 Regression 31.822 4 7.955 1.364 .252b Residual 553.888 95 5.830 Total 585.710 99 Sumber: data olah spss Berdasarkan hasil Tabel 6 dapat diketahui nilai F hitung < F tabel (1,364 < 2,47) dengan nilai signifikansi 0,252 > 0,05 maka dapat disimpulkan bahwa tidak terdapat pengaruh secara bersama atau simultan antara Fasilitas (X1), Pelayanan (X2), Harga (X3) dan Pemberian Diskon (X4) terhadap Kepuasan Pelanggan (Y). Berdasarkan hasil Tabel 6 dapat diketahui nilai F hitung < F tabel (1,364 < 2,47) dengan nilai signifikansi 0,252 > 0,05 maka dapat disimpulkan bahwa tidak terdapat pengaruh secara bersama atau simultan antara Fasilitas (X1), Pelayanan (X2), Harga (X3) dan Pemberian Diskon (X4) terhadap Kepuasan Pelanggan (Y). Uji Koefisien Determinasi (R2) Uji Koefisien Determinasi (R2) Tabel 7 Hasil Uji Koefisien Determinasi Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate 1 .233a .054 .015 2.415 Sumber: data olah spss Pada Tabel 7 terlihat bahwa nilai R square 0,054. Hal ini menjelaskan bahwa Fasilitas (X1), Pelayanan (X2), Harga (X3) dan Pemberian Diskon (X4) terhadap Kepuasan Pelanggan (Y) sebesar 5,4% dan sisanya 94,6% dijelaskan oleh faktor-faktor lain diluar variabel yang digunakan dalam penelitian ini. Faktor-faktor lain yang dimaksud seperti Store Atmosphere, Loyalitas Pelanggan, Promosi, Lokasi, WOM dan sebagainya. Tabel 7 Hasil Uji Koefisien Determinasi Pada Tabel 7 terlihat bahwa nilai R square 0,054. Hasil dan Pembahasan Error Beta 1 (Constant) 7.634 3.118 2.448 .016 FASILITAS .283 .140 .217 2.028 .045 PELAYANAN -.082 .120 -.076 -.684 .495 HARGA .197 .146 .142 1.346 .181 PEMBERIAN DISKON -.020 .238 -.009 -.085 .933 Sumber: data olah spss Berdasarkan Tabel di atas penjelasan masing-masing variabel terhadap keputusan pembelian adalah sebagai berikut: Berdasarkan Tabel di atas penjelasan masing-masing variabel terhadap keputusan pembelian adalah sebagai berikut: c Variabel Fasilitas g c. Variabel Fasilitas Nilai thitung variabel fasilitas sebesar 2,028 dimana t hitung > t tabel (2,028 >1,985) dengan nilai signifikansi 0,045 <0,05. Maka H0 ditolak dan Ha diterima. Sehingga dapat disimpulkan bahwa fasilitas berpengaruh secara positif dan signifikan terhadap kepuasan pelanggan. d. Variabel Pelayanan Nilai thitung variabel pelayanan sebesar -0,684 dimana t hitung < t tabel (-0,684 <1,985) dengan nilai signifikansi 0,495 >0,05. Maka H0 diterima dan Ha ditolak. Sehingga dapat disimpulkan bahwa pelayanan tidak berpengaruh terhadap kepuasan pelanggan. Karena kurangnya pekerja pada warnet @N-Kom Net sehingga membuat pelanggan menunggu. d. Variabel Pelayanan Nilai thitung variabel pelayanan sebesar -0,684 dimana t hitung < t tabel (-0,684 <1,985) dengan nilai signifikansi 0,495 >0,05. Maka H0 diterima dan Ha ditolak. Sehingga dapat disimpulkan bahwa pelayanan tidak berpengaruh terhadap kepuasan pelanggan. Karena kurangnya pekerja pada warnet @N-Kom Net sehingga membuat pelanggan menunggu. e. Variabel Harga g Nilai thitung variabel diskon sebesar 1,346 dimana t hitung < t tabel (1,346 <1,985) dengan nilai signifikansi 0,181 >0,05. Maka H0 diterima dan Ha ditolak. Sehingga dapat disimpulkan bahwa Harga tidak berpengaruh terhadap kepuasan pelanggan. Karena harga yang diberikan oleh warnet @N-Kom Net sama dengan warnet- warnet pada umumnya sehingga pelanggan tidak merasa puas dengan harga yang diberikan. f. Variabel Pemberian Diskon Nilai thitung variabel diskon sebesar -0,085 dimana t hitung < t tabel (-0,085 <1,985) dengan nilai signifikansi 0,933 >0,05. Maka H0 diterima dan Ha ditolak. Sehingga dapat disimpulkan bahwa pemberian diskon tidak berpengaruh terhadap kepuasan pelanggan. Dikarenakan pemberian diskon hanya diberikan kepada 647 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 9 (3) Juni Tahun 2023, Hal 642-649. orang-orang tertentu tidak untuk semua pelanggan sehingga pemberian diskon dianggap tidak berpengaruh terhadap kepuasan pelanggan. orang-orang tertentu tidak untuk semua pelanggan sehingga pemberian diskon dianggap tidak berpengaruh terhadap kepuasan pelanggan. Uji Simultan (Uji f) Hal ini menjelaskan bahwa Fasilitas (X1), Pelayanan (X2), Harga (X3) dan Pemberian Diskon (X4) terhadap Kepuasan Pelanggan (Y) sebesar 5,4% dan sisanya 94,6% dijelaskan oleh faktor-faktor lain diluar variabel yang digunakan dalam penelitian ini. Faktor-faktor lain yang dimaksud seperti Store Atmosphere, Loyalitas Pelanggan, Promosi, Lokasi, WOM dan sebagainya. Kesimpulan Secara Parsial Fasilitas berpengaruh Positif dan Signifikan terhadap Kepuasan Pelanggan pada Warnet @N-Kom Net. Secara Parsial Pelayanan tidak berpengaruh terhadap Kepuasan Pelanggan pada Warnet @N- Kom Net karena kurangnya jumlah pekerja pada warnet @N-Kom Net membuat kinerja pegawai menjadi lambat. Secara Parsial Harga tidak berpengaruh terhadap Kepuasan Pelanggan pada Warnet @N-Kom Net karena harga yang diberikan oleh Warnet @N-Kom Net cenderung sama dengan harga yang terdapat pada warnet lainnya. Secara Parsial Pemberian Diskon tidak berpengaruh terhadap Kepuasan Pelanggan pada Warnet @N- Kom Net karena pemberian diskon hanya diberikan kepada orang-orang tertentu dan warnet @N-Kom Net tidak pernah memberikan diskon secara terang-terangan. Secara Simultan Variabel Fasilitas, Pelayanan, Pemberian Diskon, dan Harga secara bersama tidak memiliki pengaruh signifikan terhadap Kepuasan Pelanggan pada Warnet @N-Kom Net. 648 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 9 (3) Juni Tahun 2023, Hal 642-649. Referensi Armstrong, G., Adam, S., Denize, S., & Kotler, P. (2014). Principles of marketing. Pearson Australia. Baskara, I. B. (2015). Pengaruh potongan harga (discount) terhadap pembelian tidak terencana Buying)(studi pada pengunjung Matahari Department Store Johar Plaza Jember). Manajemen Bisn Bhuwana, R., Bagus, M., & Sudiksa, I. B. (2013). Pengaruh Kualitas Layanan dan Kepuasan Pelanggan Terhadap Niat Pemakaian Ulang Jasa Service Pada Bengkel Toyota Auto 2000 Denpasar. Udayana University. Fandy, T., & Gregorius, C. (2016). Service, Quality dan Satisfaction. Yogyakarta: Penerbit ANDI. & (2021) ( ) ( S) Firdaus, I., & Kasmir, A. N. (2021). Pengaruh price earning (PER), earning per share (EPS), debt to equity ratio (DER) terhadap harga saham. Jurnal Manajemen Dan Bisnis, 1(1), 40–57. Firdaus, I., & Kasmir, A. N. (2021). Pengaruh price earning (PER), earning per share (EPS), de (DER) terhadap harga saham. Jurnal Manajemen Dan Bisnis, 1(1), 40–57. ( ) p g j ( ) Kotler, P., & Keller, K. L. (2016). A framework for marketing management. Pearson Boston, MA. Kusnawan, A., Diana, S., Andy, A., & Tjong, S. (2019). Pengaruh Diskon pada Aplikasi e-Wallet terhadap Pertumbuhan Minat Pembelian Impulsif Konsumen Milenial di Wilayah Tangerang. Sains Manajemen, 5(2). Oetama, S., & Sari, D. H. (2017). Pengaruh Fasilitas Dan Kualitas Pelayanan Terhadap Kepuasan Nasabah Pada Pt Bank Mandiri (Persero) Tbk Di Sampit Jurnal Terapan Manajemen Dan Bisnis 3(1) 195527 Kusnawan, A., Diana, S., Andy, A., & Tjong, S. (2019). Pengaruh Diskon pada Aplikasi e-Wallet terhadap Pertumbuhan Minat Pembelian Impulsif Konsumen Milenial di Wilayah Tangerang. Sains Manajemen, 5(2). Oetama, S., & Sari, D. H. (2017). Pengaruh Fasilitas Dan Kualitas Pelayanan Terhadap Kepuasan Pada Pt. Bank Mandiri (Persero) Tbk Di Sampit. Jurnal Terapan Manajemen Dan Bisnis, 3(1), 1955 Ramli, S. (2013). Bacaan Wajib Para Praktisi Pengadaan Barang/Jasa Pemerintah. Visimedia. Rangkuti, F. (2011). Dongkrak Penjualan Melalui Strategy & Competitive Positioning. Gramedia Pustaka Riyanto, A. (2018). Implikasi Kualitas Pelayanan Dalam Meningkatkan Kepuasan Pelanggan Pada PDAM Cibadak Sukabumi. Jurnal Ecodemica Jurnal Ekonomi Manajemen Dan Bisnis, 2(1), 117–124. Siboro, R. M., & Suhardi, S. (2020). Pengaruh Kepuasan Pelanggan, Kepercayaan Pelanggan, Dan Kualitas Pelayanan Terhadap Loyalitas Pelanggan Dalam Penggunaan Tokopedia Di Batam. Jurnal Ekuivalensi, 6(1), 118–132. ( ) Sonata, I. (2019). Pengaruh Price Discount Dan in-Store Display Terhadap Keputusan Impulse Produk Miniso. JRMB (Jurnal Riset Manajemen & Bisnis), 4(1). Produk Miniso. JRMB (Jurnal Riset Manajemen & Bisnis), 4(1). Tjiptono, F. (2014). Pemasaran jasa–prinsip, penerapan, dan penelitian. Yogyakarta: Andi Offset. ( ) ( ) Tjiptono, F. Referensi (2014). Pemasaran jasa–prinsip, penerapan, dan penelitian. Yogyakarta: Andi Offset. 649
https://openalex.org/W3159770304	https://drops.dagstuhl.de/opus/volltexte/2017/8003/pdf/LIPIcs-DISC-2017-7.pdf	English	null	Near-Optimal Approximate Shortest Paths and Transshipment in Distributed and Streaming Models	SIAM journal on computing	2,021	cc-by	10,939	Ruben Becker1, Andreas Karrenbauer2, Sebastian Krinninger3, and Christoph Lenzen4 Ruben Becker1, Andreas Karrenbauer2, Sebastian Krinninger3, and Christoph Lenzen4 1 MPI for Informatics, Saarland Informatics Campus, Saarbrücken, Germany ruben.becker@mpi-inf.mpg.de 2 MPI for Informatics, Saarland Informatics Campus, Saarbrücken, Germany andreas.karrenbauer@mpi-inf.mpg.de 3 Faculty of Computer Science, University of Vienna, Austria sebastian.krinninger@univie.ac.at 4 MPI for Informatics, Saarland Informatics Campus, Saarbrücken, Germany christoph.lenzen@mpi-inf.mpg.de 1 MPI for Informatics, Saarland Informatics Campus, Saarbrücken, Germany ruben.becker@mpi-inf.mpg.de 2 MPI for Informatics, Saarland Informatics Campus, Saarbrücken, Germany andreas.karrenbauer@mpi-inf.mpg.de 3 Faculty of Computer Science, University of Vienna, Austria sebastian.krinninger@univie.ac.at 4 MPI for Informatics, Saarland Informatics Campus, Saarbrücken, Germany christoph.lenzen@mpi-inf.mpg.de 1 MPI for Informatics, Saarland Informatics Campus, Saarbrücken, Germany ruben.becker@mpi-inf.mpg.de 2 MPI for Informatics, Saarland Informatics Campus, Saarbrücken, Germany andreas.karrenbauer@mpi-inf.mpg.de 3 Faculty of Computer Science, University of Vienna, Austria sebastian.krinninger@univie.ac.at 4 MPI for Informatics, Saarland Informatics Campus, Saarbrücken, Germany christoph.lenzen@mpi-inf.mpg.de 1 We use ˜O(·) to hide polylogarithmic factors in n. Leibniz International Proceedings in Informatics ∗Full version available at https://arxiv.org/abs/1607.05127. 1 W ˜O( ) hid l l i h i f i dréa W. Richa; Article No. 7; pp. 7:1–7:16 1 We use ˜O(·) to hide polylogarithmic factors in n. © Ruben Becker, Andreas Karrenbauer, Sebastian Krinninger, and Christoph Lenzen; licensed under Creative Commons License CC-BY 31st International Symposium on Distributed Computing (DISC 2017). Editor: Andréa W. Richa; Article No. 7; pp. 7:1–7:16 Leibniz International Proceedings in Informatics Schloss Dagstuhl – Leibniz-Zentrum für Informatik, Dagstuhl Publishing, Germany © Ruben Becker, Andreas Karrenbauer, Sebastian Krinninger, and Christoph Lenzen; licensed under Creative Commons License CC-BY 31st International Symposium on Distributed Computing (DISC 2017). Editor: Andréa W. Richa; Article No. 7; pp. 7:1–7:16 Leibniz International Proceedings in Informatics Schloss Dagstuhl – Leibniz-Zentrum für Informatik, Dagstuhl Publishing, Germany © Ruben Becker, Andreas Karrenbauer, Sebastian Krinninger, and Christoph Lenzen; licensed under Creative Commons License CC-BY y Richa; Article No. 7; pp. 7:1–7:16 g Schloss Dagstuhl – Leibniz-Zentrum für Informatik 1 Introduction Single-source shortest paths (SSSP) is a fundamental and well-studied problem in computer science. Thanks to sophisticated algorithms and data structures [20, 23, 41], it has been known for a long time how to obtain (near-)optimal running time in the RAM model. This is not the case in non-centralized models of computation, which become more and more relevant in a big-data world. Despite certain progress for exact SSSP algorithms [6, 7, 9, 15, 28, 30, 39, 40], there remain large gaps to the strongest known lower bounds. Close-to-optimal running times have so far only been achieved by eﬃcient approximation schemes [10, 17, 25, 32]. For instance, in the CONGEST model of distributed computing, the state of the art is as follows: Exact SSSP on weighted graphs can be computed in O(D1/3(n log n)2/3) rounds [15], where D is the (hop) diameter of the graph, and (1 + ε)-approximate SSSP can be computed in (√n + D) · 2O(√ log n log (ε−1 log n)) rounds [25].2 Even for constant ε, the latter exceeds the strongest known lower bound of Ω(√n/ log n + D) rounds [13] by a super-polylogarithmic factor. As a consequence of the techniques developed in this paper, we make a qualitative algorithmic improvement for (1+ε)-approximate SSSP in this model: we solve the problem in (√n + D) · ε−O(1) polylog n rounds. We thus narrow the gap between upper and lower bound signiﬁcantly and additionally improve the dependence on ε. Our new approach achieves its superior running time by leveraging techniques from continuous optimization. It is inherent to our approach that we actually tackle a problem that seems more general than SSSP. In the shortest transshipment problem, we seek to ﬁnd a cheapest routing for sending units of a single good from sources to sinks along the edges of a graph meeting the nodes’ demands. Equivalently, we want to ﬁnd the minimum-cost ﬂow in a graph where edges have unlimited capacity. The special case of SSSP can be modeled as a shortest transshipment problem by setting the demand of the source to −n+1 (thus supplying −n+1 units) and the demand of every other node to 1. Unfortunately, this relation breaks when we consider approximation schemes: A (1 + ε)-approximate solution to the transshipment problem merely yields (1 + ε)-approximations to the distances on average. 2 Note that these running times refer to weighted graphs. In unweighted graphs, the SSSP problem can easily be solved in O(D) rounds by performing a BFS tree computation. Abstract We present a method for solving the shortest transshipment problem—also known as uncapa- citated minimum cost ﬂow—up to a multiplicative error of 1 + ε in undirected graphs with non-negative integer edge weights using a tailored gradient descent algorithm. Our gradient des- cent algorithm takes ε−3 polylog n iterations, and in each iteration it needs to solve an instance of the transshipment problem up to a multiplicative error of polylog n, where n is the number of nodes. In particular, this allows us to perform a single iteration by computing a solution on a sparse spanner of logarithmic stretch. Using a careful white-box analysis, we can further extend the method to ﬁnding approximate solutions for the single-source shortest paths (SSSP) problem. As a consequence, we improve prior work by obtaining the following results: 1. Broadcast CONGEST model: (1 + ε)-approximate SSSP using ˜O((√n + D) · ε−O(1)) rounds,1 where D is the (hop) diameter of the network. 1. Broadcast CONGEST model: (1 + ε)-approximate SSSP using ˜O((√n + D) · ε−O(1)) rounds,1 where D is the (hop) diameter of the network. 2. Broadcast congested clique model: (1+ε)-approximate shortest transshipment and SSSP using ˜O(ε−O(1)) rounds. 2. Broadcast congested clique model: (1+ε)-approximate shortest transshipment and SSSP using ˜O(ε−O(1)) rounds. 3. Multipass streaming model: (1+ε)-approximate shortest transshipment and SSSP using ˜O(n) space and ˜O(ε−O(1)) passes. 3. Multipass streaming model: (1+ε)-approximate shortest transshipment and SSSP using ˜O(n) space and ˜O(ε−O(1)) passes. The previously fastest SSSP algorithms for these models leverage sparse hop sets. We bypass the hop set construction; computing a spanner is suﬃcient with our method. The above bounds assume non-negative integer edge weights that are polynomially bounded in n; for general non- negative weights, running times scale with the logarithm of the maximum ratio between non-zero weights. In case of asymmetric costs for traversing an edge in opposite directions, running times scale with the maximum ratio between the costs of both directions over all edges. 1998 ACM Subject Classiﬁcation I.1.2 Algorithms, G.1.6 Optimization 1998 ACM Subject Classiﬁcation I.1.2 Algorithms, G.1.6 Optimization Keywords and phrases Shortest Paths, Shortest Transshipment, Undirected Min-cost Flow, Gradient Descent, Spanner Keywords and phrases Shortest Paths, Shortest Transshipment, Undirected Min-cost Flow, Gradient Descent, Spanner Keywords and phrases Shortest Paths, Shortest Transshipment, Undirected Min-cost Flow, Gradient Descent, Spanner Digital Object Identiﬁer 10.4230/LIPIcs.DISC.2017.7 Digital Object Identiﬁer 10.4230/LIPIcs.DISC.2017.7 Digital Object Identiﬁer 10.4230/LIPIcs.DISC.2017.7 7:2 Our Contributions and Results. We summarize our technical and conceptual contributions as follows: (C1) We give a problem-speciﬁc gradient descent algorithm for approximating the shortest transshipment, which requires access to an oracle computing an α-approximate dual solution for any given demand vector.4 To compute a (1+ε)-approximation, the algorithm performs ˜O(ε−3α2) oracle calls. If the oracle returns primal solutions, so does our algorithm. (C1) We give a problem-speciﬁc gradient descent algorithm for approximating the shortest transshipment, which requires access to an oracle computing an α-approximate dual solution for any given demand vector.4 To compute a (1+ε)-approximation, the algorithm performs ˜O(ε−3α2) oracle calls. If the oracle returns primal solutions, so does our algorithm. (C2) We provide an additional analysis of the gradient descent algorithm that allows us to extend the method to solving SSSP in order to achieve a per-node approximation guarantee. (C3) We observe that spanners can be used to obtain an eﬃcient shortest transshipment oracle with approximation guarantee α ∈O(log n). (C3) We observe that spanners can be used to obtain an eﬃcient shortest transshipment oracle with approximation guarantee α ∈O(log n). By implementing our method in speciﬁc models of computation, we obtain the following concrete algorithmic results in graphs with non-negative polynomially bounded5 integer edge weights: (R1) We give faster algorithms for computing (1 + ε)-approximate SSSP: 1. Broadcast CONGEST model: We obtain a deterministic algorithm for computing (1 + ε)-approximate SSSP using ˜O((√n + D) · ε−O(1)) rounds. This improves upon the previous best upper bound of (√n + D) · 2O(√ log n log (ε−1 log n)) rounds [25]. For ε−1 ∈O(polylog n), we match the lower bound of Ω(√n/ log n + D) [13] (applying to any (randomized) (poly n)-approximation of the distance between two ﬁxed nodes in a weighted undirected graph) up to polylogarithmic factors in n. (√ ) [ ] ε−1 ∈O(polylog n), we match the lower bound of Ω(√n/ log n + D) [13] (applying to any (randomized) (poly n)-approximation of the distance between two ﬁxed nodes in a weighted undirected graph) up to polylogarithmic factors in n. 2. Broadcast congested clique model: We obtain a deterministic algorithm for computing (1 + ε)-approximate SSSP using ˜O(ε−O(1)) rounds. This improves upon the previous best upper bound of 2O(√ log n log (ε−1 log n)) rounds [25]. 3. Multipass streaming model: We obtain a deterministic algorithm for computing (1 + ε)- approximate SSSP using ˜O(ε−O(1)) passes and O(n log n) space. 4 Note that dual feasibility is crucial here. In particular, this rules out an oracle based on tree embeddings [2, 16], as such trees might have stretch Ω(n) on individual edges. ] ( ) 5 For general non-negative weights, running times scale by a multiplicative factor of log R, where R is the maximum ratio between non-zero edge weights. 3 Also known as the node-CONGEST model. 4 R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen Our method is widely applicable among a plurality of non-centralized models of com- putation in a rather straightforward way. We obtain the ﬁrst non-trivial algorithms for approximate undirected shortest transshipment in the broadcast CONGEST,3 broadcast congested clique, and multipass streaming models. As a further, arguably more important, consequence, we improve upon prior results for computing approximate SSSP in these models. Our approximate SSSP algorithms are the ﬁrst to be provably optimal up to polylogarithmic factors. Our Contributions and Results. We summarize our technical and conceptual contributions as follows: Our Contributions and Results. We summarize our technical and conceptual contributions as follows: 1 Introduction In the special case of SSSP, however, one is interested in obtaining a (1 + ε)-approximation to the distance for each single node and we show how to extend our algorithm to provide such a guarantee as well. Techniques from continuous optimization have been key to recent breakthroughs in the combinatorial realm of graph algorithms [8, 11, 12, 27, 31, 33, 37]. In this paper, we apply this paradigm to computing primal and dual (1 + ε)-approximate solutions to the shortest transshipment problem in undirected graphs with non-negative edge weights. Accordingly, we perform projected gradient descent for a suitable norm-minimization formulation of the problem, where we approximate the inﬁnity norm by a diﬀerentiable soft-max function. To make this general approach work in our setting, we need to add signiﬁcant problem-speciﬁc tweaks. In particular, we develop a gradient descent algorithm that reduces the problem of computing a (1 + ε)-approximation to the more relaxed problem of computing, e.g., an O(log n)-approximation. We then exploit that an O(log n)-approximation can be computed very eﬃciently by solving the problem on a sparse spanner, and that it is well-known how to compute sparse spanners eﬃciently. To obtain the aforementioned per-node guarantee in the approximate SSSP problem, we additionally exploit speciﬁc properties of our gradient descent algorithm. Further eﬀort is required to extract an approximate shortest-path tree (i.e., a primal solution) from the dual solution (i.e., estimated distances to the source). 7:3 R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen 4 Note that dual feasibility is crucial here. In particular, this rules out an oracle based on tree embeddings [2, 16], as such trees might have stretch Ω(n) on individual edges. 5 For general non-negative weights, running times scale by a multiplicative factor of log R, where R is the 4 Note that dual feasibility is crucial here. In particular, this rules out an oracle based on tree embeddings [2, 16], as such trees might have stretch Ω(n) on individual edges. 5 For general non-negative weights, running times scale by a multiplicative factor of log R, where R is the maximum ratio between non-zero edge weights. Near-Optimal Approximate Shortest Paths and Transshipment 7:4 2) We give fast algorithms for computing (1 + ε)-approximate shortest transshipment: 1. Broadcast CONGEST model: A deterministic algorithm using ˜O(ε−3n) rounds. 1. Broadcast CONGEST model: A deterministic algorithm using ˜O(ε−3n) rou ˜ 2. Broadcast congested clique model: A deterministic algorithm using ˜O(ε−3) rounds. ˜ 2. Broadcast congested clique model: A deterministic algorithm using ˜O(ε−3) rounds. ˜ 3. Multipass streaming model: A deterministic algorithm using ˜O(ε−3) passes and O(n log n) space. 3. Multipass streaming model: A deterministic algorithm using ˜O(ε−3) passes and O(n log n) space. ( g ) No non-trivial upper bounds were known before in these three models. In the case of SSSP, we can deterministically compute a (1 + ε)-approximation to the distance from the source for every node. Using a randomized procedure, we can additionally compute (with high probability within the same asymptotic running times) a tree on which every node has a path to the source that is within a factor of (1 + ε) of its true distance. In the case of shortest transshipment, we can (deterministically) return (1+ε)-approximate primal and dual solutions. We can further extend the results to asymmetric weights on undirected edges, where each edge can be used in either direction at potentially diﬀerent costs. Denoting by λ ≥1 the maximum over all edges of the cost ratio between traversing the edge in diﬀerent directions, our algorithms give the same guarantees if the number of rounds or passes, respectively, is increased by a factor of λ4 log λ. Related Work on Shortest Transshipment. Shortest transshipment is a classic problem in combinatorial optimization [29, 36]. The classic algorithms for directed graphs with non-negative edge weights in the RAM model run in time O(n(m + n log n) log n) [35] and O((m + n log n)B) [14], respectively, where B is the sum of the nodes’ demands (when they are given as integers) and the term m + n log n comes from SSSP computations. Our Contributions and Results. We summarize our technical and conceptual contributions as follows: This improves upon the previous best upper bound of (2 + 1/ε)O(√ log n log log n) passes and O(n log2 n) space [17]. By setting ε small enough, we can compute distances up to the value log n exactly in integer-weighted graphs using polylog n passes and O(n log n) space. Thus, up to polylogarithmic factors in n, our result matches a lower bound of n1+Ω(1/p)/ poly p space for all algorithms that decide in p passes if the distance between two ﬁxed nodes in an unweighted undirected graph is at most 2(p + 1) for any p = O(log n/ log log n) [22]. DISC 2017 6 Goldberg’s running time bound holds for integer-weighted graphs with most negative weight −N. R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen the one developed by Shi and Spencer in a PRAM algorithm [40]. Elkin’s main technical contribution lies in showing how to compute this hop set without constructing the overlay network explicitly. Roughly speaking, in these algorithms, both h and ρ enter the running time of the corresponding SSSP algorithms, in addition to the time needed to construct the hop set. The concept of hop sets has been introduced by Cohen in the context of PRAM algorithms for approximate SSSP [10]. The increased interest in hop sets and their applications in the last years [5, 17, 24, 25, 32] has culminated in the construction of (h, ε)-hop sets of size O(n1+ 1 2k+1 −1) for h = O ( k ε )k [18, 26]. Recent lower bounds by Abboud et al. [1] show that this trade-oﬀis essentially tight: any construction of (h, ε)-hop sets of size ≤n 1+ 1 2k−1 −δ must have h = Ωk ( 1 ε)k (where k ≥1 is an integer and δ > 0). This implies that the hop set based algorithms, as long as the factor ρ has to be paid in the running time for construction hop sets of size nρ, will never be able to achieve a running time comparable to our SSSP algorithm exclusively by ﬁnding better hop sets. Spanners. In our approach we use a spanner to obtain an eﬃcient shortest transshipment oracle. ▶Deﬁnition 1 (Spanner). Given G = (V, E, w) and α ≥1, an α-spanner of G is a subgraph (V, E′, w\|E′), E′ ⊆E, in which distances are at most by factor α larger than in G. In other words, a spanner removes edges from G while approximately preserving distances. It is well-known that for every undirected graph we can eﬃciently compute an α-spanner of size O(n log n) with α = O(log n) [3]. Structure of this paper. In the following section, we will ﬁrst describe the gradient descent algorithm for the case of symmetric weights. More precisely, we will describe how to obtain a primal/dual solution pair of approximation ratio (1+ε) for an oracle yielding both primal and dual solutions; if the oracle provides dual solutions only, so do our algorithms. In Section 2.2, we describe how to obtain (1 + ε)-approximate distances for every node in the SSSP case. R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen In Section 2.3, we show how to obtain a (1 + ε)-approximate primal tree solution. In Section 3, we brieﬂy describe how the above framework can be implemented in various distributed and streaming models of computation. Due to space limitations, we refer to the full paper for further details. The full version also discusses how our techniques can be generalized to asymmetric edge weights. The key observation is that, essentially, the gradient descent algorithm can be guided by basing the oracle on solving the symmetrized variant problem on an (undirected) spanner. The additional inaccuracy of the approximation slows down the progress of the algorithm by a factor of λ4 log λ. However, while this generalization does not aﬀect our approach structurally, some technical obstacles need to be overcome. For the sake of a streamlined presentation, we thus conﬁne the discussion to the symmetric problem. 7 Note that excluding 0 as an edge weight is a only a mild restriction, because we can always generate new weights w′ with w′ e = 1 + ⌈n/ε⌉· we while preserving at least one of the shortest paths between Near-Optimal Approximate Shortest Paths and Transshipment If the graph contains negative edge weights, then these algorithms require an additional preprocessing step to compute SSSP in presence of negative edge weights, for example in time O(mn) using the Bellman-Ford algorithm [4, 19] or in time O(m√n log N) using Goldberg’s algorithm [21].6 The weakly polynomial running time was ﬁrst improved to ˜O(m3/2 polylog R) [12] and then to ˜O(m√n polylog R) in a recent breakthrough for minimum-cost ﬂow [31], where R is the ratio between the largest and the smallest edge weight. Independent of our work, Sherman [38] obtained a randomized algorithm for computing a (1+ε)-approximate shortest transshipment in weighted undirected graphs in time O(ε−2m1+o(1)) using a generalized-preconditioning approach. We refer the reader to the full paper for a detailed comparison of Sherman’s and our approach. We are not aware of any non-trivial algorithms for computing (approximate) shortest transshipment in non-centralized models of computation, such as distributed and streaming models. Comparison to Hop Set Based SSSP Algorithms. The state-of-the art SSSP algorithms in the distributed CONGEST model follow the framework developed in [34], where (1) the problem of computing SSSP is reduced to an overlay network of size N = ˜O(√n) and (2) a sparse hop set is constructed to speed up computing SSSP on the overlay network. An (h, ε)- hop set is a set of weighted edges that, when added to the original graph, provides suﬃcient shortcuts to approximate all pairwise distances using paths with only h edges (“hops”). In the algorithm by Nanongkai et al. [25], the upper bound of (√n + D) · 2O(√ log n log (ε−1 log n)) on the number of rounds is achieved by constructing an (h, ε)-hop set of size O(Nρ) where h ≤2O(√ log n log (ε−1 log n)) and ρ ≤2O(√ log n log (ε−1 log n)). Elkin’s algorithm [15], which takes O(D1/3(n log n)2/3) rounds, uses an exact (N/ρ, 0) hop set of size O(Nρ) similar to 7:5 Near-Optimal Approximate Shortest Paths and Transshipment W.l.o.g., we restrict to feasible and non-trivial instances, i.e., bT 1 = 0 and b ̸= 0.8 A common approach to model the undirected shortest transshipment problem as a linear program considers the node-arc-incidence matrix A ∈{−1, 0, 1}n×2m of the corresponding bidirected graph,9 where we substitute each edge e by a forward and a backward arc with the same weight we in both directions. While this may seem redundant, it is convenient in terms of notation and generalizes to the case of asymmetric edge weights considered in the full paper. With W being the 2m × 2m diagonal matrix containing the weights, we obtain the following primal/dual pair of linear programs: min{∥Wx∥1 : Ax = b} = max{bT y : ∥W −1AT y∥∞≤1}, (1) min{∥Wx∥1 : Ax = b} = max{bT y : ∥W −1AT y∥∞≤1}, (1) where for z ∈Rd we write ∥z∥1 = Pd i=1 \|zi\| and ∥z∥∞= maxi∈[d]{\|zi\|}. The primal (left) program asks to “ship” the ﬂow given by b from sources (negative demand) to sinks (positive demand) along the edges of the graph, minimizing the cost of the ﬂow, i.e., P e∈E we\|xe\|. Note that, without changing that Ax = b or aﬀecting the objective, we can remove “negative” ﬂow xvw < 0 by increasing xwv by \|xvw\| and setting xvw = 0. Thus, w.l.o.g., we may assume that x ≥0; in particular, an optimal solution sends ﬂow only in one direction over any edge. The dual (right) program asks for potentials y such that for each edge e = (v, w) ∈E, \|yv −yw\| ≤we, maximizing bT y. Note that, because bT 1 = 0, shifting the potential by r × 1 for any r ∈R does neither change bT y nor yv −yw for any v, w ∈V . The goal of the dual is thus to maximize the diﬀerences in potential of sources and sinks (weighted according to b), subject to the constraint that the potentials of neighbors must not diﬀer by more than the weight of their connecting edge. y p g y 8 Here 1 denotes the all-ones vector and thus bT 1 = 0 simply means that the positive demands equal the negative demands (i.e., the supplies). 9 Near-Optimal Approximate Shortest Paths and Transshipment In the special case of SSSP with source s ∈V , we have that (i) bs = −n + 1 and bv = 1 for all v ̸= s, (ii) an optimal primal solution x∗is given by routing, for each s ̸= v ∈V , one unit of ﬂow along a shortest path from s to v, and (iii) optimal potentials y∗are given by setting y∗ v to the distance from s to v. 2 General Approach for Solving Shortest Transshipment and SSSP Let G = (V, E) be a (w.l.o.g. connected) undirected graph with n nodes, m edges, and positive7 integral edge weights w ∈Zm ≥1. Furthermore, let b ∈Zn be a vector of demands. DISC 2017 7:6 ( ) 9 The incidence matrix A of a directed graph contains a row for every node and a column for every arc and Ai,j is −1 if the j-th arc leaves the i-th vertex, 1 if it enters the vertex, and 0 otherwise. each pair of nodes as well as (1 + ε)-approximations. As we assume edge weights to be integer we can assume that ε ≥1/(n∥w∥∞) (as otherwise it is required to compute an exact solution) and thus our asymptotic running time bounds are not aﬀected by this modiﬁcation. p ( ) pp g g g assume that ε ≥1/(n∥w∥∞) (as otherwise it is required to compute an exact solution) and thus our asymptotic running time bounds are not aﬀected by this modiﬁcation. R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen via f(π) := π/∥W −1AT π∥∞. Similarly, feasible solutions y of the dual program in (1) that satisfy bT y > 0 are mapped to feasible solutions of (2) via g(y) := y/bT y. Moreover, the map f(·) preserves the approximation ratio. Namely, for any ε > 0, if π is a solution of (2) within factor 1 + ε of the optimum, then f(π) is feasible for (1) and within factor 1 + ε of the optimum. In particular, f(π∗) is an optimal solution of (1). We would like to apply gradient descent to (2). However, this is not readily possible, since the objective is not diﬀerentiable. Hence, we will change the problem another time by using the so-called soft-max function (a.k.a. log-sum-exp or lse for short), which is a suitable approximation for the maximum entry (v)max := max{vi : i ∈[d]} of a vector v ∈Rd.10 It is deﬁned as lseβ (v) := 1 β ln P i∈[d] eβvi , where β > 0 is a parameter that controls the accuracy of the approximation of the maximum at the expense of smoothness. We note that lseβ (·) is a convex function for any β > 0 and provides the following additive approximation of the maximum: (x)max = 1 β ln eβ·(x)max ≤lseβ(x) ≤1 β ln X i∈[d] eβ·(x)max = ln(d) β + (x)max. (3) (3) A trade-oﬀin the choice of β arises because β also controls the smoothness of the lse-function. Formally, lseβ is β-Lipschitz smooth (i.e., its gradient is β-Lipschitz continuous) w.r.t. to the pair 1-norm/∞-norm: ∥Φβ(x) −Φβ(y)∥1 ≤β∥x −y∥∞. (4) ∥Φβ(x) −Φβ(y)∥1 ≤β∥x −y∥∞. (4) Using the soft-max function, we deﬁne the potential function Using the soft-max function, we deﬁne the potential function Using the soft-max function, we deﬁne the potential function Φβ(π) := lseβ W −1AT π . Φβ(π) := lseβ W −1AT π . Recalling that A was deﬁned to represent each edge of the graph by a forward and backward arc, we see that (W −1AT π)max = ∥W −1AT π∥∞, i.e., Φβ(π) is indeed a smooth approximation of the objective of (2). In order to control the approximation error, β is adapted in the course of the algorithm such that the additive error ln(2m)/β is always at most ε 4Φβ(π). 10 Note the diﬀerence to the ∞-norm, which is deﬁned as the maximum of the absolute values of the entries of a vector. 2.1 Gradient Descent We now describe a gradient descent method that, given an oracle that computes α-approximate primal and dual solutions to the undirected shortest transshipment problem for any speciﬁed demand vector ˜b, returns primal and dual feasible solutions x and y to the undirected shortest transshipment problem that are (1 + ε)-close to optimal, i.e., fulﬁll ∥Wx∥1 ≤(1 + ε)bT y, using O(ε−3α2 log α log n) calls to the oracle. We then provide an oracle with α ∈polylog(n). For ease of notation, we assume that log α ∈polylog n throughout this paper. As our ﬁrst step, we relate the dual of the shortest transshipment problem to its “reciprocal” linear program that normalizes the objective to 1 and seeks to minimize ∥W −1AT y∥∞: min{∥W −1AT π∥∞: bT π = 1}. (2) min{∥W −1AT π∥∞: bT π = 1}. (2) We denote by π∗an optimal solution to this problem, whereas y∗denotes an optimal solution to the dual of the original problem (1). It is easy to see that feasible solutions π of (2) that satisfy ∥W −1AT π∥∞> 0 are mapped to feasible solutions of the dual program in (1) We denote by π∗an optimal solution to this problem, whereas y∗denotes an optimal solution to the dual of the original problem (1). It is easy to see that feasible solutions π of (2) that satisfy ∥W −1AT π∥∞> 0 are mapped to feasible solutions of the dual program in (1) 7:7 R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen Thus, we maintain a multiplicative approximation of the dual objective function of (2), i.e., ∥W −1AT π∥∞≤Φβ(π) ≤∥W −1AT π∥∞ 1 −ε/4 . (5) (5) Our gradient descent algorithm, see Algorithm 1 for a pseudo-code implementation, ﬁrst computes a starting solution π that is an α-approximate (dual) solution to (2) and an initial β that is appropriate for π as discussed above. This can be done, e.g., by solving the problem on an α-spanner and scaling down (by at most a factor of α) to obtain a feasible solution for the original graph. In each iteration, it updates the potentials π using an α-approximate solution to a shortest transshipment problem with a modiﬁed demand vector ˜b that depends on the gradient. Depending on the objective value of this approximation, the algorithm either performs an update to π or terminates, see the check for the value of δ in the algorithm. The intuition behind the algorithm is the following. As the potential function is diﬀeren- tiable, its gradient exists everywhere and it points in the opposite direction of the steepest descent. However, our update steps must maintain the constraint bT π = 1, i.e., they must lie in the orthogonal complement of b. To this end, we consider the projection of the gradient DISC 2017 Near-Optimal Approximate Shortest Paths and Transshipment Algorithm 1: gradient_transship (G, b, ε) 1 Compute α-approximation π to min{∥W −1AT π∥∞: bT π = 1}. // use oracle 2 Determine β so that 4 ln(2m) ≤εβΦβ(π) ≤5 ln(2m). 3 repeat 4 Set ˜b := P T ∇Φβ(π), where P := I −πbT . // project to maintain bT π = 1 5 if ˜b = 0 then return π // Special case: optimal solution found 6 Determine ˜h with ∥W −1AT ˜h∥∞= 1 and ˜bT ˜h ≥1 α max{˜bT h : ∥W −1AT h∥∞≤1}. // ˜h can be obtained from the oracle with demand vector ˜b = P T ∇Φβ(π) 7 Set δ := ˜bT ˜h ∥W −1AT P ˜h∥∞. // δ measures closeness to optimality 8 if δ > ε 8α then π ←π − δ 2β∥W −1AT P ˜h∥∞P˜h. // project to maintain bT π = 1 9 while 4 ln(2m) ≥εβΦβ(π) do β ←5 4β. R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen // ﬁnd appropriate β 10 until δ ≤ ε 8α 11 return π Near-Optimal Approximate Shortest Paths and Transshipment Near-Optimal Approximate Shortest Paths and Transshipment 7:8 P T ∇Φβ(π).11 Because the gradient, and hence the direction of the steepest descent, changes when we move away from our current solution, we use an adaptive step width restricting the update to a region for which we know that the gradient does not vary too much. If we had a suﬃciently good guarantee on the Lipschitz smoothness of Φβ(·), using the gradient itself (resp. its projection) as the update direction h (i.e., performing the update π ←π −ηh for an appropriate step width η) would decrease the objective Φβ(π) fast enough. However, we only have such a guarantee on the Lipschitz smoothness of lseβ (·). By the convexity of the objective Φβ(π), we can argue that the (normalized) progress of an update direction h is the ratio P T ∇Φβ(π)/∥W −1AT h∥∞, which suggests ﬁnding h by max{∇Φβ(π)T Ph : ∥W −1AT h∥∞≤1}. Note that this linear program is precisely of the form (1), with demand vector ˜b := P T ∇Φβ(π), and is thus not easier to solve as the original problem. However, ﬁnding an approximately optimal update direction only mildly aﬀects the number of iterations, i.e., querying the oracle for an α-approximate dual solution with demand ˜b yields the desired guarantee. We then use the projection P to derive a feasible update and rescale so that the gradient does not change too much, enabling us to prove a suﬃciently strong progress guarantee – unless the current solution is already close to the optimum. This is captured by δ, which is guaranteed to be large in case signiﬁcant progress still can be made. Conversely, a small δ implies that we are close to the optimum. Accordingly, at termination π is a near-optimal solution of (2), and rescaling to y = π/∥W −1AT π∥∞yields a near-optimal dual solution of (1). Here, scaling up β as the potential decreases ensures that the incurred approximation error is suﬃciently small. On the other hand, using large β and having the guarantee that δ is large as long as we are not close to the optimum guarantees that the potential function decreases rapidly and only a small number of iterations is required. We proceed by formalizing this intuition. 11 As bT π = 1, we have that bT Ph = bT (I −πbT )h = bT h −bT πbT h = 0 for all h. R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen First, we show that a primal-dual pair that is (1 + ε)-close to optimal in (1) can be constructed from the output potentials π and α-approximate primal and dual solutions, say ˜f and ˜h, to the transshipment problem that was solved in the last iteration of the algorithm. If one is only interested in a dual solution to (1), then the α-approximate dual solution ˜h is enough and thus only an oracle providing 11 As bT π = 1, we have that bT Ph = bT (I −πbT )h = bT h −bT πbT h = 0 for all h. 7:9 R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen 12 Here, we omit the special case ˜b = 0, which guarantees optimality. See full version for details. R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen Moreover, ∥W −1AT y∥∞= 1 follows directly from the deﬁnition of y. It remains to show that ∥Wx∥1 ≤(1+ε)bT y.12 It can be shown (see full paper for details) that convexity of Φβ(·) and the guarantee on β yield Proof. First note that A ˜f = ˜b and ˜b = P T ∇Φβ(π) = ∇Φβ(π) −bπT ∇Φβ(π). Thus Ax = ∇Φβ(π)−˜b πT ∇Φβ(π) = b. Moreover, ∥W −1AT y∥∞= 1 follows directly from the deﬁnition of y. Ax = ∇Φβ(π) b πT ∇Φβ(π) = b. Moreover, ∥W −1AT y∥∞= 1 follows directly from the deﬁnition of y. It remains to show that ∥Wx∥1 ≤(1+ε)bT y.12 It can be shown (see full paper for details) that convexity of Φβ(·) and the guarantee on β yield πT ∇Φβ(π) , ∥ y∥∞ y y It remains to show that ∥Wx∥1 ≤(1+ε)bT y.12 It can be shown (see full paper for details) that convexity of Φβ(·) and the guarantee on β yield πT ∇Φβ(π) ≥ 1 −ε 4 Φβ(π) ≥ 1 −ε 4 ∥W −1AT π∥∞> 0. (6) Hence, \|πT ∇Φβ(π)\| = πT ∇Φβ(π). Moreover, ∥W ˜x∥1 ≤1 + ε/8 by assumption and thus ∥Wx∥1 ∆-ineq. ≤ 1 + ε 8 + ∥W ˜f∥1 πT ∇Φβ(π) α-approx. ≤ 1 + ε 8 + α˜bT ˜h πT ∇Φβ(π) = 1 + ε 8 + αδ∥W −1AT P˜h∥∞ πT ∇Φβ(π) , πT ∇Φβ(π) ≥ 1 −ε 4 Φβ(π) ≥ 1 −ε 4 ∥W −1AT π∥∞> 0. (6) (6) ∥Wx∥1 ∆-ineq. ≤ 1 + ε 8 + ∥W ˜f∥1 πT ∇Φβ(π) α-approx. ≤ 1 + ε 8 + α˜bT ˜h πT ∇Φβ(π) = 1 + ε 8 + αδ∥W −1AT P˜h∥∞ πT ∇Φβ(π) , where δ = ˜bT ˜h ∥W −1AT P ˜h∥∞as in Algorithm gradient_transship. By the deﬁnition of P = I −πbT and the triangle inequality for the inﬁnity norm, we obtain ∥W −1AT P˜h∥∞≤ ∥W −1AT ˜h∥∞+\|bT ˜h\|∥W −1AT π∥∞. Using the upper bound \|bT ˜h\| ≤bT y∗from the optimality of y∗and ∥W −1AT ˜h∥∞≤1, we obtain ∥W −1AT P˜h∥∞≤1 + ∥W −1AT π∥∞bT y∗. Using (6) for the denominator, this yields where δ = ˜bT ˜h ∥W −1AT P ˜h∥∞as in Algorithm gradient_transship. By the deﬁnition of P = I −πbT and the triangle inequality for the inﬁnity norm, we obtain ∥W −1AT P˜h∥∞≤ ∥W −1AT ˜h∥∞+\|bT ˜h\|∥W −1AT π∥∞. R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen a dual solution is required, as done in Algorithm 1. A primal solution can be obtained from ˜f and the vector ˜x := W −1∇lseβ W −1AT π . This choice of ˜x is obtained by applying the chain rule of diﬀerentiation to ∇Φβ(π), i.e., ∇Φβ(π) = AW −1∇lseβ W −1AT π . In the correctness proof we allow a more general choice of ˜x, which we will exploit later on for ﬁnding a tree solution for approximate SSSP. a dual solution is required, as done in Algorithm 1. A primal solution can be obtained from ˜f and the vector ˜x := W −1∇lseβ W −1AT π . This choice of ˜x is obtained by applying the chain rule of diﬀerentiation to ∇Φβ(π), i.e., ∇Φβ(π) = AW −1∇lseβ W −1AT π . In the correctness proof we allow a more general choice of ˜x, which we will exploit later on for ﬁnding a tree solution for approximate SSSP. ▶Lemma 2 (Correctness). Let 0 < ε ≤1/2, ▶Lemma 2 (Correctness). Let 0 < ε ≤1/2, π ∈Rn and β ∈R denote the return values of Algorithm gradient_transship, ˜f ∈R2m and ˜h ∈Rn be the α-approximate pair returned by the oracle in the last iteration of Algorithm gradient_transship, and f g g _ p, ˜x ∈R2m be such that A˜x = ∇Φβ(π) and ∥W ˜x∥1 ≤1 + ε/8. ˜ ˜ f f g g _ p, ˜x ∈R2m be such that A˜x = ∇Φβ(π) and ∥W ˜x∥1 ≤1 + ε/8. Then x := ˜x−˜ f πT ∇Φβ(π), y := π ∥W −1AT π∥∞is a (1+ε)-approximate pair, i.e., it holds that Ax = b, ∥W −1AT y∥∞≤1, and ∥Wx∥1 ≤(1 + ε)bT y. ˜x ∈R2m be such that A˜x = ∇Φβ(π) and ∥W ˜x∥1 ≤1 + ε/8. Then x := ˜x−˜ f πT ∇Φβ(π), y := π ∥W −1AT π∥∞is a (1+ε)-approximate pair, i.e., it holds that Ax = b, ∥W −1AT y∥∞≤1, and ∥Wx∥1 ≤(1 + ε)bT y. Proof. First note that A ˜f = ˜b and ˜b = P T ∇Φβ(π) = ∇Φβ(π) −bπT ∇Φβ(π). Thus Ax = ∇Φβ(π)−˜b πT ∇Φβ(π) = b. Moreover, ∥W −1AT y∥∞= 1 follows directly from the deﬁnition of y. Proof. First note that A ˜f = ˜b and ˜b = P T ∇Φβ(π) = ∇Φβ(π) −bπT ∇Φβ(π). Thus Ax = ∇Φβ(π)−˜b πT ∇Φβ(π) = b. ∥∥p∥∥q p q ∞ 14 Here, we omit the technical argument that the condition ∥W −1AT P ˜h∥∞> 0 of Lemma 3 is always fulﬁlled when we apply the lemma. See full version for details. 13 Hölder’s inequality states that xT y ≤∥x∥p∥y∥q for p, q satisfying 1 p + 1 q = 1, assuming 1 ∞= 0. 14 Here, we omit the technical argument that the condition ∥W −1AT P ˜h∥∞> 0 of Lemma 3 is always fulﬁlled when we apply the lemma. See full version for details. R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen Using the upper bound \|bT ˜h\| ≤bT y∗from the optimality of y∗and ∥W −1AT ˜h∥∞≤1, we obtain ∥W −1AT P˜h∥∞≤1 + ∥W −1AT π∥∞bT y∗. Using (6) for the denominator, this yields ∥Wx∥1 ≤1 + ε 8 + αδ(1 + ∥W −1AT π∥∞bT y∗) (1 −ε 4)∥W −1AT π∥∞ ≤ 1 + ε 8 + ε 8(1 + ∥W x∥1 bT y ) (1 −ε 4) bT y ∥Wx∥1 ≤1 + ε 8 + αδ(1 + ∥W −1AT π∥∞bT y∗) (1 −ε 4)∥W −1AT π∥∞ ≤ 1 + ε 8 + ε 8(1 + ∥W x∥1 bT y ) (1 −ε 4) bT y, since bT y∗≤∥Wx∥1 by weak duality, ∥W −1AT π∥∞= 1/bT y, and δ ≤ ε 8α at termination of the algorithm. Thus (1 + ε 4)/(1 −3ε 8 ) ≤(1 + ε 4)/(1 −ε 2) ≤(1 + ε) yields the result. ◀ ∥ ∥ (1 −ε 4)∥W −1AT π∥∞ (1 −ε 4) y, since bT y∗≤∥Wx∥1 by weak duality, ∥W −1AT π∥∞= 1/bT y, and δ ≤ ε 8α at termination of the algorithm. Thus (1 + ε 4)/(1 −3ε 8 ) ≤(1 + ε 4)/(1 −ε 2) ≤(1 + ε) yields the result. ◀ since bT y∗≤∥Wx∥1 by weak duality, ∥W −1AT π∥∞= 1/bT y, and δ ≤ ε 8α at termination of the algorithm. Thus (1 + ε 4)/(1 −3ε 8 ) ≤(1 + ε 4)/(1 −ε 2) ≤(1 + ε) yields the result. ◀ since bT y∗≤∥Wx∥1 by weak duality, ∥W −1AT π∥∞= 1/bT y, and δ ≤ ε 8α at termination of the algorithm. Thus (1 + ε 4)/(1 −3ε 8 ) ≤(1 + ε 4)/(1 −ε 2) ≤(1 + ε) yields the result. ◀ It remains to show a bound on the number of iterations until termination. To this end, we establish that the potential function decreases by a multiplicative factor in each iteration. ▶Lemma 3 (Multiplicative Decrement of Φβ). Let π ∈Rn, let β satisfy εβΦβ(π) ≤5 ln(2m), and let ˜h satisfy ∥W −1AT P˜h∥∞> 0, where P = I −πbT . Then, for δ := ˜bT ˜h ∥W −1AT P ˜h∥∞, where ˜b = P T ∇Φβ(π), it holds that Φβ π − δ 2β∥W −1AT P˜h∥∞ P˜h ≤ 1 − εδ2 20 ln(2m) Φβ(π). DISC 2017 13 Hölder’s inequality states that xT y ≤∥x∥p∥y∥q for p, q satisfying 1 p + 1 q = 1, assuming 1 ∞= 0. ˜ Near-Optimal Approximate Shortest Paths and Transshipment 7:10 Near-Optimal Approximate Shortest Paths and Transshipment Proof. Let us denote h := δ 2β∥W −1AT P ˜h∥∞ ˜h. Recall that Φβ(·) is convex, thus Proof. Let us denote h := δ 2β∥W −1AT P ˜h∥∞ ˜h. Recall that Φβ(·) is convex, thus Φβ(π −Ph) −Φβ(π) Convexity ≤ −∇Φβ π −Ph T Ph + ∇Φβ(π)T Ph −∇Φβ(π)T Ph = ∇lseβ W −1AT π −∇lseβ W −1AT (π −Ph) T W −1AT Ph −˜bT h Hölder ≤ ∥∇lseβ W −1AT π −∇lseβ W −1AT (π −Ph) ∥1∥W −1AT Ph∥∞−˜bT h β-Lipschitz ≤ β∥W −1AT Ph∥2 ∞−˜bT h, where we used Hölder’s inequality13 and then the fact that the lseβ-function is β-Lipschitz smooth (see (4)). Using the deﬁnitions of h and δ yields Φβ(π−Ph)−Φβ(π) ≤δ2 4β −δ2 2β = −δ2 4β . Using the upper bound on β yields the result. ◀ where we used Hölder’s inequality13 and then the fact that the lseβ-function is β-Lipschitz smooth (see (4)). Using the deﬁnitions of h and δ yields Φβ(π−Ph)−Φβ(π) ≤δ2 4β −δ2 2β = −δ2 4β . Using the upper bound on β yields the result. ◀ This progress guarantee is suﬃcient to show the following bound on the number of iterations. ▶Lemma 4 (Number of Iterations). Suppose that 0 < ε ≤1/2. Then, it holds that Algorithm gradient_transship terminates within O(ε−3α2 log α log n) iterations. Proof. Note that for all x ∈Rn, ∇β lseβ(x) ≤0, i.e., lseβ is decreasing as a function of β and thus the while-loop that scales β up does not increase Φβ(π). Denote by β0 and π0 the initial values of β and π, respectively, and by β and π the values at termination. By Lemma 3 and the fact that the algorithm ensures δ > ε/(8α) as long as it does not terminate, the potential decreases by a factor of 1 − εδ2 20 ln(2m) ≤1 − ε3 1280α2 ln(2m).14 Hence, the number of iterations k can be bounded by k ≤log Φβ(π) Φβ0(π0) log 1 − ε3 1280α2 ln(2m) −1 ≤log Φβ0(π0) Φβ(π) 1280α2 ln(2m) ε3 . As ln(2m) ∈O(log n), it remains show that Φβ0(π0) Φβ(π) ∈O(α). Using that π0 is an α- approximate solution and that β0 is such that 4 ln(2m) ≤εβ0Φβ0(π0), we obtain that As ln(2m) ∈O(log n), it remains show that Φβ0(π0) Φβ(π) ∈O(α). 2.2 Single-Source Shortest Paths In the special case of SSSP, we have bv = 1 for all v ∈V \ {s} and bs = 1 −n for the source s. In fact, it is the combination of n −1 shortest s-t-path problems. Let π be the potentials returned by Algorithm gradient_transship and let us assume, w.l.o.g., that πs = 0 (otherwise shift π ←π −πs1). Recall that in an optimal solution π∗with π∗ s = 0 the value of π∗ v for any v denotes the distance from s to v. Thus the approximation guarantee from Theorem 5 yields that for the potentials π, it holds that P v̸=s πv ≤(1 + ε) P v̸=s π∗ v, i.e., the distances merely approximate the optimal distances on average over all sink-nodes, which is unsatisfactory. However, we can obtain potentials π such that for every v, it holds that πv ≤(1 + ε)π∗ v and equivalently y∗≥yv ≥y∗ v/(1 + ε) for the s-v-distances. Using the tools proposed above, we can show that when running the gradient descent algorithm with higher precision, we can determine “good” nodes for which we know the distance with suﬃcient accuracy by checking, for every node v, whether the gradient would allow further progress for the s-v shortest path problem. We then argue that a constant fraction of the nodes will be “good” when the algorithm is ﬁnished. We then concentrate on the other nodes by adapting the demand vector b accordingly, i.e., setting bv = 0 for all good nodes v. We iterate until all nodes are good. The pseudocode is given in Algorithm sssp. ▶Theorem 6. Let y∗∈Rn denote the distances of all nodes from the source node s. Algorithm sssp computes a vector y ∈Rn with ∥W −1AT y∥∞≤1 such that y∗ v/(1+ε) ≤yv ≤ y∗ v holds for each v ∈V , using polylog(n, ∥w∥∞) calls to Algorithm gradient_transship. Near-Optimal Approximate Shortest Paths and Transshipment // ˜h can be obtained from the oracle with demand vector ˜b = P T ∇Φβ(π) 8 Set δ := ˜bT ˜h ∥W −1AT P ˜h∥∞. 9 if δ ≤ ε 8α then set bv = 0, ˆyv = yv −ys and bs ←bs + 1 10 return ˆy R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen Algorithm 2: sssp (G, s, ε) 1 Let ˆy = 0, b = 1 −n1s, and ε′ = ε3 3840α2 ln(2m). 2 while bs < 0 do 3 Set π = gradient_transship G, b, ε′ and y = π ∥W −1AT π∥∞. 4 Determine β so that 4 ln(2m) < ε′βΦβ(y) ≤5 ln(2m) and compute ∇Φβ(y). 5 for each v ∈V with bv = 1 do 6 Set ˜b := P T ∇Φβ(y), where P := [I − y (1v−1s)T y(1v −1s)T ]. 7 Compute ˜h with ∥W −1AT ˜h∥∞= 1 and ˜bT ˜h ≥1 α max{˜bT h : ∥W −1AT h∥∞≤1}. // ˜h can be obtained from the oracle with demand vector ˜b = P T ∇Φβ(π) 8 Set δ := ˜bT ˜h ∥W −1AT P ˜h∥∞. 9 if δ ≤ ε 8α then set bv = 0, ˆyv = yv −ys and bs ←bs + 1 10 return ˆy 10 return ˆy Near-Optimal Approximate Shortest Paths and Transshipment Using that π0 is an α- approximate solution and that β0 is such that 4 ln(2m) ≤εβ0Φβ0(π0), we obtain that Φβ0(π0) = lseβ0 W −1AT π0 (3) ≤∥W −1AT π0∥∞+ ln(2m) β0 ≤α∥W −1AT π∗∥∞+ εΦβ0(π0) 4 Φβ0(π0) = lseβ0 W −1AT π0 (3) ≤∥W −1AT π0∥∞+ ln(2m) β0 ≤α∥W −1AT π∗∥∞+ εΦβ0(π0) 4 and thus Φβ0(π0) ≤α∥W −1AT π∗∥∞/(1−ε/4). On the other hand, Φβ(π) ≥∥W −1AT π∥∞≥ 0 and thus Φβ0(π0) ≤α∥W −1AT π∗∥∞/(1−ε/4). On the other hand, Φβ(π) ≥∥W −1AT π∥∞≥ ∥W −1AT π∗∥∞and thus Φβ0(π0) Φβ(π) ≤ α 1−ε/4 = O(α) and the bound follows. ◀ and thus Φβ0(π0) ≤α∥W −1AT π∗∥∞/(1−ε/4). On the other hand, Φβ(π) ≥∥W −1AT π∥∞≥ ∥W −1AT π∗∥∞and thus Φβ0(π0) Φβ(π) ≤ α 1−ε/4 = O(α) and the bound follows. ◀ and thus Φβ0(π0) ≤α∥W −1AT π∗∥∞/(1−ε/4). On the other hand, Φβ(π) ≥∥W −1AT π∥∞≥ ∥W −1AT π∗∥∞and thus Φβ0(π0) Φβ(π) ≤ α 1−ε/4 = O(α) and the bound follows. ◀ We remark that one can ﬁrst run the gradient descent algorithm with ε = 1/2 and then switch to the desired accuracy. Using this trick, the above bound slightly improves to O((ε−3 + log α)α2 log n). From the discussion so far, we obtain the following result. ▶Theorem 5. Given an oracle that computes α-approximate solutions to the undirected transshipment problem, using Algorithm gradient_transship, we can compute primal and dual solutions x, y to the shortest transshipment problem satisfying ∥Wx∥1 ≤(1 + ε)bT y with ˜O(ε−3α2) oracle calls. If the oracle only returns α-approximate dual solutions, then Algorithm gradient_transship computes a (1 + ε)-approximate dual solution. R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen 7:11 R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen Algorithm 2: sssp (G, s, ε) 1 Let ˆy = 0, b = 1 −n1s, and ε′ = ε3 3840α2 ln(2m). 2 while bs < 0 do 3 Set π = gradient_transship G, b, ε′ and y = π ∥W −1AT π∥∞. 4 Determine β so that 4 ln(2m) < ε′βΦβ(y) ≤5 ln(2m) and compute ∇Φβ(y). 5 for each v ∈V with bv = 1 do 6 Set ˜b := P T ∇Φβ(y), where P := [I − y (1v−1s)T y(1v −1s)T ]. 7 Compute ˜h with ∥W −1AT ˜h∥∞= 1 and ˜bT ˜h ≥1 α max{˜bT h : ∥W −1AT h∥∞≤1}. 3 Implementation in Various Models of Computation 3 Common to all our implementations is the use of sparse spanners. An optimal solution of an instance of the shortest transshipment problem on an α-spanner of the input graph is an α-approximate solution to the original problem. Thus, whenever our gradient descent algorithm asks the oracle for an α-approximate solution to a subproblem, we solve the subproblem on a spanner to get an approximation with α = O(log n). Broadcast Congested Clique. In the broadcast congested clique model, the system consists of n fully connected nodes labeled by unique O(log n)-bit identiﬁers. Computation proceeds in synchronous rounds, where in each round, nodes may perform arbitrary local computations, broadcast (send) an O(log n)-bit message to the other nodes, and receive the messages from other nodes. The input is distributed among the nodes. The ﬁrst part of the input of every node consists of its incident edges (given by their endpoints’ identiﬁers) and their weights. The second part of the input is problem speciﬁc: for the transshipment problem, every node v knows its demand bv and for SSSP v knows whether or not it is the source s. In both cases, every node knows 0 < ε ≤1/2 as well. Each node needs to compute its part of the output. For shortest transshipment, every node in the end needs to know a (1 + ε)-approximation of the optimum value, and for SSSP every node needs to know a (1 + ε)-approximation of its distance to the source. The complexity of the algorithm is measured in the worst-case number of rounds until the computation is complete. Implementing our approach in this model is straightforward. The key observations are: Every node can locally aggregate information about its incident edges (e.g. concerning the “stretches” under the potential of the current solution π) and make it known to all other nodes in a single communication round. Thus, given β > 0 and π ∈Rn, it is rather straightforward to evaluate Φβ(π) and ∇Φβ(π) in a constant number of rounds. An O(log n)-spanner of the input graph can be computed and made known to all nodes quickly, following the algorithm of Baswana and Sen [3] (see full paper for details). Local computation then suﬃces to solve (sub)problems on the spanner optimally. In particular, O(log n)-approximations to transshipment problems can be computed easily. Near-Optimal Approximate Shortest Paths and Transshipment tree solution xT in the graph G′ = (V, T1 ∪S) consisting of the tree T1 and the edges of the spanner S. As E[∥Wx(T1)∥1] = 1, using Markov’s inequality we get that, with probability Ω(ε), the tree solution x(T1) corresponding to T1 satisﬁes ∥Wx(T1)∥1 ≤1 + ε/8. Repeating the sampling O(ε log n) times and taking the best result, we obtain such a solution with high probability. Thus, using Lemma 2, we can conclude that the optimal tree solution xT in G′ is (1 + ε)-approximate for the problem. To obtain an approximate tree solution in the case of SSSP, we repeat this sampling after every call of the gradient descent algorithm, obtaining a tree Ti in the i-th call. We can then ﬁnd the approximate shortest path tree in the graph G′ = (V, S i Ti ∪S) combining the sampled edges of each iteration and the initial spanner. Note that, since the number of calls to the gradient descent algorithm is polylog(n, ∥w∥∞), the resulting graph is still of size O(n polylog(n, ∥w∥∞)) for a spanner of size O(n log n). 2.3 Finding a Primal Tree Solution In the following, we explain how to obtain primal tree solutions, for a speciﬁc implementation of the transshipment oracle from Section 3, where we solve the subproblem on spanner. ˜ Recall that, as shown in Lemma 2, x := x−f πT ∇Φβ(π) is a (1+ϵ)-approximate primal solution, where ˜f is the primal solution computed by the oracle in the last iteration of the algorithm and ˜x := W −1∇lseβ(W −1AT π). To also obtain a (1 + ϵ)-approximate primal tree solution, we ﬁrst sample a tree, say T1, from ˜x by sampling for each node among its incident edges a parent edge with probabilities proportional to the values in ˜x. Then we compute an optimal DISC 2017 7:12 ▶Theorem 7. For any 0 < ε ≤1/2, in the broadcast congested clique model a deterministic (1 + ε)-approximation to the shortest transshipment problem in undirected graphs with non- negative edge weights can be computed in ε−3 polylog n rounds. R. Becker, A. Karrenbauer, S. Krinninger, and C. Lenzen ▶Theorem 8. For any 0 < ε ≤1, in the broadcast congested clique model a deterministic (1 + ε)-approximation to single-source shortest paths in undirected graphs with non-negative edge weights can be computed in ε−9 polylog n rounds. To compute a tree solution, the main observation is that the sampling of the tree can be performed locally at every node. Broadcast CONGEST Model. The broadcast CONGEST model diﬀers from the broadcast congested clique in that communication is restricted to edges that are present in the input graph. That is, node v receives the messages sent by node w if and only if {v, w} ∈E. All other aspects of the model are identical to the broadcast congested clique. We stress that this restriction has signiﬁcant impact, however: Denoting the hop diameter of the input graph (i.e., the diameter of the unweighted graph G = (V, E)) by D, it is straightforward to show that Ω(D) rounds are necessary to solve the SSSP problem. Moreover, it has been established that Ω(√n/ log n) rounds are required even on graphs with D ∈O(log n) [13]. Both of these bounds apply to randomized approximation algorithms. Our main result for this model is that we can nearly match the above lower bounds for approximate SSSP computation. The solution is based on combining a known reduction to an overlay network on ˜Θ(ε−1√n) nodes, simulating the broadcast congested clique on this overlay, and applying Theorem 8. Simulating a round of the broadcast congested clique for k nodes is done by pipelining each of the k messages over a breadth-ﬁrst search tree of the underlying graph, taking O(D + k) rounds. ▶Corollary 9. For any 0 < ε ≤1, in the broadcast CONGEST model a deterministic (1 + ε)-approximation to single-source shortest paths in undirected graphs with non-negative weights can be computed in ˜O((√n + D) · ε−9) rounds. ▶Corollary 9. For any 0 < ε ≤1, in the broadcast CONGEST model a deterministic (1 + ε)-approximation to single-source shortest paths in undirected graphs with non-negative weights can be computed in ˜O((√n + D) · ε−9) rounds. Multipass Streaming Model. In the streaming model the input graph is presented to the algorithm edge by edge as a “stream” without repetitions. The goal is to design algorithms that use as little space as possible. 3 Implementation in Various Models of Computation It suﬃces to communicate the demand vector; in cases where the demand vector is known a priori (e.g. when strengthening the approximation guarantee from average to worst-case for each node in the SSSP problem), even this is not necessary. ▶Theorem 7. For any 0 < ε ≤1/2, in the broadcast congested clique model a deterministic (1 + ε)-approximation to the shortest transshipment problem in undirected graphs with non- negative edge weights can be computed in ε−3 polylog n rounds. ▶Theorem 7. For any 0 < ε ≤1/2, in the broadcast congested clique model a deterministic (1 + ε)-approximation to the shortest transshipment problem in undirected graphs with non- negative edge weights can be computed in ε−3 polylog n rounds. 7:13 References 1 Amir Abboud, Greg Bodwin, and Seth Pettie. A hierarchy of lower bounds for sublinear additive spanners. In Symposium on Discrete Algorithms (SODA), pages 568–576, 2017. doi:10.1137/1.9781611974782.36. 2 Yair Bartal. On approximating arbitrary metrices by tree metrics. In Symposium on the Theory of Computing (STOC), pages 161–168, 1998. doi:10.1145/276698.276725. 3 Surender Baswana and Sandeep Sen. A simple and linear time randomized algorithm for computing sparse spanners in weighted graphs. 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For graph algorithms, the usual assumption is that the edges of the graph are presented to the algorithm in arbitrary order. The main observation is that we can apply the same approach as before with O(n log n) space: this enables us to store a spanner throughout the entire computation, and we can keep track of intermediate (node) state vectors. Computations on the spanner are thus “free,” while Φβ(π) and ∇Φβ(π) can be evaluated in a single pass by straightforward aggregation. It follows that ε−O(1) polylog n passes suﬃce for completing the computation. ▶Theorem 10. For any 0 < ε ≤1/2, in the multipass streaming model a deterministic (1+ε)- approximation to the shortest transshipment problem in undirected graphs with non-negative weights can be computed in ε−3 polylog n passes with O(n log n) space. ▶Theorem 11. 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https://openalex.org/W4286616175	https://e-journal.metrouniv.ac.id/index.php/an-nabighoh/article/download/4762/2791	Arabic	null	Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni	An Nabighoh/An-Nabighoh	2,022	cc-by-sa	6,473	TA’ALUM AL-LUGHAH AL-ARABIYYAH LI AGHRADL KHASHAH: AKADIMI WA MIHNI كلمات أساسية :تعلم اللغة العربية؛ أغراض خاصة؛ أكادميي؛ ومهين. AN NABIGHOH P-ISSN: 1907-1183 E-ISSN: 2581-2815 Vol. 24, No. 1 (2022): 81-92 http://e-journal.metrouniv.ac.id/index.php/an-nabighoh DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762 AN NABIGHOH P-ISSN: 1907-1183 E-ISSN: 2581-2815 Vol. 24, No. 1 (2022): 81-92 http://e-journal.metrouniv.ac.id/index.php/an-nabighoh DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762 تعلم اللغة العربية ألغراض خاصة :أكادميي ومهين Abstract Arabic has a function and essence for the life of Islamic communication, but because of its nature which is in the midst of an ongoing educational tradition today, it requires various innovations, as a logical consequence of the development of science and technology. The development of science and technology is so rapid for human life, it is necessary to make efforts on Arabic language teaching technicians who adapt to their respective fields and competencies. Then the purpose of learning Arabic with this specific purpose is to develop the professional abilities and academic potential of students.This type of research is descriptive qualitative research, namely a research conducted systematically by using library data using data collection tools, namely, observation, and documentation. This study provides a goal-based Arabic learning model to be achieved which is called Arabic learning for special purposes. This learning is more directed at learning Arabic for a certain scope and context, for example for work or professions and also in the academic field. Article History: Received: February 2022 Revised: May 2022 Accepted: June 2022 Published: June 2022 Article History: Received: February 2022 Revised: May 2022 Accepted: June 2022 Published: June 2022 Corresponding Author: Name: Nawang Wulandari Email: nawangtaufiq@gmail.com Copyright © 2022, Novita Rahmi et al. This is an open-access article under the CC–BY-SA license Keywords: Arabic Learning; Special Purposes; Academic; Work. مستخلص البحث Copyright © 2022, Novita Rahmi et al. This is an open-access article under the CC–BY-SA license Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762. Copyright © 2022, Novita Rahmi et al. This is an open-access article under the CC–BY-SA license Keywords: Arabic Learning; Special Purposes; Academic; Work. TA’ALUM AL-LUGHAH AL-ARABIYYAH LI AGHRADL KHASHAH: AKADIMI WA MIHNI Novita Rahmi1, Albarra Sarbaini2, J. Sutarjo3, Nawang Wulandari4 1,2,3,4 Institut Agama Islam Negeri Metro, Indonesia Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762. Article Info Abstract Article History: Received: February 2022 Revised: May 2022 Accepted: June 2022 Published: June 2022 *Corresponding Author: Name: Nawang Wulandari Email: nawangtaufiq@gmail.com Arabic has a function and essence for the life of Islamic communication, but because of its nature which is in the midst of an ongoing educational tradition today, it requires various innovations, as a logical consequence of the development of science and technology. The development of science and technology is so rapid for human life, it is necessary to make efforts on Arabic language teaching technicians who adapt to their respective fields and competencies. Then the purpose of learning Arabic with this specific purpose is to develop the professional abilities and academic potential of students.This type of research is descriptive qualitative research, namely a research conducted systematically by using library data using data collection tools, namely, observation, and documentation. This study provides a goal-based Arabic learning model to be achieved which is called Arabic learning for special purposes. This learning is more directed at learning Arabic for a certain scope and context, for example for work or professions and also in the academic field. Copyright © 2022, Novita Rahmi et al. This is an open-access article under the CC–BY-SA license Keywords: Arabic Learning; Special Purposes; Academic; Work. مستخلص البحث اللغة العربية هلا وظيفة وجوهر حلياة التواصل ،اإلسالمي ولكن بسبب طبيعتها اليت هي يف خضم تقليد تعليمي مستمر ،اليوم فهي تتطلب ابتكارات ،خمتلفة كنتيجة منطقية لتطور العلوم والتكنولوجيا .إن تطور العلوم والتكنولوجيا سريع للغاية ابلنسبة حلياة اإلنسا،ن ومن الضروري بذل جهود لفنيي تدريس اللغة العربية الذين يتأقلمون مع جماالت ختصصهم وكفاءاهتم .مث الغرض من تعلم اللغة العربية هلذا الغرض احملدد هو تطوير القدرات املهنية واإلمكاانت األكادميية للطالب .هذا النوع من البحث هو حبث نوعي ،وصفي أي البحث الذي يتم إجرا ؤه بشكل منهجي ابستخدام بياانت املكتبة ابستخدام أدوات مجع ،البياانت وهي املالحظة والتوثيق .تقدم هذه الدراسة منوذجًا لتعلم اللغة العربية قائمًا على اهلدف املطلوب حتقيقه وهو ما يسمى تعلم اللغة العربية ألغراض خاصة .هذا التعلم موجه أكثر حنو تعلم اللغة العربية يف نطاق وسياق معينني ، على سبيل املثال للعمل أو املهن وأيضًا يف اجملال األكادميي. 1 Mauidlotun Nisa and Syamsul Arifin, “Bahasa Arab untuk Tujuan Khusus di Indonesia dan Malaysia: Tinjauan Normatif dan Empiris,” Buletin Al-Turas 26, no. 1 (2020): 37–53, https://doi.org/10.15408/bat.v26i1.13303. Abstract مستخلص البحث اللغة العربية هلا وظيفة وجوهر حلياة التواصل ،اإلسالمي ولكن بسبب طبيعتها اليت هي يف خضم تقليد تعليمي مستمر ،اليوم فهي تتطلب ابتكارات ،خمتلفة كنتيجة منطقية لتطور العلوم والتكنولوجيا .إن تطور العلوم والتكنولوجيا سريع للغاية ابلنسبة حلياة اإلنسا،ن ومن الضروري بذل جهود لفنيي تدريس اللغة العربية الذين يتأقلمون مع جماالت ختصصهم وكفاءاهتم .مث الغرض من تعلم اللغة العربية هلذا الغرض احملدد هو تطوير القدرات املهنية واإلمكاانت األكادميية للطالب .هذا النوع من البحث هو حبث نوعي ،وصفي أي البحث الذي يتم إجرا ؤه بشكل منهجي ابستخدام بياانت املكتبة ابستخدام أدوات مجع ،البياانت وهي املالحظة والتوثيق .تقدم هذه الدراسة منوذجًا لتعلم اللغة العربية قائمًا على اهلدف املطلوب حتقيقه وهو ما يسمى تعلم اللغة العربية ألغراض خاصة .هذا التعلم موجه أكثر حنو تعلم اللغة العربية يف نطاق وسياق معينني ، على سبيل املثال للعمل أو املهن وأيضًا يف اجملال األكادميي. كلمات أساسية :تعلم اللغة العربية؛ أغراض خاصة؛ أكادميي؛ ومهين. Arabic Learning; Special Purposes; Academic; Work. اللغة العربية هلا وظيفة وجوهر حلياة التواصل ،اإلسالمي ولكن بسبب طبيعتها اليت هي يف خضم تقليد تعليمي مستمر ،اليوم فهي تتطلب ابتكارات ،خمتلفة كنتيجة منطقية لتطور العلوم والتكنولوجيا .إن تطور العلوم والتكنولوجيا سريع للغاية ابلنسبة حلياة اإلنسا،ن ومن الضروري بذل جهود لفنيي تدريس اللغة العربية الذين يتأقلمون مع جماالت ختصصهم وكفاءاهتم .مث الغرض من تعلم اللغة العربية هلذا الغرض احملدد هو تطوير القدرات املهنية واإلمكاانت األكادميية للطالب .هذا النوع من البحث هو حبث نوعي ،وصفي أي البحث الذي يتم إجرا ؤه بشكل منهجي ابستخدام بياانت املكتبة ابستخدام أدوات مجع ،البياانت وهي املالحظة والتوثيق .تقدم هذه الدراسة منوذجًا لتعلم اللغة العربية قائمًا على اهلدف املطلوب حتقيقه وهو ما يسمى تعلم اللغة العربية ألغراض خاصة .هذا التعلم موجه أكثر حنو تعلم اللغة العربية يف نطاق وسياق معينني ، على سبيل املثال للعمل أو املهن وأيضًا يف اجملال األكادميي. كلمات أساسية :تعلم اللغة العربية؛ أغراض خاصة؛ أكادميي؛ ومهين. Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah L Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762. Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762. 82 \| Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari املقدمة إن ال لغة العربية هلا الدور الكبري يف عامل االتصال، وخاصة ابلنسبة للمسلمني، جيعل هذه اللغة غالبًا ما تدرس يف خمتلف مستوايت التعليم، سواء يف التعليم الرمسي أو غري الرمسي. يف التعليم الرمسي، يتم تضمني اللغة العربية يف املواد الدراسية من املرحلة االبتدائية إىل املرحلة الثانوية. يف التعليم غري الرمسي، تُستخدم اللغة العربية كمادة تعليمية يف مؤسسات الدورة أ و مؤسسات التدريب. ابإلضافة إىل ذلك، هناك أوقات يهدف فيها تعلم اللغة العربية بشكل عام إىل زايدة اكتساب اللغة للطالب يف جمال اللغة العربية وأحياانً يتم دراسة اللغة العربية ألغراض خاصة. تنقسم هذه األهداف اخل اص إىل أهداف أك ادميية وأهداف مهنية. يف البداية ، كان الدافع وراء فكرة تعلم اللغات هلذا الغرض اخلاص هو مطالب التعليم وتدفق العمالة إىل خمتلف البلدان. ميكن أن حيدث هذا أيضًا بسبب االختالفات يف الظروف اجلغرافية للبلد واليت هلا أتثري على .االختالفات يف املوارد الطبيعية اللغة اليت تُدرس غالبًا ألغراض خاصة هي اللغ ة اإلجنليزية( Language For Spesific Porpose/LSP .) ا ستخد م ا ا ملؤلفات الكثري ة من اللغة اإلجنليزية ، ويساهم يف لغة العامل. أحد األدلة هو أن قسمLSP نفسه مت اعتماده من فئةESP وفقًا لروبنسون. ينقسمLSP إىل جزأين ، مهاLOP (لغة لألغراض املهنية) ألغراض العمل ولغة LAP .(لغة لألغراض األكادميية) لألغراض األكادميية1 مث يستمر مفهومLSP .هذا يف لغات أخرى مبا يف ذلك العربية AN NABIGHOH P ISSN 1907 1183 اللغة اليت تُدرس غالبًا ألغراض خاصة هي اللغ ة اإلجنليزية( Language For Spesific Porpose/LSP .) ا ستخد م ا ا ملؤلفات الكثري ة من اللغة اإلجنليزية ، ويساهم يف لغة العامل. أحد األدلة هو أن قسمLSP نفسه مت اعتماده من فئةESP وفقًا لروبنسون. املقدمة ينقسمLSP إىل جزأين ، مهاLOP (لغة لألغراض املهنية) ألغراض العمل ولغة LAP .(لغة لألغراض األكادميية) لألغراض األكادميية1 مث يستمر مفهومLSP .هذا يف لغات أخرى مبا يف ذلك العربية دراسة تعلم اللغة العربية أبهداف عامة من قبل الطالب يف جمال اللغة ،العربية و دراسة تعلم اللغة العربية ألغراض خاصة من قبل الطالب الذين ليسوا من جمال لغة ال ،العربية على الصعيدين األكادميي واملهين .إعداد تعلم اللغة العربية هلذا الغرض اخلاص للطالب الذين يرغبون يف إتقان اللغة وظيفيًا وعمليًا يف نطاق حمدود نسبيًا .يف برانمج تعلم اللغة العربية هلذا ،الغرض يتم تنظيم املواد التعليمية واملناهج الدراسية بشكل خاص ومنهجي من خالل مراعاة اخللفيات واالحتياجات املتنوعة للطالب .على الرغم من أن تطور اللغة العربية يف إندونيسيا ليس بنفس سرعة تطور اللغات األجنبية األخرى مثل اإلجنليزية واملاندرين 1 Mauidlotun Nisa and Syamsul Arifin, “Bahasa Arab untuk Tujuan Khusus di Indonesia dan Malaysia: Tinjauan Normatif dan Empiris,” Buletin Al-Turas 26, no. 1 (2020): 37–53, https://doi.org/10.15408/bat.v26i1.13303. دراسة تعلم اللغة العربية أبهداف عامة من قبل الطالب يف جمال اللغة ،العربية و دراسة تعلم اللغة العربية ألغراض خاصة من قبل الطالب الذين ليسوا من جمال لغة ال ،العربية على الصعيدين األكادميي واملهين .إعداد تعلم اللغة العربية هلذا الغرض اخلاص للطالب الذين يرغبون يف إتقان اللغة وظيفيًا وعمليًا يف نطاق حمدود نسبيًا .يف برانمج تعلم اللغة العربية هلذا ،الغرض يتم تنظيم املواد التعليمية واملناهج الدراسية بشكل خاص ومنهجي من خالل مراعاة اخللفيات واالحتياجات املتنوعة للطالب .على الرغم من أن تطور اللغة العربية يف إندونيسيا ليس بنفس سرعة تطور اللغات األجنبية األخرى مثل اإلجنليزية واملاندرين 1 Mauidlotun Nisa and Syamsul Arifin, “Bahasa Arab untuk Tujuan Khusus di Indonesia dan Malaysia: Tinjauan Normatif dan Empiris,” Buletin Al-Turas 26, no. 1 (2020): 37–53, https://doi.org/10.15408/bat.v26i1.13303. Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni \| 83 83 ،والياابنية إال أن العربية خبصائصها ومميزاهتا ومكانتها كلغة ،دولية تلهم معظم موا طين ،العامل خاصة ،املثقفني ملواصلة التعلم وإتقاهنا. بدأ تعلم اللغة العربية يف إندونيسيا مع انتشار اإلسالم من قبل األوصياء .يبدأ التعليم الديين اإلسالمي بتقدمي أساسيات العقيدة والتعاليم اليت يسهل فهمها وتنفيذها .على مستوى ،أعلى تقدمي حنو و صرف اجلديدان كجزء مهم من تعلم اللغة العربية ألن حنو و صرف مدرجان يف قواعد /قواعد اللغة العربية. 2 ابإلضافة إىل ،ذلك يف خمتلف جماالت ،احلياة ال مفر من إتقان اللغة ،العربية مثل اجملال ،األكادميي واجملال ،املهين وغريها .هلذا ،السبب هناك حاجة لربامج تعلم اللغة العربية ألغراض خا،صة مع اعتبارات وأسباب خمتلفة جتعل هذا الربانمج التعليمي خمتلفًا عن برامج تعلم اللغة العربية بشكل عام. Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762. املقدمة تدريس اللغة ألغراض ،خاصة هو التدريس الذي حيتوي على مواد تعليمية حمددة واسرتاتيجيات وتقييمات مصممة خصيصًا ألهداف واحتياجات املتعلمني /الطالب .املدخل املست خدم يف تدريس هذه اللغة ًبناء على خصائص املتعلمني وأهدافهم من تعلم اللغة العربية مع الوضع (السياق )الذي سيستخدمون فيه .يف هذا ،الصدد يرى حممود أسيوط أن تعلم اللغة العربية ألغراض خاصة له خاصية استخدام اللغة لتقوية مهنة ،الفرد لذلك غالبًا ما حيدث هذا التعلم يف شخص لديه جمال معريف خمتلف عن اللغة ،العربية أو أولئك الذين لديهم مهنة مثل .الصحفيني واحملامني واألطباء وغريهم. جيب أن يكون الصحفيون قادرين على إتقان لغة أجنبية ملصلحتهم يف استخراج املعلومات من ،األجانب وجيب على اخلرباء القانونيني واألطباء تعميق نظريتهم و معرفتهم من خالل قراءة الكتب ابللغات األجنبية .ستناقش يف هذه املقالة أمهية تعلم اللغة العربية ألغراض خاصة على الصعيدين األكادميي واملهين. 2 Zuhairini, Sejarah Pendidikan Islam (Jakarta: Bumi Aksara, 1997), 211. 3 Nur Halim, “Bahasa Arab Dengan Tujuan Khusus Berbasis Komunikatif Wisata Travelling,” Bintang : Jurnal Pendidikan Dan Sains 2, no. 3 (2020): 230, https://ejournal.stitpn.ac.id/index.php/bintang/article/view/960. املناقشة تعليم اللغة العربية الغرض اخلاص ميكن تعريف تدريس اللغة العربية ألغراض خاصة على أنه التعلم يف الدورات النوعية ا لكيفية اليت يعتمد تصميمها على االحتياجات اللغوية للمتعلمني الذين ي تضمن ون يف جماالت معرفية خبصائص لغوية 2 Zuhairini, Sejarah Pendidikan Islam (Jakarta: Bumi Aksara, 1997), 211. Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah L Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. h //d / / b h h 84 \| Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari .وأمناط لغوية معينة3 يهدف هذا التعلم إىل متكني الطالب ابملهارات والعناصر اللغوية لتحسني أدائهم األكادميي االحرتايف والتواصلي، على سبيل املثال دليل الس فر أو من حيث احلج والعمرة مثل املطوف عندما يكونون يف مكة واملزور عندما يكونون يف املدينة املنورة. ميكن تفسري مصطلح األغراض احملددة :(خنازير حمددة) على النحو التايل أ - خصوصية حتفيز الطالب العرب ألغراض خاصة هم الدبلوماسيون والطب واملهندسون واملهنيون واملمر ضات ورجال األعمال الذين يرغبون يف التواصل مع العرب لديهم رغبات مهنية ووظيفية ختتلف عن الدوافع األكادميية للطالب األجانب العرب، و دوافع خمتلفة عن متعلمي اللغة العربية .لألغراض العامة ب - اخلصوصية إن اللغة اليت أتيت من جمال املعرفة تتطلب توفري توازن معني يف املفردات والرتاكيب اليت .تليب احتياجات التواصل اللغوي للطالب ج - خصوصية الكفاءة اللغوية من خصوصية احتياجات الطالب اللغوية إلكمال مهامهم املهنية أو .األكادميية ج - خصوصية الكفاءة اللغوية من خصوصية احتياجات الطالب اللغوية إلكمال مهامهم املهنية أو .األكادميية د - تصميم مساق يهدف إىل التدريس للطالب واحتياجاهتم اللغوية مما يتطلب تصميم الدروس واألنشطة واإل.هلام الذي حيتاجه الطالب د - تصميم مساق يهدف إىل التدريس للطالب واحتياجاهتم اللغوية مما يتطلب تصميم الدروس واألنشطة واإل.هلام الذي حيتاجه الطالب هـ - خصوصية منهجية ل تصميم مقررات اللغة العربية ألغراض حمددة ، لتنوع ومنهجية تصميم هذه الدورات على دورات اللغة العربية لألغراض احلياتية. Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762. 4 Radliyah Zaenuddin, Metodologi & Strategi Alternatif Pembelajaran Bahasa Arab (Yogyakarta: Pustaka Rihlah Group, 2005), 46. 5 Juwairiyah Dahlan, Metode Belajar Mengajar Bahasa Arab (Surabaya: Al-Ikhlas, 1992), 29. p ) 5 Juwairiyah Dahlan, Metode Belajar Mengajar Bahasa Arab (Surabaya: Al-Ikhlas, 1992), 29. 4 Radliyah Zaenuddin, Metodologi & Strategi Alternatif Pembelajaran Bahasa Arab (Yogya Pustaka Rihlah Group, 2005), 46. املناقشة وقدم حممود حتليالً مطوالً ملراحل تطور تعليم اللغة ألغراض خاصة ، بعد أن استعرض العديد من املراج.ع األجنبية ، وفيما يلي ذكر هذه املراحل يبدأ تعلم اللغة ألغراض خاصة بتحليل احتياجات ،الطالب وليس من خالل تطوير التدريب الذي يتم إجراؤه حول حتليل اللغة .يشري كالمها إىل أن هذا التوجه أصبح مصدر اهتمام جديد يف تعلم اللغة ألغراض ،خاصة حيث جتاهل معظم خمططي م تعلي اللغة والتدريب هذه اخلطوة ذات األولوية املنطقية واملهمة .لذلك فإن الطالب ذوي االختالفات املختلفة لديهم احتياجات لغوية خمتلفة .وما سيتعلمونه أن 3 Nur Halim, “Bahasa Arab Dengan Tujuan Khusus Berbasis Komunikatif Wisata Travelling,” Bintang : Jurnal Pendidikan Dan Sains 2, no. 3 (2020): 230, https://ejournal.stitpn.ac.id/index.php/bintang/article/view/960. Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni \| 85 يقتصر على ما حيتاجون إليه .وهذه االحتياجات حمدودة قدر اإلمكان ، إذا كان من املمكن معرفة ،ذلك فيجب أن تكون د املوا اخلاصة بكل جلسة حمدودة أيضًا. فيجب أن تكون د املوا اخلاصة بكل جلسة حمدودة أيضًا. إن كان الغرض من تعلم اللغة العربية هو إتقان املهارات األكادميية يف خمتلف املؤسسات التعليمية واملنتدايت ،الرمسية فيجب أن تكون أولوية التعلم هي اللغة العربية الرمسية (الفصحى)، ولكن إن كانت احلاجة إىل إتقان اللغة هي جمرد أن ح تصب قوة عاملة أو تتواصل معها عامة الناس يف الشارع أو يف األماكن ،العامة فالعلم هو اللغة العربية غري الرمسية .لكن بشكل ،عام مييل تعلم اللغة العربية إىل استخدام اللغة العربية الرمسية (الفصحى)، ألن الفصحى العربية هلا قيمة أكرب من اللغة العربية .ومن مزاايها أهنا تستخدم لتحسني اجلودة الروحية ،للعبادة بسبب فهم التعاليم الدينية املستمدة من القرآن ،واحلديث ًفضال عن الكنوز الفكرية اإلسالمية املكتوبة ابللغة العربية الفصحى. 4 يف حبث عن تدريس اللغة العربية ألغراض خاصة ، عديد من األشياء اليت أصبحت القضااي الرئيسية .مثل :اذا مل حنتاج إىل تدريس لغة عربية ألغراض خاصة؟ هل ميكن تدريس اللغة العربية دون الرجوع إىل الغرض من تعلم اللغة العربية نفسها؟ و آخر ، هل ميكننا تعليم اللغة العربية بشكل عام للطالب دون اتباع األهداف احملددة للغة العربية ًبناء على جمال الدراسة أو برانمج الدراسة ل لطالب أو الطالب حيث سيطبقون أنفسهم ما تعلموه يف جماالت ختصصهم. املناقشة أهداف تعلم اللغة العربية لتقرير املدخل و الطروق واألساليب وتقنيات التعلم .لذلك ، جيب صياغة أهداف تعلم اللغة العربية بطريقة جتعل االجتاه املقصود صحيحًا على اهلدف .تنقسم أهداف تعلم اللغة العربية إىل أهداف عامة وأهداف خاصة .األهداف العامة هي كما يلي :١ )الطالب قادرون على فهم القرآن واحلديث كمصدر للشريعة اإلسالمية وتعاليمها؛ ٢ )قادر على فهم الكتب الدينية والثقافية اإلسالمية املكتوبة ابللغة العربية؛ ٣ )قادر على التحدث والتأليف ابللغة العربية؛ ٤ )للغة ا العربية مستخدمة ملساعدة املهارات األخرى .بينما األهداف اخلاصة لتعلم اللغة العربية هي :١ )الغرض من االستخدام ،العملي الكتساب الكفاءة على اخلربة يف جمال الدراسة أو لدعم علم أو مهنة معينة؛ ٢ )الغرض من االستخدام ،التطبيقي هو اكتساب مهارات االتصال ابللغة ا،لعربية ًكتابة ،ًوحتداث ومتقبلة ومنتجة. 5 إن كان الغرض من تعلم اللغة العربية هو إتقان املهارات األكادميية يف خمتلف املؤسسات التعليمية واملنتدايت ،الرمسية فيجب أن تكون أولوية التعلم هي اللغة العربية الرمسية (الفصحى)، ولكن إن كانت احلاجة إىل إتقان اللغة هي جمرد أن ح تصب قوة عاملة أو تتواصل معها عامة الناس يف الشارع أو يف األماكن ،العامة فالعلم هو اللغة العربية غري الرمسية .لكن بشكل ،عام مييل تعلم اللغة العربية إىل استخدام اللغة العربية الرمسية (الفصحى)، ألن الفصحى العربية هلا قيمة أكرب من اللغة العربية .ومن مزاايها أهنا تستخدم لتحسني اجلودة الروحية ،للعبادة بسبب فهم التعاليم الدينية املستمدة من القرآن ،واحلديث ًفضال عن الكنوز الفكرية اإلسالمية املكتوبة ابللغة العربية الفصحى. 4 ربي ر مي (ص ى) أ ص ى ربي ه ي رب ن ربي و ن زاي ه م لتحسني اجلودة الروحية ،للعبادة بسبب فهم التعاليم الدينية املستمدة من القرآن ،واحلديث ًفضال عن الكنوز الفكرية اإلسالمية املكتوبة ابللغة العربية الفصحى. 4 يف حبث عن تدريس اللغة العربية ألغراض خاصة ، عديد من األشياء اليت أصبحت القضااي الرئيسية .مثل :اذا مل حنتاج إىل تدريس لغة عربية ألغراض خاصة؟ هل ميكن تدريس اللغة العربية دون الرجوع إىل الغرض من تعلم اللغة العربية نفسها؟ و آخر ، هل ميكننا تعليم اللغة العربية بشكل عام للطالب دون اتباع األهداف احملددة للغة العربية ًبناء على جمال الدراسة أو برانمج الدراسة ل لطالب أو الطالب حيث سيطبقون أنفسهم ما تعلموه يف جماالت ختصصهم. املناقشة أهداف تعلم اللغة العربية لتقرير املدخل و الطروق واألساليب وتقنيات التعلم .لذلك ، جيب صياغة أهداف تعلم اللغة العربية بطريقة جتعل االجتاه املقصود صحيحًا على اهلدف .تنقسم أهداف تعلم اللغة العربية إىل أهداف عامة وأهداف خاصة .األهداف العامة هي كما يلي :١ )الطالب قادرون على فهم القرآن واحلديث كمصدر للشريعة اإلسالمية وتعاليمها؛ ٢ )قادر على فهم الكتب الدينية والثقافية يف حبث عن تدريس اللغة العربية ألغراض خاصة ، عديد من األشياء اليت أصبحت القضااي الرئيسية .مثل :اذا مل حنتاج إىل تدريس لغة عربية ألغراض خاصة؟ هل ميكن تدريس اللغة العربية دون الرجوع إىل الغرض من تعلم اللغة العربية نفسها؟ و آخر ، هل ميكننا تعليم اللغة العربية بشكل عام للطالب دون اتباع األهداف احملددة للغة العربية ًبناء على جمال الدراسة أو برانمج الدراسة ل لطالب أو الطالب حيث سيطبقون أنفسهم ما تعلموه يف جماالت ختصصهم. أ ل ل ل لم لط أل ل ل جلذل ا مياج مخ أهداف تعلم اللغة العربية لتقرير املدخل و الطروق واألساليب وتقنيات التعلم .لذلك ، جيب صياغة أهداف تعلم اللغة العربية بطريقة جتعل االجتاه املقصود صحيحًا على اهلدف .تنقسم أهداف تعلم اللغة العربية إىل أهداف عامة وأهداف خاصة .األهداف العامة هي كما يلي :١ )الطالب قادرون على فهم القرآن واحلديث كمصدر للشريعة اإلسالمية وتعاليمها؛ ٢ )قادر على فهم الكتب الدينية والثقافية اإلسالمية املكتوبة ابللغة العربية؛ ٣ )قادر على التحدث والتأليف ابللغة العربية؛ ٤ )للغة ا العربية مستخدمة ملساعدة املهارات األخرى .بينما األهداف اخلاصة لتعلم اللغة العربية هي :١ )الغرض من االستخدام ،العملي الكتساب الكفاءة على اخلربة يف جمال الدراسة أو لدعم علم أو مهنة معينة؛ ٢ )الغرض من االستخدام ،التطبيقي هو اكتساب مهارات االتصال ابللغة ا،لعربية ًكتابة ،ًوحتداث ومتقبلة ومنتجة. 5 86 \| Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari قال رشدي أمحد ،طعيمة أن برامج تعليم اللغة العربية لغري العرب تنقسم بشكل عام إىل نوعني: أ - برانمج تعلم اللغة العربية مدى احلياة ، وهو برانمج عام يتضمن خصائص املتعلم ، يهدف إىل التواصل مع العرب يف مواقف احلياة املخت لفة .هذا الربانمج هو برانمج عام يغطي تنوع املهن اجملتمعية وخصائصها واهتماماهتا .يتعلمون اللغة العربية لتلبية احتياجاهتم يف احلياة بشكل عام .كما هو احلال يف السوق ، يف املؤسسة ، يف املنزل ، أثناء التنقل ، وما إىل ذلك. 6 Halim, “Bahasa Arab Dengan Tujuan Khusus Berbasis Komunikatif Wisata Travelling.” Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762. املناقشة ب - برامج تعلم اللغة العربية ألغراض صة خا ، وهي برامج التعلم اليت تشمل املتعلمني من ذوي االحتياجات اخلاصة واحملددة .من بني تلك املدرجة يف هذا النوع هي التعلم لألغراض األكادميية ، والتعلم ألغراض العمل ، وألغراض العمل ، وألغراض هيئة التدريس ، ولألغراض الصحية والطبية ، ر ومراحل تطوير تعلم لغة ال ألغراض حمددة أو خاصة هي كما يلي : أ - اخرت الكتاب املناسب حسب الظروف اليت تستخدم فيها اللغة ، مث تركيز على بناء برانمج لتحليل اللغوايت واالعتماد على ما هو مناسب للتعليم أو اخللفية التعليمية للطالب. ب - حتليل عينة من الكتب املدرسية املتعلقة بتخصص ني مع على أساس أن اللغة يف اهلندسة ختتلف عن تلك املوجودة يف الطب ، مما يوفر ًتوازان ، من مفردات عالية الرتدد ، وأصعبها تركز القواعد على منطقة اللغة يف معني االنضباط ، وترك ما ليس لديك جانبًا .العالقة ابلتخصص . ًبناء على هذه البياانت ، ظهر مفهوم لغوي خاص من الرب انمج الذي مت تصميمه وفقًا الحتياجات الطالب الستخدامها يف أماكن معينة. ج - انتقل املعاجل من حتليل الكتاب إىل حتليل اخلطاب ، ًمؤكدا أنه من املهم االنتباه لفهم املعىن ، وأن التفاعل أمر ال بد منه ، وأنه حيدث بني وحدات املعىن ، ودرجة الرتابط بينها اليت تؤدي إىل فهم النص .أن تقرأ أو تسمع .جيادل أبن هذه املرحلة تتطلب اهتمامًا خاصًا بوظائفها وسياقاهتا البالغية املختلفة. د - حتديد أولوايت حتليل السمات اللغوية العامة ، وحتليل احتياجات املتعلمني من حيث أهداف االتصال ، ويستهدف الربانمج الطالب املؤهلني الستخدام اللغة يف جماالت مهنية معينة ، واالعتماد على قياس األداء مبستوى من الدقة والطالقة. Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni \| 87 ه - ًبناء على احتياجات املتعلمني ، املهتمني ابملعىن ، واملهتمني ابالسرتاتيجيات التعليمية ومواءمتها مع أهداف التعلم اليت هتدف إىل الطالقة والدقة يف لغة شاملة ، والسياق الذي مت فيه ا كتساب اللغة ، مت تصميم اإلجراءات التعليمية وفقًا الحتياجات املتعلمني. 6 أنواع تعلم اللغة العربية ألغراض خاصة تعلم اللغة ألغراض خاصة من قبل املؤسسات ،التعليمية سواء املؤسسات الرمسية أو غري الرمسية .يتم تنفيذ غالبية هذا التعلم من قبل مؤسسات التعليم العايل .ذا ه يعين أن تعلم اللغة العربية ألغراض خاصة هو أكثر عملية للطالب رفيعي املستوى .مبا يف ذلك طالب اجلامعات .هذا ألن التعليم العايل موجه حنو التوظيف .الوظيفة اليت هتتم هبا هلا أتثري على اجملال /القسم املختار .ومع ،ذلك ابلنسبة للطالب خبالف الطالب الذين يرغبون يف ممارسة ،مهنة فمن املمكن أيضًا املشاركة يف هذا ،التعلم طاملا أن اللغة العربية تدعم مهنتهم وجماهلم. املناقشة ٢ )اجملال املهين احملادثة عن تعلم اللغة العربية ألغراض خاصة يف اجملال املهين ، فإن إتقان اللغة العربية يهدف إىل دعم املهنة اليت يعمل هبا شخص ما ابلفعل .على سبيل املثال :مجهور إندونيسي يعمل كمرافق للحج والعمرة .على غرار تعلم اللغة العربية يف اجملال األكادميي ، تتطلب هذه ملهنة ا أيضًا ختطيطًا تعليميًا دقيقًا ، مثل املناهج وخطط الدروس وإعداد املواد التعليمية والوسائط واألساليب والنهج والتقييمات يف وقت حمدد ، مع هدف اإلجناز ، ال ميكن جتاهله كمعلم أو منفّذ يف التدريس ، إذا مل يتم التخطيط له بعناية ، فسيحصل على نتائج غري مثالية. ومع ذلك ، فإن ما مييز هذا التعلم هو أن املواد اليت يتم تدريسها مركزة وحمدودة ، وهي مصممة خصيصًا الحتياجات كل طالب .فيما يلي شرح ألدلة احلج والعمرة وكذلك األحاديث العربية املتعلقة هبذه املهن. يف كل رحلة عمرة أو حج جند دائمًا مطوفًا سريافق احلجاج ًليال رًا وهنا .دور املطوّف مهم جدًا لدعم حسن سري العبادة أثناء تواجده يف األرض ،املقدسة مثل :مرافقة املصلني إلظهار مكان ،العبادة تقدمي التوجيه واإلرشاد بشأن تنفيذ احلج أو ،العمرة مرافقة املصلني .املصلني إىل مناطق ،التسوق ومساعدة احلجاج ،الضائعني واملساعدة يف حل مشاك ل اجلماعة اليت حتدث أثناء احلج.يف األرض ،املقدسة رواية القصص عن األنبياء والرسل واألرض ،املقدسة توفري معلومات عن اللغة والثقافة ،العربية إرسال املرضى املصلني إىل ،املستشفيات إخل. أمثلة من األحاديث املتعلقة مبهنة أدالء احلج والعمرة: السؤال اجلواب )+( َا مَاذ ُتـَفْعَل ِيف املَدِيـْنَةِ؟ (- ) ُأَزُوْر َاملَسْجِد النـَبَوِي )+( َأَيْن تـَقْضِي َأَاي م العَيْدِ؟ (- )أَقْضِيـْهَا ِيف َمَك ة ِأَو ِيف ِاملَدِيـْنَة. )+(وَمَاذَا ُتـَفْعَل ِيف َمَك ة ؟ (- ) ُأَطُوْف َحَوْل ِالكَعْبَة َسَبـْعَة ، ٍأَشْوَاط ُمث ِّي أَصَل ِْرَكْعَتـَني َخَلْف ِمَقَام َإِبـْرَاهِيْم. )+( َكَيْف َاعْتَمَرْت َاي أَيبِ ؟ (- ) ُوَصَلْت َإِىل َاملْيـَقَات َعِنْد ِالظُّهْر. إذا مت تدريس اللغة العربية للطالب غري ،الرتبويني فإن املادة اليت يتم تدريسها تكون يف شكل مصطلحات عربية حيتاجون إليها لدر اسة األدب واملصطلحات العربية املستخدمة يف جماهلم .على سبيل املثال :تدرس كلية الطب اللغة العربية لفهم الكتب العربية البن سينا والفارايب .تدرس كلية الشريعة اللغة العربية لفهم استخدام املصطلحات العربية مثل املضاربة واملعاملة واملشاركة وغريها. املناقشة ،لروبنسون ينقسم تعلم اللغة ألغراض خاصة إىل ،قسمني مها لغة لألغراض ،املهنية ومها اللغة ألغراض العمل واللغة لألغراض ،األكادميية أي اللغة لألغراض األكادميية. ١) اجملال األكادميي جاأمأ أنواع تعلم اللغة العربية ألغراض خاصة أنواع تعلم اللغة العربية ألغراض خاصة تعلم اللغة ألغراض خاصة من قبل املؤسسات ،التعليمية سواء املؤسسات الرمسية أو غري الرمسية .يتم تنفيذ غالبية هذا التعلم من قبل مؤسسات التعليم العايل .ذا ه يعين أن تعلم اللغة العربية ألغراض خاصة هو أكثر عملية للطالب رفيعي املستوى .مبا يف ذلك طالب اجلامعات .هذا ألن التعليم العايل موجه حنو التوظيف .الوظيفة اليت هتتم هبا هلا أتثري على اجملال /القسم املختار .ومع ،ذلك ابلنسبة للطالب خبالف الطالب الذين يرغبون يف ممارسة ،مهنة فمن املمكن أيضًا املشاركة يف هذا ،التعلم طاملا أن اللغة العربية تدع هنته جماهل ،لروبنسون ينقسم تعلم اللغة ألغراض خاصة إىل ،قسمني مها لغة لألغراض ،املهنية ومها اللغة ألغراض العمل واللغة لألغراض ،األكادميية أي اللغة لألغراض األكادميية. أمجم تعلم اللغة العربية لألغراض األكادميية تتم دراسته من قبل الطالب الذين ليسوا من جمال اللغة العربية .يتم دراسة اللغة العربية بشكل رمسي ألن اللغة العربية هي إحدى املواد اإلجبارية يف مؤسسة التعليم العايل ، وخاصة يف PTKI / PTKIN .مادة اللغة الع ربية اليت يتم تدريسها مصممة لتناسب جماهلم .على سبيل املثال :تعلم اللغة العربية لكليات الطب ، والقانون ، والشريعة ، وبرامج دراسة PIAUD ، و PAI ، وعلم األحياء Tadris ، والدراسات االجتماعية ، إخل .عادة ، ابلنسبة لكليات الرتبية ، يتم تدريس اللغة العربية يف شكل مواد سيقومون بتدريسها على مستوايت PAUD و SD و SLTP و SLTA وفقًا لسياق جمال معرفتهم .يتم اختيار املوضوعات وفقًا لسياق معرفتهم .على سبيل املثال :مت تصميم برانمج دراسة اللغة العربية لربانمج PIAUD لتعلم اللغة العربية اليت سيقومون بتدريسها الحقًا ملرحلة الطفول ة املبكرة .مت تصميم برانمج دراسة اللغة العربية يف PAI لدعم موضوعات القرآن واحلديث النبوي اليت سيدرسوهنا يف املدارس اإلعدادية والثانوية. 88 \| Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari \| , , J j , g إذا مت تدريس اللغة العربية للطالب غري ،الرتبويني فإن املادة اليت يتم تدريسها تكون يف شكل مصطلحات عربية حيتاجون إليها لدر اسة األدب واملصطلحات العربية املستخدمة يف جماهلم .على سبيل املثال :تدرس كلية الطب اللغة العربية لفهم الكتب العربية البن سينا والفارايب .تدرس كلية الشريعة اللغة العربية لفهم استخدام املصطلحات العربية مثل املضاربة واملعاملة واملشاركة وغريها. املناقشة )+(وَمَاذَا ُنـَفْعَل َبـَعْد ذَلِكَ؟ (- ) ُنَذْهَب َإِىل َمِىن َقـَبْل ِشُرُوْق ِالش مْس وَنـَرْمِي َاجلَمْرَة ،الكُبـْرَى ُثم ُنَذْبَح َاهلَدْي ُوَحنَْلِق رُؤُوْسَنَا. )+( َوَمَت ُنَطُوْف َطَوَاف الوَدَاعِ؟ (- ) َبـَعْد ِرَمْي ِاجلَمَرَات ِيف ِاليـَوْم ِالث ان ،َعَشَر ِأَو َالث الِث عَشَر. اأ )+(مَاذَا َفـَعَلْت ِيف ِاملِيـْقَات ؟ (- ) ُخَلَعْت ، ِثـَوْيب ُوَلَبِسْت َمَالَ بِس ،ِاإلِحْرَام ُوَلَبـ يْت ِابِلعُمْرَة. )+( َوَمَت َوَصَلْت َإِىل ِاملَسْجِد ِاحلَرَام ؟ (- ) ُوَصَلْت َبـَعْد ِالعَصْر. )+( ْكَم شَوْطًا َطُفْت َحَوْل الكَعْبَةِ؟ (- ) ُطُفْت َسَبـْعَة ٍأَشْوَاط ، ُمث ُصَل يْت ِْرَكْعَتـَني َخَلْف ِمَقَام َإِبـْرَاهِيْم. )+(وَمَاذَا َفـَعَلْت َبـَعْد ذَلِكَ؟ (- ) َسَع ُيْت َْبـَني الص فَا َوَاملَرْوَة َسَبـْعَة ٍأَشْوَاط ، ُمث ُحَلَقْت رَأْسِي. )+( َمَت ُيـَبْدَأ ُالوُقـُوْف بِعَرَفَةَ؟ (- ) ُيـَبْدَأ غَدًا ِيف ِاليـَوْم ِالت اسِع َبـَعْد ِالز وَال ُعَدْانَن. )+( َوَمَت سينتهي؟ (- )ينـْتَهِي َعِنْد الفَجْر. )+( َمَت ُنَذْهَب َإِىل مُزْدَلِفَةَ؟ (- ) ُنَذْهَب َبـَعْد ِغُرُوْب ِالش مْس وَنُصَلِّي فِيـْهَا َاملَغْرِب َوَالعِشَاء مجَْعًا وَقَصْرًا. )+(وَمَاذَا ُنـَفْعَل َبـَعْد ذَلِكَ؟ (- ) ُنَذْهَب َإِىل َمِىن َقـَبْل ِشُرُوْق ِالش مْس وَنـَرْمِي َاجلَمْرَة ،الكُبـْرَى ُثم ُنَذْبَح َاهلَدْي ُوَحنَْلِق رُؤُوْسَنَا. )+( َوَمَت ُنَطُوْف َطَوَاف الوَدَاعِ؟ (- ) َبـَعْد ِرَمْي ِاجلَمَرَات ِيف ِاليـَوْم ِالث ان ،َعَشَر ِأَو َالث الِث عَشَر. أهداف وغاايت تعلم اللغة العربية ألغراض خاصة أهداف وغاايت تعلم اللغة العربية ألغراض خاصة تعلم اللغة العربية للتواصل حسب خلفية أنشطة ا ملتعلم .يف تعلم اللغة العربية لغرض ،معني يتميز إبعداد برانمج أو منهج مبواصفات معينة .يتطلب النظر يف تنوع اجملتمع يف البيئة ،األكادميية ،والوظيفة ،واملهنة جمموعة متنوعة من الربامج بسبب االحتياجات املختلفة .الغرض من تعلم اللغة العربية هلذا الغرض احملدد يوجه املتعلمني ليكونوا قادرين على التواصل حيث يتم وضعهم وفقًا الحتياجاهتم .سواء يف احلياة اليومية أو يف املنتدايت العلمية أو يف املنتدايت الرمسية األخرى. يناسب هذا اهلدف مببدأ ،االتصال ألن املتعلمني لديهم خياران ،فقط ومها العمل كطرف ،نشط أي املتحدثني ،والكتاب وأ كأطراف ،سلبية أي كمستمعني وقراء . ،لذلك جيب النظر يف استخدام منوذج التعلم لغرض حمدد (خاص .)يهدف التعلم ابستخدام هذا النموذج إىل توفري مهارات اللغة العربية الوظيفية والعملية للطالب الذين يدرسوهنا .على سبيل ،املثال دورة يف اللغة العربية مصممة خصيصًا للعمال احملتملني الذين سيتم تعيينهم يف الشرق ،األوسط أو لضباط احلج ،احملتملني وما إىل ذلك .رضلية زين الدين له اجتاهني ألهداف تعلم اللغة العربية ، ومها: ١ )اللغة العربية كهدف - إتقان املهارات اللغوية؛ ٢ ) اللغة العربية إلتقان املعرفة األخرى ابستخدام مركبات اللغة ال عربية .بعض األسباب اليت تكمن وراء تطوير Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762. املناقشة احملادثة عن تعلم اللغة العربية ألغراض خاصة يف اجملال املهين ، فإن إتقان اللغة العربية يهدف إىل دعم املهنة اليت يعمل هبا شخص ما ابلفعل .على سبيل املثال :مجهور إندونيسي يعمل كمرافق للحج والعمرة .على غرار تعلم اللغة العربية يف اجملال األكادميي ، تتطلب هذه ملهنة ا أيضًا ختطيطًا تعليميًا دقيقًا ، مثل املناهج وخطط الدروس وإعداد املواد التعليمية والوسائط واألساليب والنهج والتقييمات يف وقت حمدد ، مع هدف اإلجناز ، ال ميكن جتاهله كمعلم أو منفّذ يف التدريس ، إذا مل يتم التخطيط له بعناية ، فسيحصل على نتائج غري مثالية. ومع ذلك ، فإن ما مييز هذا التعلم هو أن املواد اليت يتم تدريسها مركزة وحمدودة ، وهي مصممة خصيصًا الحتياجات كل طالب .فيما يلي شرح ألدلة احلج والعمرة وكذلك األحاديث العربية املتعلقة هبذه املهن. أمهم يف كل رحلة عمرة أو حج جند دائمًا مطوفًا سريافق احلجاج ًليال رًا وهنا .دور املطوّف مهم جدًا لدعم حسن سري العبادة أثناء تواجده يف األرض ،املقدسة مثل :مرافقة املصلني إلظهار مكان ،العبادة تقدمي التوجيه واإلرشاد بشأن تنفيذ احلج أو ،العمرة مرافقة املصلني .املصلني إىل مناطق ،التسوق ومساعدة احلجاج ،الضائعني واملساعدة يف حل مشاك ل اجلماعة اليت حتدث أثناء احلج.يف األرض ،املقدسة رواية القصص عن األنبياء والرسل واألرض ،املقدسة توفري معلومات عن اللغة والثقافة ،العربية إرسال املرضى املصلني إىل ،املستشفيات إخل. أمثلة من األحاديث املتعلقة مبهنة أدالء احلج والعمرة: السؤال اجلواب )+( َا مَاذ ُتـَفْعَل ِيف املَدِيـْنَةِ؟ (- ) ُأَزُوْر َاملَسْجِد النـَبَوِي )+( َأَيْن تـَقْضِي َأَاي م العَيْدِ؟ (- )أَقْضِيـْهَا ِيف َمَك ة ِأَو ِيف ِاملَدِيـْنَة. )+(وَمَاذَا ُتـَفْعَل ِيف َمَك ة ؟ (- ) ُأَطُوْف َحَوْل ِالكَعْبَة َسَبـْعَة ، ٍأَشْوَاط ُمث ِّي أَصَل ِْرَكْعَتـَني َخَلْف ِمَقَام َإِبـْرَاهِيْم. )+( َكَيْف َاعْتَمَرْت َاي أَيبِ ؟ (- ) ُوَصَلْت َإِىل َاملْيـَقَات َعِنْد ِالظُّهْر. Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni \| 89 )+(مَاذَا َفـَعَلْت ِيف ِاملِيـْقَات ؟ (- ) ُخَلَعْت ، ِثـَوْيب ُوَلَبِسْت َمَالَ بِس ،ِاإلِحْرَام ُوَلَبـ يْت ِابِلعُمْرَة. )+( َوَمَت َوَصَلْت َإِىل ِاملَسْجِد ِاحلَرَام ؟ (- ) ُوَصَلْت َبـَعْد ِالعَصْر. )+( ْكَم شَوْطًا َطُفْت َحَوْل الكَعْبَةِ؟ (- ) ُطُفْت َسَبـْعَة ٍأَشْوَاط ، ُمث ُصَل يْت ِْرَكْعَتـَني َخَلْف ِمَقَام َإِبـْرَاهِيْم. )+(وَمَاذَا َفـَعَلْت َبـَعْد ذَلِكَ؟ (- ) َسَع ُيْت َْبـَني الص فَا َوَاملَرْوَة َسَبـْعَة ٍأَشْوَاط ، ُمث ُحَلَقْت رَأْسِي. )+( َمَت ُيـَبْدَأ ُالوُقـُوْف بِعَرَفَةَ؟ (- ) ُيـَبْدَأ غَدًا ِيف ِاليـَوْم ِالت اسِع َبـَعْد ِالز وَال ُعَدْانَن. )+( َوَمَت سينتهي؟ (- )ينـْتَهِي َعِنْد الفَجْر. )+( َمَت ُنَذْهَب َإِىل مُزْدَلِفَةَ؟ (- ) ُنَذْهَب َبـَعْد ِغُرُوْب ِالش مْس وَنُصَلِّي فِيـْهَا َاملَغْرِب َوَالعِشَاء مجَْعًا وَقَصْرًا. 7 Imam Makruf, Strategi Pembelajaran Bahasa Arab Aktif (Semarang: Need’s Press, 2009), 67–68 8 Muhbib Abdul Wahab, Epistemologi dan Metodologi Pembelajaran Bahasa Arab (UIN Jakar Press, 2019), 130, https://repository.uinjkt.ac.id/dspace/handle/123456789/44533. , ), , p // p y j / p / / / 9 Rusydi Ahmad Thu’aimah, Ta’lim Al-Lughoh Li Aghrodh Khosoh: Mafahimah Wa Manhajiya (Khartoum: Ma’had Khartoum Ad-Dauly, 2003), 10. 7 Imam Makruf, Strategi Pembelajaran Bahasa Arab Aktif (Semarang: Need s Press, 2009), 67–68. 8 Muhbib Abdul Wahab, Epistemologi dan Metodologi Pembelajaran Bahasa Arab (UIN Jakarta Press, 2019), 130, https://repository.uinjkt.ac.id/dspace/handle/123456789/44533. 9 Rusydi Ahmad Thu’aimah, Ta’lim Al-Lughoh Li Aghrodh Khosoh: Mafahimah Wa Manhajiyah 7 Imam Makruf, Strategi Pembelajaran Bahasa Arab Aktif (Semarang: Need’s Press, 2009), 67–68. 8 Muhbib Abdul Wahab, Epistemologi dan Metodologi Pembelajaran Bahasa Arab (UIN Jakarta Press, 2019), 130, https://repository.uinjkt.ac.id/dspace/handle/123456789/44533. 7 Imam Makruf, Strategi Pembelajaran Bahasa Arab Aktif (Semarang: Need’s Press, 2009), 67–68. 8 Muhbib Abdul Wahab, Epistemologi dan Metodologi Pembelajaran Bahasa Arab (UIN Jakarta Press, 2019), 130, https://repository.uinjkt.ac.id/dspace/handle/123456789/44533. 9 Rusydi Ahmad Thu’aimah, Ta’lim Al-Lughoh Li Aghrodh Khosoh: Mafahimah Wa Manhajiyah (Khartoum: Ma’had Khartoum Ad-Dauly, 2003), 10. املناقشة Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762. 90 \| Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari هذا التعلم ، هي :١ )يوجد حتليل متعمق الحتياجات الطالب يف التواصل؛ ٢ )لتحسني قدرة لغة معينة عميقة؛ ٣ )عدم حاجة الستخدام طرق معينة يف التعلم. 7 منوذج تعلم اللغة العربية لغرض خاص له عملية ختتلف عن منوذج تعلم اللغة العربي ة بشكل عام .ومن أجل حتسني مهارات ،االتصال هناك حاجة إىل أربع كفاءات أساسية على األقل جيب أن تكون موجهة لتعلم اللغة ، وهي :١ )الكفاءة النحوية - حنو و صرف - واجلوانب الصوتية؛ ٢ )الكفاءة اللغوية االجتماعية؛ ٣ )اختصاص حتليل اخلطاب؛ ٤ )الكفاءة االسرتاتيجية. 8 ال ط قة ال يُطلب من املعلم اتباع طريقة معينة يف عملية التعلم هلذا النموذج .الشاغل الرئيسي هو كيف ميكن تسليم املواد وفقًا لألهداف اليت سيحققها املتعلم .وابلتايل ، فإن عملية التعلم تتم مبرونة ، بل ومرنة . تعتمد كفاءة الوقت املطلوبة على ضرورة حتقيق التعلم ، حت لو مت تنفيذه ابملصادفة عندما يريد املتعلم تسليم مواد معينة .ميكن اختيار تعلم اللغة هلذا الغرض اخلاص مع عدة اعتبارات ، مبا يف ذلك :١ )الوقت للطالب حمدود للغاية؛ ٢ )استخدام اللغة املدروسة على نطاق معني؛ ٣ )الطالب الذين يدرسون اللغة ال يتقنون اللغة العربية دًا؛ جي ٤ )ميكن تنفيذه بشكل مكثف حبيث يكون أكثر فعالية وكفاءة .بينما اخلصائص الرئيسية لربامج تعلم اللغة ألغراض خاصة من بني أمور أخرى :١ )إعداد هذا الربانمج لتلبية االحتياجات اخلاصة للمتعلمني؛ ٢ )املواد املقدمة مالئمة مبجال العمل؛ ٣ )تركيز اختيار اللغة على األ نشطة السابقة ، سواء من حيث القواعد (اللغة)، واملفردات (املفرودات)، والكتب املدرسية (الكتاب)، ،واملعىن وغريها؛ ٤ )خيتلف عن تعلم اللغة العربية بشكل عام. 9 عديد من برامج تعلم اللغة العربية ألغراض حمددة .يرجع عدد القضية اليت حتدث يف الدول العربية املتعلقة بعام لني من إندونيسيا بشكل أكرب إىل عدم قدرة العمال على التواصل ابللغة العربية .لذلك ، من املهم للعمال اإلندونيسيني أن يشاركوا يف تعلم اللغة العربية كتوفري للتواصل ، على األقل أمية العربية اليت ) p p y j p 9 Rusydi Ahmad Thu’aimah, Ta’lim Al-Lughoh Li Aghrodh Khosoh: Mafahimah Wa Manhajiyah hartoum: Ma’had Khartoum Ad-Dauly, 2003), 10. AN NABIGHOH Vol. 24, No. 1 (2022) Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni \| 91 تستخدم على نطاق واسع يف احلياة اليومية أكثر من الفصحى العربية .وابل تايل ، ميكن جتنب األشياء غري املتوقعة ، مثل سوء الفهم يف التواصل. 10 Mohammad Arif Setyabudi, Syamsuddin, and Hanifuddin, “Pembelajaran Bahasa Arab Untuk Tujuan Khusus,” EL-FUSHA: Jurnal Bahasa Arab Dan Pendidikan 1, no. 1 (2020): 90–98, http://ejournal.unhasy.ac.id/index.php/alfusha/article/view/1220. Please cite this article as Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari “Ta’alum Al-Lughah Al-Arabiyyah Li Aghradl Khashah: Akadimi Wa Mihni.” An Nabighoh 24, No. 1 (2022): 81-92. DOI: https://doi.org/10.32332/an-nabighoh.v24i1.4762. املناقشة اجلمهور يف الدول العربية إىل استخدام لغة العامية يف حياهتم اليومية .تنقسم لغة العامية أيضًا إىل عدة هلجات حسب كل بلد .مثل اللهجة العربية الشرقية ، واللهجة ،اخلليجية واللهجة ،املصرية واللهجة ،اليمنية واللهجة ،السودانية وغريها .اللغة العربية للعمال اإلندونيسيني هي مطلب جيب إعداده يف أقرب وقت ممكن .يتم إعطاء األولوية إلعداد املواد التعليمية على جوانب االتصال والسياق الذي حيدث فيه ،االتصال وليس املواد اليت حتتوي على نظرايت اللغة . ،لذلك يف ،امللحق يتم تقدمي بعض األمثلة املتعلقة ابللغة العربية واليت يتم تدريسها خصيصًا للعمال اإلندونيسيني ،احملتملني كل من الفصحى العربية واألمية العربية .يتم تنفيذ هذا التعلم حبيث يكون لدى العمال اإلندونيسيني األحكام ،واملعرفة ًفضال عن القدرة على التحدث ابللغة العربية بطريقة تواصلية. 10 اخلالصة اخ الوعي بتعلم اللغة العربية لغري العرب على أساس االحتياجات املتنوعة . ،لذلك متت صياغة منوذج تعلم اللغة العربية القائم على اهلدف والذي يسمى تعلم اللغة العربية ألغراض خاصة .هذا التعلم موجه أكثر حنو تعلم اللغة العربية يف نطاق وسياق معينني، على سبيل املثال للعمل .تعلم اللغة العربية لغرض خاصة مربمج لتوجيه املتعلمني ليكونوا قادرين على التواصل حيث يتم وضعهم وفقًا الحتياجاهتم .هناك نوعان من األهداف يف هذه ،الدراسة ومها األهداف األكادميية واألهداف املهنية .تعلم اللغة العربية لألغراض األكادميية رمسيًا للطالب الذين ليسوا من جمال اللغة العربية .يدرسون اللغة العربية لدعم خرباهتم العلمية يف جماهلم ، مثل دراسة األدب العريب األصلي أو استخدام املصطلحات يف جمال معرفتهم .يتم تقدمي تعلم اللغة العربية لألغراض املهنية لدعم عمل ،الفرد مثل مرشدي احلج والعمرة لذين ا يتم توجيههم للتواصل ابللغة العربية. 92 \| Novita Rahmi, Albarra Sarbaini, J. Sutarjo, Nawang Wulandari املراجع Dahlan, Juwairiyah. Metode Belajar Mengajar Bahasa Arab. Surabaya: Al-Ikhlas, 1992. Dahlan, Juwairiyah. Metode Belajar Mengajar Bahasa Arab. Surabaya: Al-Ikhla 1992. Halim, Nur. “Bahasa Arab Dengan Tujuan Khusus Berbasis Komunikatif Wisata Travelling.” Bintang : Jurnal Pendidikan Dan Sains 2, no. 3 (2020): 230. https://ejournal.stitpn.ac.id/index.php/bintang/article/view/960. Makruf, Imam. Strategi Pembelajaran Bahasa Arab Aktif. Semarang: Need’s Press, 2009. Nisa, Mauidlotun, and Syamsul Arifin. “Bahasa Arab untuk Tujuan Khusus di Indonesia dan Malaysia: Tinjauan Normatif dan Empiris.” Buletin Al-Turas 26, no. 1 (2020): 37–53. https://doi.org/10.15408/bat.v26i1.13303. Setyabudi, Mohammad Arif, Syamsuddin, and Hanifuddin. “Pembelajaran Bahasa Arab Untuk Tujuan Khusus.” EL-FUSHA: Jurnal Bahasa Arab Dan Pendidikan 1, no. 1 (2020): 90–98. http://ejournal.unhasy.ac.id/index.php/alfusha/article/view/1220. Thu’aimah, Rusydi Ahmad. Ta’lim Al-Lughoh Li Aghrodh Khosoh: Mafahimah Wa Manhajiyah. Khartoum: Ma’had Khartoum Ad-Dauly, 2003. Wahab, Muhbib Abdul. Epistemologi dan Metodologi Pembelajaran Bahasa Arab. UIN Jakarta Press, 2019. https://repository.uinjkt.ac.id/dspace/handle/123456789/44533. Press, Zaenuddin, Radliyah. Metodologi & Strategi Alternatif Pembelajaran Bahasa Arab. Yogyakarta: Pustaka Rihlah Group, 2005. Zuhairini. Sejarah Pendidikan Islam. Jakarta: Bumi Aksara, 1997. AN NABIGHOH Vol. 24, No. 1 (2022) P-ISSN : 1907-1183 E-ISSN : 2581-2815 https://doi.org/10.32332/an-nabighoh.v24i1.4762
https://openalex.org/W4319348360	https://revistas.univalle.edu/index.php/jlsc/article/download/361/369	es		Course - Awareness Workshop on Climate Change: Expected learning results that each participant must acquire.	Journal of Latin American sciences and culture	2,022	cc-by	2,433	JLASC Journal of Latin American Sciences and Culture, 4(6) Journal of Latin American Sciences and Culture Opinion Article Curso - Taller de sensibilización frente al cambio climático: Resultados de aprendizaje esperados que debe adquirir cada participante Diego Fernando Chicaiza Burbano1 , Cristian Andrés Ruiz Terán², Alejandro Ocampo López³, Mary Luz Ojeda Solarte⁴ , José Gabriel Pérez Canencio5 1 2 3 4 5 Unidad Central del Valle del Cauca; diego.chicaiza01@uceva.edu.com Unidad Central del Valle del Cauca; cristian.ruiz03@uceva.edu.com Unidad Central del Valle del Cauca; alejandro.ocampo01@uceva.edu.com Unidad Central del Valle del Cauca; mojeda@uceva.edu.com Unidad Central del Valle del Cauca; jperez@uceva.edu.com Resumen: En algunas universidades del mundo se están impartiendo cátedras de cuidado del medio ambiente con especial interés en los temas de manejo de residuos sólidos y cuidado de los recursos naturales, sin embargo, hace falta complementar estas temáticas con acciones que los seres humanos podemos realizar para disminuir emisiones de gases efecto invernadero mediante las buenas prácticas tecnológicas verdes considerados en el Green It. Este fundamento especial lo ha tratado la ciencia como un valioso aporte para el cuidado del ambiente. Citation: Chicaiza, D. F., Ruiz, C. A., Ocampo, A., Ojeda, M. L., & Pérez, J. G. (2022). Curso – Taller de sensibilización frente al cambio climatico: Resultados de aprendizaje esperados que debe adquirir cada participante. Journal of Latin American Sciences and Culture, 4(6), 61-67. https:// doi.org/10.52428/27888991. v4i6.361 Received: October 8, 2022 Accepted: December 15, 2022 Published: December 28, 2022 Publisher’s Note: JLASC stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Submitted for open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/ by/4.0/). En una Universidad Colombiana se ha empezado a impartir la cátedra de Sensibilización frente al Cambio Climático con énfasis en las buenas prácticas tecnológicas verdes Green It y la eficiencia energética. Por ser un curso académico, se hace importante definir los resultados de aprendizaje esperados por quienes tomen este estudio, precisamente, este es el tema que se aborda en el presente texto al igual que la rúbrica para la evaluación de la asimilación de conocimiento. Palabras clave: Sensibilización; Green IT; Cambio climático; Competencias, Resultados de Aprendizaje, Rúbricas. 1. Introducción El auge de la tecnología trae consigo un gran impacto y con ello se ve el aumento de nuevas herramientas que nos permiten automatizar, controlar y administrar sistemas de información. No está mal mencionar que estas nuevas herramientas tecnológicas traen consigo problemas para el medio ambiente generando impactos como la huella de carbono, altos consumos energéticos, entre otros. Por ello, los fabricantes de hardware y software buscan optimizar procesos que permitan reducir los daños que se le causa al medio ambiente en el ciclo de vida de los productos, igualmente, se buscan estrategias como la utilización de sistemas de información los cuales nos permitan optimizar funciones en las empresas para disminuir el esfuerzo en 61 Journal of Latin American Sciences and Culture, 4(6) los procesos, la utilización de papel y optar por el procesamiento en la nube para la toma de decisiones y el almacenamiento de datos aplicando buenas prácticas verdes tecnológicas denominadas Green IT. Pensar la formación universitaria basada en un modelo orientado por competencias, hace necesario un cambio en el enfoque centrado en el profesor a uno centrado en el estudiante. Por lo tanto, la atención debe dirigirse a evidenciar aquellos aprendizajes que los alumnos deben ser capaces de hacer como resultado de un proceso formativo. En este escenario, el modelo orientado por competencias define que para dar cuenta de los desempeños alcanzados por los estudiantes, deben establecerse cierto número de resultados de aprendizaje que orienten los procesos de evaluación educativa (Parejo & Clemenza, 2022). Los resultados de aprendizaje son enunciados acerca de lo que se espera que el estudiante sea capaz de hacer, comprender y/o sea capaz de demostrar una vez terminado un proceso de aprendizaje (Ministerio de Educación Nacional, 2021) (Jerez Yáñez & others, 2012). Deben dar cuenta de la tridimensionalidad del saber: conceptual, procedimental y actitudinal; en este sentido, hablamos, por lo tanto, del desempeño de una competencia o de una porción de esa competencia (MasapantaCarrión & Velázquez-Iturbide, 2017). Actualmente en diversas instituciones de educación tanto media como superior se orientan cátedras dedicadas a enseñar a sus estudiantes sobre el cuidado del medio ambiente; sin embargo no se encuentran asignaturas cuyo contenido permita, exclusivamente, formar a los estudiantes tanto en su responsabilidad individual y colectiva con el medio ambiente y también con el compromiso de aplicar prácticas que por medio de la tecnología disminuyan los impactos que se están generando con las acciones diarias sobre el medio ambiente, los recursos naturales, la biodiversidad y el desarrollo sostenible de los pueblos. Una cátedra específica deberá de ir acompañada de sus aspectos metodológicos y epistemológicos que conduzcan tanto al docente como al estudiante a la obtención de resultados de aprendizaje enriquecidos por contenidos, temáticas, rubricas, cuyo fundamento se base en la aplicación de una taxonomía definida de manera institucional. He aquí la importancia de definir los resultados de aprendizaje que se espera que un individuo alcance al finalizar un curso en la temática de GREEN IT y Sensibilización Frente al Cambio Climático cuyo fundamento sea derivado de la ciencia ciudadana -Citizen Science (Fraisl et al., 2022). 2. Métodos. Una Aproximación al Micro currículo de Sensibilización Frente al Cambio Climático - GREEN IT En la Unidad Central del Valle del Cauca, UCEVA - Universidad pública de formación profesional ubicada en el Departamento del Valle del Cauca, Colombia, se está implementando la cátedra de 62 Journal of Latin American Sciences and Culture, 4(6) Sensibilización Frente al Cambio Climático con el propósito de que los estudiantes adquieran conocimientos en aspectos sociales, ambientales y económicos del desarrollo sostenible, la tecnología (Ambrosi, 2018), la eficiencia energética, los ODS, la huella de carbono, las cumbres por el cambio climático y las buenas prácticas verdes tecnológicas para reducir emisiones de gases efecto invernadero – Green IT, con especial referencia al curso de Green It que se oferta en otra Universidad Latinoamericana (Velasquez, 2019). Los temas principales que se han establecido para el curso en la Uceva, son: Tabla 1. Temas del curso Green It. Unidad 1 E Unidad 2 Unidad 3 Unidad 4 Unidad 5 Unidad 6 Unidad 7 Unidad 8 Unidad 9 Unidad 10 Unidad 11 Unidad 12 Unidad 13 l calentamiento global El cambio climático y la biodiversidad Gases de efecto invernadero Objetivos de desarrollo sostenible Huella de carbono de alcance 2 Matriz energética colombiana Consumo energético Proceso de Sensibilización Green It Mitigación Compensación Comprobación de conocimiento 2.1. Competencias y resultados de aprendizaje sugeridos para el currículo de Sensibilización Frente al Cambio Climático Las principales competencias que se esperan en un estudiante al finalizar el curso son: • Trabajar de manera individual y colectivamente en la búsqueda de soluciones para los problemas relacionados directamente con la administración eficiente del consumo energético por ser generador de emisiones de gases efecto invernadero. • Realizar análisis del entorno teniendo sensibilidad y conciencia sobre los problemas derivados del mal uso de la tecnología consumidora de energía eléctrica, actuando con mayor conciencia ambiental y tener uso eficiente del recurso energético por ser generador de emisiones de gases efecto invernadero. • Adquirir la capacidad para educar a otros en el tema del cuidado del medio ambiente y la aplicación de buenas prácticas tecnológicas verdes (Green IT) para disminuir emisiones de CO2 derivadas del consumo energético. 63 Journal of Latin American Sciences and Culture, 4(6) 3. Resultados esperados. Proceso de evaluación La implementación de la rúbrica se hace con el propósito de que las personas tengan una retroalimentación casi inmediata sobre las temáticas para acortar sustancialmente el retorno de información (Valverde Berrocoso & others, 2014). Tabla 2. Rúbrica para evaluar los resultados de aprendizaje Nivel de desempeño Criterio de Evaluación Reconocer Peso del Criterio 25% Relacionar Peso del Criterio 25% Peso del Criterio 25% 64 Adquirido Parcialmente Adquirido No Adquirido Reconoce los diferentes elementos y da solución a problemas básicos de ingeniería con soporte en el conocimiento sobre el calentamiento global, cambio climático, Gases de efecto invernadero, Reconoce, pero requiere de más documentación y apoyo para solución a problemas básicos de ingeniería con soporte en el conocimiento sobre el calentamiento global, cambio climático, Gases de efecto invernadero, No reconoce ni da a solución problemas básicos de ingeniería con soporte en el conocimiento sobre el calentamiento global, cambio climático, Gases de efecto invernadero, y otros aplicando técnicas de GREEN IT. Relaciona los diferentes elementos para dar solución a problemas básicos de ingeniería con soporte en el conocimiento sobre el calentamiento global, cambio climático, gases de efecto invernadero, matriz energética colombiana, Huella de carbono de alcance 2. aplicando técnicas de GREEN IT. I diferentes elementos y da solución a problemas básicos de ingeniería con soporte en el conocimiento sobre el calentamiento global, cambio climático, mitigación, aplicando técnicas de GREEN IT. aplicando técnicas de GREEN IT. Relaciona, pero requiere de más documentación y apoyo para solución a problemas básicos de ingeniería con soporte en el conocimiento sobre el calentamiento global, cambio climático, gases de efecto invernadero, matriz energética colombiana, huella de carbono de alcance 2. aplicando técnicas de GREEN IT. No relaciona ni da solución a problemas básicos de ingeniería con soporte en el conocimiento sobre el calentamiento global, cambio climático, Gases de efecto invernadero, matriz energética colombiana, huella de carbono de alcance 2. aplicando técnicas de GREEN IT. de más documentación y apoyo para solución a problemas básicos de ingeniería con soporte en el conocimiento sobre el calentamiento global, cambio climático, Mitigación, compensación, gases de solución a problemas básicos de ingeniería con soporte en el conocimiento sobre el calentamiento global, cambio climático, mitigación, Peso del Criterio 25% Crear Peso del Criterio 25% energética carbono de alcance 2. colombiana, huella carbono de colombiana, Huella aplicando técnicas de de Sciences and Culture, 4(6) alcance 2. aplicando de carbono de alcance GREEN IT.Journal of Latin American técnicas de GREEN 2. aplicando técnicas IT. de GREEN IT. I diferentes elementos de más documentación solución a y da solución a y apoyo para solución a problemas básicos problemas básicos de problemas básicos de de ingeniería con ingeniería con ingeniería con soporte soporte en el soporte en el en el conocimiento sobre conocimiento sobre conocimiento sobre el el calentamiento global, el calentamiento calentamiento global, cambio climático, global, cambio cambio climático, Mitigación, climático, mitigación, compensación, gases de mitigación, compensación, gases efecto invernadero, compensación, de efecto matriz energética gases de efecto invernadero, matriz colombiana, huella de invernadero, matriz energética carbono de alcance 2. energética colombiana, Huella aplicando técnicas de colombiana, huella de carbono de alcance GREEN IT. de carbono de 2. aplicando técnicas alcance 2. aplicando de GREEN IT. técnicas de GREEN IT. Crea los diferentes Crea, pero requiere de No crea ni da elementos y da más documentación, solución a solución a problemas apoyo y entrenamiento problemas básicos básicos de ingeniería para solución a de ingeniería con el de forma práctica con problemas básicos de soporte en soporte en el ingeniería con soporte conocimiento sobre calentamiento conocimiento sobre el en el conocimiento sobre el cambio calentamiento global, el calentamiento global, global, cambio climático, cambio climático, climático, mitigación, mitigación, mitigación, compensación, gases compensación, gases de compensación, de efecto efecto invernadero, gases de efecto invernadero, matriz matriz energética invernadero, matriz energética colombiana, huella de energética colombiana, huella de carbono de alcance 2. colombiana, huella carbono carbono de alcance 2. aplicando técnicas de de de aplicando técnicas de GREEN IT. alcance 2. aplicando GREEN IT. técnicas de GREEN IT. Los resultados de aprendizaje se lleven a una calificación individual. Se calificarán de acuerdo con un nivel de desempeño, así: 65 Journal of Latin American Sciences and Culture, 4(6) Tabla 3. Escala de calificación de los resultados de aprendizaje De esta manera, se espera que, al finalizar el curso, cada estudiante alcance niveles de desempeño altos y sea capaz de transferir conocimiento a las comunidades en las cuales se encuentre inmerso. Este curso hará parte del programa Ingeniería de Sistemas que se ofrece en la Unidad Central del Valle del Cauca – Uceva. Quedará incluido en el área de las asignaturas de humanidades. Como ya se mencionó, se incluye la taxonomía de Bloom en la construcción de los resultados de aprendizaje. Esto no constituye un estándar sino el modelo de una nueva asignatura para la Universidad. 4. Conclusiones Hace mucho tiempo que la tecnología de la información es parte de nuestra vida, teniendo un crecimiento exponencial en su evolución, satisfaciendo nuestras necesidades, pero así mismo, ocasionando más daño a nuestro medio ambiente aumentando la huella de carbono que se genera día a día. Es nuestro deber tener conciencia ambiental y empezar a aplicar buenas prácticas de GREEN IT para disminuir el impacto generado por las emisiones de CO2, precisamente un curso de este tema nos puede aportar la información necesaria para lograrlo. El aprendizaje sobre sensibilización frente al cambio climático debería ser de vital importancia es muchas universidades, para brindar una perspectiva a la ciudadanía sobre las consecuencias que podremos tener a futuro si no decidimos realizar cambios en nuestros hábitos sobre el uso energético y otras acciones que empeoran nuestro medio ambiente, así mismo, mostrando la manera en la que debemos actuar frente a esta problemática y aplicarla desde nuestros hogares, sitios de trabajo, espacios públicos o cualquier lugar del planeta. 66 Journal of Latin American Sciences and Culture, 4(6) Referencias Ambrosi, V. M. (2018). Informe de personal de apoyo: Ambrosi, Viviana Miriam (2017-2018). https://digital.cic.gba.gob.ar/ handle/11746/8828 Velasquez, A. (2019). PLAN DE ASIGNATURA Y PLAN DE UNIDAD DE APRENDIZAJE CAMBIO CLIMATICO. Fraisl, D., Hager, G., Bedessem, B., Gold, M., Hsing, P.-Y., Danielsen, F., Hitchcock, C. B., Hulbert, J. M., Piera, J., Spiers, H., & others. (2022). Citizen science in environmental and ecological sciences. Nature Reviews Methods Primers, 2(1), 1-20. DOI https://doi.org/10.1038/s43586-022-00144-4 Jerez Yáñez, Ó. & others. (2012). Los resultados de aprendizaje en la Educación Superior por competencias. Granada: Universidad de Granada. Masapanta-Carrión, S., & Velázquez-Iturbide, J. Á. (2017). Una revisión sistemática del uso de la taxonomía de Bloom en la enseñanza de la informática. Atas do XIX Simpósio Internacional de Informática Educativa e VIII Encontro do CIED–III Encontro Internacional, 294-299. Ministerio de Educación Nacional, C. (2021). ¿Cómo formular e implementar los resultados de aprendizaje? Mineducación. Parejo, N. F. H., & Clemenza, C. (2022). Evaluación de los aprendizajes por competencias: Una mirada teórica desde el contexto colombiano. Revista de ciencias sociales, 28(1), 106-122. Valverde Berrocoso, J. & others. (2014). El uso de e-rúbricas para la evaluación de competencias en estudiantes universitarios: Estudio sobre fiabilidad del instrumento. Red U: revista de docencia universitaria. DOI https://doi.org/10.4995/ redu.2014.6415 67
https://openalex.org/W2899017358	https://www.biogeosciences.net/16/1685/2019/bg-16-1685-2019.pdf	English	null	Grazing-related nitrous oxide emissions: from patch scale to field scale	Biogeosciences	2,019	cc-by	17,236	Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale Received: 2 October 2018 – Discussion started: 29 October 2018 Revised: 22 February 2019 – Accepted: 15 March 2019 – Published: 25 April 2019 system M and 0.83 ± 0.29 % for system G. These EF val- ues were thus signiﬁcantly smaller compared to the default EF of 2 % provided by the IPCC guidelines for cattle excreta deposited on pasture. The measurements showed that urine patch emission dominated the ﬁeld-scale ﬂuxes (57 %), fol- lowed by signiﬁcant background emissions (38 %), and only a small contribution of dung patch emission (5 %). The re- sulting source-speciﬁc EFs exhibited a clear difference be- tween urine (1.12 ± 0.43 %) and dung (0.16 ± 0.06 %), sup- porting a disaggregation of the grazing-related EFs by exc- reta type in emission inventories. The study also highlights the advantage of a N-optimized diet, which resulted in re- duced N2O emissions from animal excreta. Abstract. Grazed pastures are strong sources of the green- house gas nitrous oxide (N2O). The quantiﬁcation of N2O emissions is challenging due to the strong spatial and tem- poral variabilities of the emission sources and so N2O emis- sion estimates are very uncertain. This study presents N2O emission measurements from two grazing systems in western Switzerland over the grazing season of 2016. The 12 dairy cows of each herd were kept in an intensive rotational graz- ing management. The diet for the two herds of cows con- sisted of different protein-to-energy ratios (system G: grass only diet; system M: grass with additional maize silage) re- sulting in different nitrogen (N) excretion rates. The N in the excretion was estimated by calculating the animal nitrogen budget taking into account the measurements of feed intake, milk yield, and body weight of the cow herds. Directly after the rotational grazing phases, background and urine patches were identiﬁed based on soil electric conductivity measure- ments while fresh dung patches were identiﬁed visually. The magnitude and temporal pattern of these different emission sources were measured with a fast-box (FB) chamber and the ﬁeld-scale ﬂuxes were quantiﬁed using two eddy covariance (EC) systems. The FB measurements were ﬁnally upscaled to the ﬁeld level and compared to the EC measurements for quality control by using EC footprint estimates of a back- ward Lagrangian stochastic dispersion model. The compari- son between the two grazing systems was performed during emission periods that were not inﬂuenced by fertilizer appli- cations. Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale This allowed the calculation of the excreta-related N2O emissions per cow and grazing hour and resulted in con- siderably higher emissions for system G compared to system M. Relating the found emissions to the excreta N resulted in excreta-related emission factors (EFs) of 0.74 ± 0.26 % for 1 Introduction Nitrous oxide (N2O) is a strong greenhouse gas (GHG) with a 265 times stronger warming potential compared to CO2 on a mass basis (IPCC, 2014). Typically an inert gas in the tro- posphere, N2O has a strong potential to destroy the ozone layer in the stratosphere (Portmann et al., 2012). The largest share of N2O emissions are attributed to nitrogen (N) fertil- ization in the agricultural sector, but livestock grazing, espe- cially by cows, can also lead to signiﬁcant direct and indi- rect N2O emissions due to excreta from the animals (Luo et al., 2017; Reay et al., 2012). The nitrogen deposited by ani- mal excreta often exceeds the N applied by fertilizer (Aarons et al., 2017). The available reactive N is used by micro- bial nitriﬁcation and denitriﬁcation processes where signif- icant amounts of N2O can be produced (Selbie et al., 2015). A nonlinear response of N2O emissions to N loading has Biogeosciences, 16, 1685–1703, 2019 https://doi.org/10.5194/bg-16-1685-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Biogeosciences, 16, 1685–1703, 2019 https://doi.org/10.5194/bg-16-1685-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Published by Copernicus Publications on behalf of the European Geosciences Union. ublished by Copernicus Publications on behalf of the European Geosciences Union. K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld sca K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1686 been shown previously (Cardenas et al., 2010), and urine patches of cattle have exceptionally high N loading rates (up to 2000 kg N ha−1), making them especially prone to high N2O losses (Selbie et al., 2015). were often performed on manually applied urine and dung patches (Bell et al., 2015; Cai and Akiyama, 2016). Addition- ally, due to the strong heterogeneity of the emissions from a pasture (Cowan et al., 2015; Flechard et al., 2007) cham- ber techniques are not ideal to compute ﬁeld-scale emis- sions for grazing systems. The eddy covariance (EC) method overcomes this problem by integrating over multiple emis- sion sources over a larger spatial domain. The EC technique was already applied successfully to quantify N2O emissions from pastures and grasslands (Jones et al., 2011). Some stud- ies also tried to compare different systems (e.g., intensive – extensive, different crops, land/lake) with one EC tower (e.g., Biermann et al., 2014; Fuchs et al., 2018) by partition- ing the ﬂuxes based on wind direction and system geometry, but typically one tower for each system is preferable. In or- der to understand and quantify the emissions of a pasture, the combined approach of EC measurements and chambers is regarded as the best solution (Cowan et al., 2015). The EC systems can be used to quantify the ﬁeld-scale emissions while the chamber approach can be used to estimate the con- tributions from single emission sources (urine patches, dung patches, and other “background” areas). 2 ( ) For inventories and live cycle assessments, the magnitude of the N2O emissions is usually calculated by applying emis- sion factors (EFs) related to the magnitude of N inputs to the agricultural ﬁelds (EF = emitted N2O-N/N input). Accord- ing to the guidelines of the Intergovernmental Panel on Cli- mate Change (IPCC, 2006) for national emission reporting, a separation is made between (i) emissions related to excreta N deposited by the grazing animals and (ii) emissions related to fertilizer applications and other N inputs. 2 Material and methods The efﬁcient use of fed N is essential to reduce the emis- sions associated with animal excreta. Studies have shown that an optimized feeding strategy can lead to less N excreted by the animals (e.g., Arriaga et al., 2010; Dijkstra et al., 2013; Yan et al., 2006). For this purpose, forage with a low N con- tent (e.g., maize) can be used as a supplement to N-rich grass and this subsequently leads to less N in the excreta, mainly in the form of less urine N. A lower amount of N input to the pasture is supposed to produce less N2O emissions, but corresponding emission experiments under real grazing con- ditions for a full season, to our knowledge, have not been reported hitherto. Published by Copernicus Publications on behalf of the European Geosciences Union. While for fertilizer- induced N2O emissions, a default value of 1 % is proposed by IPCC (2006), the default EF related to excreta of grazing cattle (denoted as EF3PRP,CPP) is 2 %. Most countries includ- ing Switzerland presently use these default values. However, the default EF3PRP,CPP value often overestimates observed pasture emissions (Bell et al., 2015; Chadwick et al., 2018) and does not take into account country-speciﬁc conditions (climate, soil, management). Therefore, some countries have developed a country-speciﬁc EF (e.g., New Zealand; Saggar et al., 2015) which is still lacking for Switzerland. Addition- ally, it has been shown that separate EFs for urine and dung might be beneﬁcial in describing the emissions and under- standing the contributions of the different emission sources on a pasture (Bell et al., 2015). A better understanding of the individual contributions would also be very helpful to reduce the emissions, as dietary changes, for example, typically af- fect the excreted urine N, which is mainly responsible for the high N2O emission associated with excreta (Dijkstra et al., 2013). However, the range and thus the uncertainty of spe- ciﬁc urine EFs is rather large (0 %–14 %, n = 40) as shown by Selbie et al. (2015) based on a survey of literature reports. Many of those studies measured the emissions on artiﬁcially applied urine or under laboratory conditions, making these results questionable with regard to the applicability within greenhouse gas inventories. In our experiment, we measured N2O emissions from two neighboring pastures simultaneously with the EC method over a full grazing season. The two pastures differed in the energy-to-protein balance of the cows’ diet. The small-scale ﬂuxes were quantiﬁed with a fast-box chamber and upscaled to match the EC ﬂux footprints for comparison. Further on, we computed the contribution of the different emission sources to the overall pasture emissions. The results were compared to default values provided by IPCC and other liter- ature values. The main goal of the study was to quantify the excreta-related emission and the corresponding EF for real grazing systems and to analyze the speciﬁc contributions of dung and urine patches. K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1687 Table 1. Near-surface soil parameters (5–10 cm depth) averaged over four locations on the pasture. The measurements are given as mean ±1 standard deviation. Table 2. Measured averages ± standard deviation of observed cow properties (ECM: energy corrected milk) and feed protein contents used by the dairy cow nitrogen budget approach for both pasture systems during the grazing season 2016. Parameter Value Pore volume (%) 57 ± 4 Bulk density (g cm−3) 1.09 ± 0.11 pH (–) 6.0 ± 0.3 Sand (%) 42.6 ± 2.5 Clay (%) 18.7 ± 1.7 Silt (%) 33.0 ± 1.3 Soil organic matter (%) 5.7 ± 0.3 Total N (%)∗ 0.38 ± 0.03 Total C (%)∗ 3.76 ± 0.20 ∗Measured at a depth of 0–10 cm. Input parameter (units) System M System G Number of cows 12 12 Milk yield, ECM (kg cow−1 day−1) 25.1 ± 2.9 24.2 ± 3.7 Animal weight (kg) 633 ± 14 633 ± 10 Grass crude protein (g kg-DM−1) 195 ± 23 196 ± 23 Maize crude protein (g kg-DM−1) 84 ± 8 n/a n/a – not applicable Input parameter (units) System M System G Number of cows 12 12 Milk yield, ECM (kg cow−1 day−1) 25.1 ± 2.9 24.2 ± 3.7 Animal weight (kg) 633 ± 14 633 ± 10 Grass crude protein (g kg-DM−1) 195 ± 23 196 ± 23 Maize crude protein (g kg-DM−1) 84 ± 8 n/a n/a – not applicable ∗Measured at a depth of 0–10 cm. spent a longer time in the barn during daytime (up to 6 h; see Fig. 2c) mainly due to high air temperatures and to a minor degree to additional experiments of other research groups. Heavy rain events in June led to very wet soil conditions, which prevented grazing between 8 June and 4 July and ne- cessitated a grass cut on 22 and 27 June (Fig. 2c). times between May and September). After the last renova- tion treatment in 2007 the ﬁeld had been used as an inten- sive pasture for cattle grazing with occasional grass cuts for maintaining a homogenous sward. In addition to the N input through excreta from the grazing animals, N had been ap- plied through fertilizer at a rate of about 120 kg N ha−1 per year between 2007 and 2015. 2.3 N input to the pasture During the grazing season, N input to the pasture mainly occurred in the form of excreta of the grazing animals and to a lesser extent as mineral fertilizer (Fig. 2d). The min- eral fertilizer was ammonium nitrate (28 kg ha−1) applied at the end of June and urea (42 kg ha−1) with a split applica- tion between the middle of August (western paddocks X.11– X.16) and early September (eastern paddocks X.21–X.25) due to concurrent grazing. In the present study we focus on the N input by grazing excreta and their effect on N2O emis- sions. The comparison between the ﬁeld-scale EC method and the small-scale chamber measurements also required es- timates of the number of dung and urine patches on the pas- ture. These numbers were calculated as described in Sect. 2.7 based on the excreted N amounts. N excretion cannot easily be measured in the ﬁeld, but it can be calculated based on the energy demand of the cows and measured N in feeds and products (e.g., milk, body weight gain). We followed the ap- proach described by Felber et al. (2016) to calculate the en- ergy and N ﬂows of the dairy cows in the experiment and to calculate daily values of excreted N per cow. Input param- eters to the budget calculation were daily measurements of milk yield, milk N content, and body weight gain as well as seasonal measurements of protein content of the grass (eight times between end of April and end of September) and of the maize silage (three times between beginning of May and be- ginning of September). The breakdown of the excreted N in urine and dung was based on work by Bracher et al. (2011). For further details see Voglmeier et al. (2018), where the cor- responding uncertainty of the total N and urine/dung N was estimated to be 15 % (2σ) for the same experiment. Seasonal statistics of the input variables are given in Table 2. 2.1 Experimental site The experiment was conducted at the research farm Agro- scope Posieux in the Prealps of Switzerland in the canton of Fribourg (46◦46′04′′ N, 7◦06′28′′ E) during the grazing season of 2016 and has already been described in detail by Voglmeier et al. (2018). The farm is located at an elevation of 642 m with an annual average temperature of 8.7 ◦C and a mean annual precipitation sum of 1075 mm (MeteoSwiss, 2018). The soil consisted mainly of a stagnic Anthrosol with a loamy texture (see Table 1). Soil proﬁle samples for anal- ysis of texture and other soil characteristics were taken at four locations on the pasture in 2013 and 2016. The vege- tation consisted of a grass–clover mixture typical for Swiss pastures (78 ± 12 % grasses and 15 ± 10 % legumes; main species: Lolium perenne and Trifolium repens, 10 sampling Historically, most studies used static chambers to quan- tify N2O emissions (Flechard et al., 2007). Chamber mea- surements are ideal to quantify emissions on a small spatial scale and to attribute the measured ﬂuxes to certain emis- sion drivers, but for excreta emissions these measurements Biogeosciences, 16, 1685–1703, 2019 www.biogeosciences.net/16/1685/2019/ 2.2 Experimental design The experiment took place at a 5.5 ha pasture, which was di- vided into two separate systems differing in feeding strat- egy of the 12 cows per system (Fig. 1a). The northern sys- tem (system M) represented a N-optimized feeding option where the diet of the cows consisted of grass with additional maize silage (roughly 20 % of the dry matter intake, DMI, fed in barn during milking periods) resulting in a demand- optimized protein content in the diet (Arriaga et al., 2010; Yan et al., 2006). This was supposed to reduce the excreta N input to the pasture. The southern system (system G) repre- sented a full grazing regime with no additional forage, which resulted in a considerable protein surplus (see Table 2). Both systems were managed as a rotational grazing system with 11 paddocks (Fig. 1a) resulting in a typical rotation period of about 20 days. The size of the paddocks was adjusted for the different feeding strategies and resulted in typical sizes of 1700 m2 for system M and 2200 m2 for system G. The rotation of both systems was managed synchronously with a new rotation starting on the westerly paddocks (X.11 to X.16 with X indicating both systems) followed by the east- erly ones (X.21 to X.25). Grazing on the paddocks started with intermittent grazing phases in March and ended in early November with the main grazing season being between the end of April and early Oc- tober. During this time period eight full rotations took place. The cows typically spent 18 to 20 h per day on the pasture and were brought to the barn twice a day (around 05:00 and 17:00 LT) for milking. However, in July and August the cows Biogeosciences, 16, 1685–1703, 2019 2.4.1 Excreta detection Small-scale emissions from urine and dung patches as well as background pasture areas were measured with a fast-box (FB) chamber (Hensen et al., 2006). The measurements took place on the paddocks X.11 and X.21 (Fig. 1a) between the beginning of July and middle of October and were therefore taken mainly during dry soil conditions (Fig. 2a, periods with VWC < 0.4). Measurements usually started after the excre- tion detection (Sect. 2.4.1) and about 1–2 days after the end of grazing (EOG). The age of the excreta patches is impor- tant for the interpretation of the measured ﬂuxes. However, the exact determination of the excreta age was not possible. Thus, the time since EOG was used as excreta age for each FB measurement. The potential age variability of a single excreta patch resulted from the sojourn time of the cows on the paddock, which typically was in the range of 1–1.5 days. h ll d 5 b The localization of fresh dung and urine patches was es- sential in this study to measure N2O emissions attributable to speciﬁc excreta sources. Intensive observation areas of 10 m × 10 m or 15 m × 15 m close to both EC towers in the paddocks X.11 and X.21, respectively (see Fig. 1a) were se- lected. Within these areas fresh dung and urine patches were mapped typically 1–3 days after grazing of the respective paddock. Dung pats were mapped visually and labeled for subsequent chamber measurements. For urine patches a di- rect visual identiﬁcation was not possible. Bates et al. (2015) demonstrated the ability of surface-soil electrical conduc- tivity measurements to detect urine patches. Using this ap- proach we mounted a soil probe (GS3, Meter Group, US; for soil moisture, temperature, and electrical conductivity mea- surements) on a handheld stick and mapped the intensive observation area on a 25 cm grid (Fig. 3). Based on pre- experimental tests, areas with conductivity values below a threshold of 0.15 mS cm−1 (dark blue areas in Fig. 3a) were considered background without recent inﬂuence of excreta. Spots with a conductivity above the threshold were marked as possible urine patches for the chamber measurements. Time series of electrical conductivity measurements (Fig. 3b) on manually applied urine patches in 2017 illustrate the long- term effect and demonstrate the possibility to distinguish be- tween background areas and urine patches more than 10 days after the application of urine. K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1688 Figure 1. (a) Measurement site with the pastures for the two herds (blue: grass diet with additional maize silage; green: full grazing regime; grey: optional pasture areas) and the division into the paddocks (M.11–M.25, G.11–G.25). Additionally the location of the two EC towers (triangles) and the area of the chamber measurements (red dashed rectangles) are shown. (b) Wind distribution for the northern sonic anemometer with the corresponding sector contributions (black dotted circles) for the period May–October 2016. The areas A and B indicate wind sectors from which advection from nearby farm building can occur. The wind distribution was overlaid on a Google Earth image of the experimental area (map data: Google, DigitalGlobe). Figure 1. (a) Measurement site with the pastures for the two herds (blue: grass diet with additional maize silage; green: full grazing regime; grey: optional pasture areas) and the division into the paddocks (M.11–M.25, G.11–G.25). Additionally the location of the two EC towers (triangles) and the area of the chamber measurements (red dashed rectangles) are shown. (b) Wind distribution for the northern sonic anemometer with the corresponding sector contributions (black dotted circles) for the period May–October 2016. The areas A and B indicate wind sectors from which advection from nearby farm building can occur. The wind distribution was overlaid on a Google Earth image of the experimental area (map data: Google, DigitalGlobe). www.biogeosciences.net/16/1685/2019/ www.biogeosciences.net/16/1685/2019/ Biogeosciences, 16, 1685–1703, 2019 2.4.1 Excreta detection The manually operated opaque 0.8 m × 0.8 m × 0.5 m box was connected to a fast response quantum cascade laser analyzer (QCL, Aerodyne Research Inc.) that was also used for the EC system on the respective ﬁeld (see below Sect. 2.5.1). The sample air was drawn continuously from the FB headspace through a 40 m 1/4′′ polyamide (PA) tube to the analyzer, allowing measurements within a radius of about 35 m on the paddocks X.11 and X.21 (see Fig. 1). The sample ﬂow rate Q was typically around 8 L min−1. The box was modiﬁed by using a deﬁned vent to ambient air through a tube of 4 cm in diameter and 1 m in length. The inlet of the vent tube was packed with a foam material over a length of 10 cm to avoid uncontrolled air exchange due to wind-induced pressure ﬂuctuations. The chamber was also equipped with a GMP343 CO2 probe (Vaisala, FI) to mea- Biogeosciences, 16, 1685–1703, 2019 www.biogeosciences.net/16/1685/2019/ K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1 Figure 2. Time series of (a) daily averaged soil temperature and moisture at a depth of 5 cm measured at system M (solid lines) and sp 1689 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1689 Figure 2. Time series of (a) daily averaged soil temperature and moisture at a depth of 5 cm measured at system M (solid lines) and spread of the four measurement locations, (b) daily air temperature at 2 m above ground and precipitation at the measurement site, and (c) grazing duration on the single paddocks of the pasture (X: both pasture systems M and G) for the study year 2016. The dashed vertical orange lines indicate the harvest event (split between X.11–X.16 and X.21–X.25) (d) N input to system M during the main grazing season in 2016. Fertilizer was applied two times (vertical red lines). The second application (dashed lines) in August was split in two parts due to concurrent rotational grazing. The grey shaded areas indicate time periods inﬂuenced by fertilization or harvest events as explained in Sect. 2.5.2. Figure 2. 2.4.1 Excreta detection Time series of (a) daily averaged soil temperature and moisture at a depth of 5 cm measured at system M (solid lines) and spread of the four measurement locations, (b) daily air temperature at 2 m above ground and precipitation at the measurement site, and (c) grazing duration on the single paddocks of the pasture (X: both pasture systems M and G) for the study year 2016. The dashed vertical orange lines indicate the harvest event (split between X.11–X.16 and X.21–X.25) (d) N input to system M during the main grazing season in 2016. Fertilizer was applied two times (vertical red lines). The second application (dashed lines) in August was split in two parts due to concurrent rotational grazing. The grey shaded areas indicate time periods inﬂuenced by fertilization or harvest events as explained in Sect. 2.5.2. sure the soil respiration, which was used for quality control purposes (Sect. 2.4.3). The increase in N2O concentration af- ter placing the chamber on the soil with a ﬂux FCham was recorded every 3 s for a time period of about 90 s (taking into account the time delay due to tube sampling). The inﬂow of the background concentration Cbg into the chamber volume V (with area A) through the vent led to lower measured con- centration values C. This can be described by the following differential equation for the chamber headspace concentra- tion C(t): C (t) = A · FCham Q 1 −e−Q V ·t + Cbg. (1b) (1b) For small values of the exponent Q/V ·t (slow chamber vol- ume exchange of about 40 min and short measurement time) as characteristic for the present fast-box measurements, the entire bracket term can be linearized with a series expansion to (Q/V · t). Inserting the resulting function for C(t) into Eq. (1a) yields For small values of the exponent Q/V ·t (slow chamber vol- ume exchange of about 40 min and short measurement time) as characteristic for the present fast-box measurements, the entire bracket term can be linearized with a series expansion to (Q/V · t). Inserting the resulting function for C(t) into Eq. (1a) yields V δC δt = A · FCham 1 + Q V t . (1c) V δC δt = A · FCham −Q C −Cbg . 2.4.1 Excreta detection (1a) (1a) (1c) This is a combination of the two equations for static cham- bers (right-hand term = 0) and for the dynamic chamber (left- hand term = 0). Solving of the equation yields the explicit time function This is a combination of the two equations for static cham- bers (right-hand term = 0) and for the dynamic chamber (left- hand term = 0). Solving of the equation yields the explicit time function With the FB dimensions and sampling ﬂow rate as given above and a maximum accumulation time t ≤2 min, the de- viation from the ideal linear increase in a fully closed static chamber was ≤5 %. The ﬂux was ﬁnally calculated by using 2.5.2 Flux calculation A customized program written in the statistical software R (R Core Team, 2016) was used to calculate EC ﬂuxes for 30 min intervals (similar to Felber, 2015, and Felber et al., 2015). The program is based on Ammann et al. (2006, 2007). In a ﬁrst step, 10 Hz data outside a plausible physical range were identiﬁed and replaced by a running mean ﬁlter with a window size of 500 data points. In a next step, wind vector components were rotated into the mean wind direction using the double coordinate rotation technique (Kaimal and Finni- gan, 1994), and concentration values were subject to linear detrending within an averaging interval of 5 min. For a direct comparison of the FB measurements on the two pasture systems, the ﬂuxes obtained on the same day were ordered based on their magnitude for each system and source class. Due to the synchronous grazing regime, the ﬂuxes represented the same excreta age (e.g., on day 3 af- ter EOG). However, synchronous FB measurements on both systems were not always performed. Resulting numbers of data pairs are 46, 54, and 40 for background, urine, and dung ﬂuxes, respectively. The EC ﬂux is deﬁned as the covariance of the vertical wind speed and the trace gas mixing ratio. Due to the long inlet tube the time series of the trace gas signals are delayed in relation to the wind measurements by a quasi-constant lag time of about 6 s for system M and 7 s for system G. Thus, the trace gas signals have to be shifted to obtain the correct covariance ﬂux (Langford et al., 2015). In a pre-evaluation, the “default lag” was determined as the most frequent posi- tion of the maximum absolute value of the cross-covariance function over periods of weeks to months (depending on in- strument maintenance). Then it was checked for each half- hour period whether the individual “dynamic” lag was within K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1690 the HMR package (Pedersen et al., 2010), which uses linear and nonlinear regression to ﬁt the measured concentration values. The uncertainty of an individual box measurement is estimated to be around 20 % (Hensen et al., 2006). abled a good footprint coverage (Fig. 4, Sect. 2.5.4) of both ﬁelds and allowed the measurement of ﬁeld-scale ﬂuxes of both systems. The two EC systems were identically equipped with an ul- trasonic anemometer–thermometer (further on named sonic, HS-50, Gill Instruments Ltd., UK) to quantify the turbu- lent mixing by measuring the three-dimensional wind ve- locity (u,v,w) and air temperature. Dry air mixing ratios of N2O were measured with closed-path quantum cascade laser spectrometers (QCL, QC-TILDAS, Aerodyne Research Inc.) that analyzed air samples drawn through a 25 m PA tube (inner diameter 6 mm) by a vacuum pump (Bluffton Mo- tor Works, ﬂow rate ca. 13 L min−1). One ﬁlter at the inlet (AcroPak, Pall Corporation, 0.2 µm) and one before the in- strument (Midisart 2000, Sartorius Stedim Biotech GmbH, 0.2 µm) were used for each system to ﬁlter out particles. The distance of the inlets of the QCL from the center of the sonic head were around 20 cm and the QCL instruments were placed in a temperature-controlled environment (trailer at system M, shelter at system G) about 20 m north (system M) or south (system G) of the EC towers. In order to relate the measured ﬂuxes to environmental driving parameters the following sensors were placed inside on the chamber: a thermocouple (type K) for air temper- ature measurement within the chamber, a GS3 probe (see Sect. 2.4.1) for soil moisture, soil temperature, and soil con- ductivity measurements (ca. 0–5 cm depth), and an ML3 ThetaProbe (Delta-T Devices Ltd., UK) for soil moisture and soil temperature observations (ca. 0–10 cm depth). All mea- sured data values were stored on a data logger mounted on top of the box and transferred to a computer in the nearby shelter or trailer. A customized LabVIEW (National Instru- ments, US) program allowed for online inspection of all mea- sured data values including the gas concentrations. 2.4.3 Quality control and system comparison FB ﬂuxes were selected for post-processing after fulﬁlling certain quality criteria. In a ﬁrst step, the R2 value of any ﬂux calculation had to exceed 0.9 (e.g., for N2O ﬂux the R2 value of N2O, CH4, or CO2 had to exceed 0.9). For urine patches, the soil conductivity had to exceed 0.25 mS cm−1 at the be- ginning of the measurements (see also Fig. 3b) in order to ex- clude possible old urine patches (of previous management ro- tations). Presumable old patches were therefore rejected for further processing. Background ﬂuxes were removed from further processing if the ﬂux value exceeded 40 µg m−2 h−1 (= 4×median value) to ensure that undetected urine patches at the chamber surroundings did not inﬂuence the ﬂux mea- surements. Finally, 360 and 293 ﬂux measurements met the criteria on systems M and G, respectively. These measure- ments were composed of 238 background ﬂuxes, 242 urine patch ﬂuxes, and 173 dung ﬂuxes. The sample frequency of the EC system was 10 Hz. A cus- tomized LabVIEW (National Instruments, US) program was used to combine the data strings of the individual instruments and store them as binary raw data for ofﬂine analyses. Addi- tionally the program visualized the measurements and ﬂuxes of the N2O concentrations and ﬂuxes, calculated with an on- line ﬂux calculation. The program also allowed us to check the EC system by remote access. www.biogeosciences.net/16/1685/2019/ Biogeosciences, 16, 1685–1703, 2019 Biogeosciences, 16, 1685–1703, 2019 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1691 1691 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1691 Figure 3. (a) Measured conductivity within a quadratic 15 m × 15 m intensive observation area on the 3 October 2016 in system G. High val- ues (> 0.15 mS cm−1) indicate urine patch locations and brown crosses indicate observed dung pats. (b) Conductivity measured continuously during a ﬁeld experiment in 2017 with four GS3 sensors. Figure 3. (a) Measured conductivity within a quadratic 15 m × 15 m intensive observation area on the 3 October 2016 in system G. High val- ues (> 0.15 mS cm−1) indicate urine patch locations and brown crosses indicate observed dung pats. (b) Conductivity measured continuously during a ﬁeld experiment in 2017 with four GS3 sensors. Figure 4. Footprint climatology for both EC towers averaged for the time period between 15 March and 15 November 2016. The legend values indicate the percentage of the total footprint weight. were quantiﬁed using the “ogive” method where the damp- ing factor was calculated by ﬁtting the normalized cumula- tive co-spectrum of N2O to the one of the sensible heat at a frequency of 0.065 Hz. In a post-processing step, these half- hourly damping factors were ﬁltered for favorable conditions e.g., low noise level of the ogive and the ﬂux. The selected values were used to compute a damping function dependent on wind speed and stability which was ﬁnally used to es- timate the damping factor. Depending mainly on the wind speed, a damping effect of 10 %–30 % was found and cor- rected for. EC ﬂuxes were measured continuously over the grazing season. Since the present study is focused on N2O emissions from grazing, time periods with strong inﬂuence of N2O emissions from fertilization and harvest events (see Fig. 2c– d) were excluded for computation of cumulative emissions and for comparisons between ﬁeld-scale and small-scale measurements. These exclusion periods were limited to the 15 days following fertilization or harvest and led to a rejec- tion of 47 days during the grazing season. The criterion is based on observed EC ﬂuxes (Sect. 3.1) and is in accordance with Jones et al. (2011). K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale The time period used for calculation of the cumulative grazing emissions is further on deﬁned as grazing-only periods (GOPs) and accumulated to 198 days. Figure 4. Footprint climatology for both EC towers averaged for the time period between 15 March and 15 November 2016. The legend values indicate the percentage of the total footprint weight. 2.5.3 Quality control and gap ﬁlling a time window of 0.61 s around the default lag. If this was the case, the dynamic lag was used, otherwise the default lag was used. In order to minimize the effect of nonstationarities in the time series, the 30 min ﬂux was ﬁnally calculated as the average over six 5 min subinterval ﬂux values. This caused a minor low-frequency spectral loss (1 %–5 %) that was quan- tiﬁed (and corrected for) using Kaimal cospectra and the the- oretical transfer function for block averaging. EC ﬂux measurements are subject to different sources of measurement problems and quality issues, which often re- sult in data loss or data rejection. These sources can be in- strument speciﬁc like power failures or malfunctioning, en- vironmental driven like measurements under nonideal condi- tions (e.g., low turbulence), or a combination of both (Papale, 2012). Power outage, instrument maintenance (only on sys- tem M), and delayed installation (only on system G) led to data losses during the GOP of 12 % and 17 % for systems M and G, respectively. Data rejection due to low friction veloc- ity (u∗< 0.07 m s−1) and large vertical tilt angle (−2 to 6◦) of the wind vector led to a further data loss of about 35 %. The ﬂuxes measured by EC systems are also subject to different high-frequency losses due to sensor separation and in case of N2O air transport through the inlet tubes (Foken et al., 2012). These damping effects can lead to a signiﬁcant underestimation of the ﬂux and must be corrected. Based on Ammann et al. (2006) the half-hourly high-frequency losses 2.5.1 Eddy covariance system For ﬁeld-scale ﬂux measurements EC towers were installed in the middle of the two pasture ﬁelds to account for the pre- dominant wind directions northeast and southwest (Fig. 1) and were fenced with a radius of 2–3 m to avoid unwanted animal contact. The measurement height was 2 m, which en- www.biogeosciences.net/16/1685/2019/ Biogeosciences, 16, 1685–1703, 2019 2.5.4 Footprint modeling Because nonstationarity of the ﬂux was already reduced by the short averaging/detrending interval of 5 min, a quality se- lection based on nonstationarity (Foken et al., 2012) had little effect and was therefore not used here. Additional rejection of wind sectors inﬂuenced by the farm facilities, trailer, or shelter and to avoid cross-inﬂuences from the other pasture system (wind dir = 280–25◦and wind dir = 97–195◦) con- tributed to an overall data loss of 64 % and 69 % for systems M and G. The resulting occurrence of data gaps showed a di- urnal pattern with stronger data loss during the night, which was driven by the wind pattern with typically stronger wind speeds during daytime and calm nights. EC measurements yield a spatially integrated ﬂux over a cer- tain area represented by the ﬂux footprint (Schmid, 2002). In the present study, this footprint typically extends over multi- ple grazing paddocks depending on wind direction and turbu- lence intensity. Therefore quantitative footprint information is needed for the comparison of the EC ﬂuxes with the up- scaled FB measurements (Sect. 2.7), and the footprint has to be checked for the spatial dimension to be sure that the measured ﬂux is mainly dominated by the area of the sys- tem and not contaminated by the neighboring systems (ei- ther the other grazing system or ﬂuxes originating from sur- rounding ﬁelds). In this study an open-source version of a backward Lagrangian stochastic dispersion footprint model (bLS) was used (Häni, 2017; Häni et al., 2018), based on Flesch et al. (2004). The ﬂux-to-emission ratio is calculated following Eq. (2): p g y g The gaps in the ﬂux time series needed to be ﬁlled in order to compute cumulative sums over a certain period of time. However, no well-established reference method for the gap ﬁlling of N2O ﬂuxes exists to date. We followed the evalu- ation of Mishurov and Kiely (2011) and used a lookup ta- ble method (LUT) with three parameters: one for the pre- ceding cumulative rainfall of the last 12 h with three classes (no rainfall, 0–2 mm, > 2 mm), one for the percentiles of the soil temperature at 5 cm depth during the GOP with four classes (0–25th percentile, > 25th percentile–median, > median–75th percentile, > 75th percentile), and one for the footprint-weighted (Sect. 2.5.4 Footprint modeling 2.5.4) averaged cow density (cows ha−1) on the single paddocks over the preceding 5 days (0, 0–2, > 2 cows ha−1). To check the sensitivity towards dif- ferent gap-ﬁlling methods, three other techniques were com- pared to the LUT approach: (I) running mean with a vari- able ﬁlter window size and at least 12 values, (II) monthly mean diurnal variation (MDV; see Zhao and Huang, 2015) with a running half-hourly window size of ﬁve in order to have more values during nighttime, and (III) seasonal MDV based on half-hourly values averaged over the whole grazing season. Due to the delayed installation of the EC tower on the southern ﬁeld, all values prior to 14 April on system G resulted from the gap-ﬁlling routine. The uncertainty of gap ﬁlling for seasonal cumulative ﬂuxes was estimated from the standard deviations of monthly cumulative ﬂuxes retrieved with the different gap-ﬁlling methods during GOP, which re- sulted in an uncertainty of 14 % and 18 % for systems M and G, respectively (1σ). It was assumed that this uncertainty re- ﬂects the sum of all important individual uncertainties of the cumulative emissions (e.g., Sects. 3.3.1 and 4). FEC Ej = 2 N nj X i=1 wi ini wio , (2) (2) where FEC is the measured EC ﬂux, Ej the surface emission of paddock (source area) j, N the total number of released particles, nj the number of touchdowns within paddock j, wi ini the vertical release velocity, and wi o the touchdown ve- locity of the particles. In order to calculate the footprint for a 30 min period, N = 80000 ﬂuid particles were released backwards in time using the wind and turbulence parameters calculated from the sonic measurements of the EC systems. The systematic uncertainty of the bLS model was estimated to about 10 % (Flesch and Wilson, 2005; Wilson et al., 2013). The half-hourly footprint fractions of the individual paddocks were used to upscale the small-scale measurements to the EC ﬂux footprint (Sect. 2.7) for intercomparison of the two ﬂux measurement methods. In addition, the seasonally integrated footprint extension was analyzed, taking into account the wind direction and u∗ ﬁltering as described in Sect. 2.5.3. The analysis showed a distinct separation of the footprint distributions for the two systems (Fig. 4) with only marginal contributions of the other system (< 2.5 %). www.biogeosciences.net/16/1685/2019/ Biogeosciences, 16, 1685–1703, 2019 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1692 2.5.4 Footprint modeling More than 80 % of the footprint contribu- tions were from the actual rotation area (without the optional areas indicated in grey in Fig. 1a). The experimental setup was expected to result in very sim- ilar systematic errors of the two EC systems; thus only the in- dependent (or random) errors have to be considered for com- paring the two neighboring systems (Ammann et al., 2009). As the cumulative ﬂuxes of both EC systems were by chance of similar magnitude (Sect. 3.1 and 3.3.1), the random uncer- tainty of the cumulative EC ﬂuxes was determined from the differences between the cumulative monthly EC ﬂuxes of the two towers and resulted in a relative uncertainty of 5 % (1σ). Biogeosciences, 16, 1685–1703, 2019 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1693 Figure 5. Flowchart of upscaling procedure to compare small-scale chamber ﬂuxes with EC ﬂuxes and to estimate the contribution of excreta emissions to the overall pasture emission. Rectangular shapes indicate time series data and other data sets. Time series data with thin frames have gaps whereas bold frames indicate complete data sets. The light blue color speciﬁes N2O ﬂux data. Other shapes show operations (e.g., ﬁt or gap-ﬁlling routines). Figure 5. Flowchart of upscaling procedure to compare small-scale chamber ﬂuxes with EC ﬂuxes and to estimate the contribution of excreta emissions to the overall pasture emission. Rectangular shapes indicate time series data and other data sets. Time series data with thin frames have gaps whereas bold frames indicate complete data sets. The light blue color speciﬁes N2O ﬂux data. Other shapes show operations (e.g., ﬁt or gap-ﬁlling routines). Soil moisture and soil temperature were measured contin- uously with two repetitions on each pasture system close to the EC towers with ML3 ThetaProbe (Delta-T Devices Ltd., UK) devices at depths of 5, 10, 20, and 40 cm. the daily N excretion rate (Sect. 2.3), the daily grazing du- ration of the cows, and a N loading of 22 g N per urination event (Misselbrook et al., 2016) and of 12.5 g N per dung pad (Cardenas et al., 2016). During the grazing season, about 12.5 dung patches d−1 cow−1 and a ratio of dung to urine patches of 1.3 for system M and 1.1 for system G were cal- culated. This compares well to values from literature (Orr et al., 2012; Oudshoorn et al., 2008; Villettaz Robichaud et al., 2011). Due to a very similar ﬁeld-scale N2O emission pat- tern (Sect. 3.1) and comparable soil measurements (Fig. 2), it was assumed that soil parameters were homogenous on the pasture and that the soil measurements on system M were representative for the whole ﬁeld. www.biogeosciences.net/16/1685/2019/ 2.6 Environmental parameters In order to relate the measured ﬂuxes to meteorological driv- ing parameters, an automated weather station (with data log- ger CR10X, Campbell Scientiﬁc Ltd., UK) was installed at the northern ﬁeld next to the sonic. A WXT520 (Vaisala, Vantaa, Finland) measured the wind speed, precipitation, temperature, and barometric pressure, and global radiation was measured with a pyranometer (CNR1, Kipp&Zonen, Delft, the Netherlands). Biogeosciences, 16, 1685–1703, 2019 www.biogeosciences.net/16/1685/2019/ K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 2.7 Upscaling of chamber measurements to eddy covariance footprint Pasture N2O emissions result from a combination of “hotspot” emissions from urine and dung patches and of background emissions from the other pasture areas. Even though the FB measurements (Sect. 2.4.2) allowed for quan- tiﬁcation of single emission sources, quantifying the contri- butions to the overall pasture emission is challenging due to the inherent heterogeneous nature of these emissions (e.g., spatial dimension, emission strength, temporal behav- ior, number of excreta patches). The EC method, in contrast, allowed us to measure the combination of all pasture sources by integrating over multiple paddocks (see footprint, Fig. 4). The FB-derived N2O emissions for the different sources were analyzed for the potential driving parameters excreta age, soil temperature, and soil moisture. For this purpose various regression models (using the statistical software R; R Core Team, 2016) were tested using different predeﬁned function types (linear, exponential, polynomial functions, sigmoidal). Based on goodness of ﬁt and statistical signiﬁ- cance of regression coefﬁcients, the most suitable relation- ships were chosen and applied to produce continuous emis- sion time series for the paddock areas (Fig. 5). FB measurements were upscaled to the EC footprint to al- low a direct comparison between the two measurement ap- proaches and to compute the contributions of the different emission sources to the overall pasture emission. The upscal- ing procedure is illustrated in Fig. 5. The number of urine and dung patches on the paddocks was estimated by using Biogeosciences, 16, 1685–1703, 2019 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1694 ated nitrous oxide emissions: from patch scale to ﬁeld scale Figure 6. Time series of half-hourly EC ﬂux measurements in both systems during the grazing season 2016. For the analysis of grazing- related emissions, only the non-shaded periods (GOP) were used. The vertical lines show the timings of fertilization (red) and harvest (orange) events. Dashed lines indicate that harvest and urea appli- cation were split for the western (X.11–X.16) and eastern (X.21– X.25) parts. The shaded areas indicating time periods inﬂuenced by fertilization events or harvest were excluded for the evaluation of grazing excreta-related emissions. One ﬂux value (29.0 g N2O- N ha−1 h−1 on system M, 30 June 2016) was skipped for better readability. 1. Background ﬂuxes were parametrized as a function of soil moisture at a depth of 5 cm using the soil proﬁle information provided in Sect. 2.6 by using a logistic re- gression. 2. Urine patch emissions were parametrized as an expo- nential decay function of excreta age. To account for different environmental conditions, the deviations of the single emissions from this temporal emission pattern were again parametrized as a function of soil temper- ature and moisture at a depth of 5 cm (Sect. 2.6). Up- scaling ﬂuxes to the paddock sizes involved additional information on the computed number density of urine patches (as mentioned previously). Figure 6. Time series of half-hourly EC ﬂux measurements in both systems during the grazing season 2016. For the analysis of grazing- related emissions, only the non-shaded periods (GOP) were used. The vertical lines show the timings of fertilization (red) and harvest (orange) events. Dashed lines indicate that harvest and urea appli- cation were split for the western (X.11–X.16) and eastern (X.21– X.25) parts. The shaded areas indicating time periods inﬂuenced by fertilization events or harvest were excluded for the evaluation of grazing excreta-related emissions. One ﬂux value (29.0 g N2O- N ha−1 h−1 on system M, 30 June 2016) was skipped for better readability. Figure 6. Time series of half-hourly EC ﬂux measurements in both systems during the grazing season 2016. For the analysis of grazing- related emissions, only the non-shaded periods (GOP) were used. 3. Dung patch emissions were parametrized as a second- order polynomial function of excreta age. Paddock emissions were calculated by applying this function to the computed number of dung patches (as previously mentioned) per paddock. 3.1 EC ﬂuxes Observed EC ﬂuxes on both pasture systems showed an almost identical temporal pattern (Fig. 6). The half-hourly ﬂuxes on each system showed considerable variation during the grazing season with clear peaks after fertilization (grey shaded areas) and after grazing phases in the nearby pad- docks (e.g., peaks in May, beginning of August). The overall highest emissions (29.0 and 24.3 g N2O-N ha−1 h−1 for sys- tems M and G) were measured directly after the fertilizer application, which followed a harvest of hay at the end of June. This harvest event also led to an increase in the mea- sured N2O ﬂuxes (0.5–3.0 g N2O-N ha−1 h−1) which lasted less than 1 day. The partial fertilizer application in middle of August resulted in higher ﬂuxes compared to the following one in early September. The relatively high emissions during 3 Results During the GOP (excluding the grey shaded fertilizer- inﬂuenced time periods in Fig. 6), the ﬂuxes were still very similar for the two pasture systems M and G with a mean and standard deviation of 0.32±0.36 and 0.33±0.37 g N2O- N ha−1 h−1, respectively. A mean diurnal cycle of the mea- sured ﬂuxes could be observed in both systems with the high- est values typically occurring in the afternoon and, on aver- age, about 10 %–20 % lower values during the night. K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale The upscaled paddock emissions were ﬁnally compared to the EC ﬂuxes by applying the computed footprint fractions of the paddocks (Sect. 2.5.4) in order to validate the FB mea- surements and to quantify the uncertainty of the upscaling process. the ﬁrst full grazing event at the beginning of May were char- acterized by high soil moisture contents (see also Fig. 2a), whereas the very wet soil conditions and the corresponding grazing break during June resulted in low ﬂuxes in both sys- tems. The small observed ﬂuxes from the middle of March until the end of April resulted mainly from background ﬂuxes and sporadic grazing (Fig. 2c). Occasional negative individ- ual ﬂux values between 0 and −1.5 g N2O-N ha−1 h−1 were observed in both systems (7 %–8 % of the cases). However, these ﬂuxes exclusively occurred in cases, when no deﬁned peak in the cross-covariance function could be identiﬁed (and thus the default lag was used, Sect. 2.5.2). Thus it can be concluded that the negative ﬂuxes were generally below the detection limit. The area-related N2O emissions for urine and dung were also converted to emissions per cow and grazing hour. For this purpose, the upscaled paddock emissions were combined over all paddocks, accumulated for the GOP, multiplied by the pasture area of each system, and divided by the number of cows and the grazing duration (Fig. 5). The resulting emis- sions associated with animal excreta were then related to the excreted N of the cows (Sect. 2.3) to obtain an excreta-related EF that is comparable to the one provided by the IPCC guide- lines (EF3PRP,CPP; IPCC, 2006). www.biogeosciences.net/16/1685/2019/ www.biogeosciences.net/16/1685/2019/ K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld sca K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 3.2.1 Comparison of pasture systems The dotted lines show the ﬁtted curves through the averaged values of urine and dung patch emissions (see also Eqs. 3 and 4). Figure 8. N2O ﬂux evolution with time for urine patches, dung pats, and background areas. The ﬂuxes were measured with the fast box and averaged over 3-day periods, and the error bars show the stan- dard error of the measurements. The standard errors for the back- ground ﬂuxes are smaller than the symbols. The dotted lines show the ﬁtted curves through the averaged values of urine and dung patch emissions (see also Eqs. 3 and 4). Figure 7. Scatterplot shows the comparison of near-simultaneous ﬂuxes for different sources measured with the fast box on the two pasture systems. The black line indicates the 1 : 1 line. riods and were related to the excreta age 1tEOG (Fig. 8), de- ﬁned as days after EOG (Sect. 2.4.2). Background ﬂuxes were on average considerably smaller than excreta ﬂuxes and showed small persisting emissions without systematic dependence on time since grazing. In contrast, for urine patch ﬂuxes a clear relation to 1tEOG was found. The highest ﬂuxes were usually observed within the ﬁrst days after the urination event. Afterwards, they rapidly decreased with time, although with a high variability that can partly be attributed to the inﬂuence of environmental condi- tions (see Sect. 3.2.3). The age-dependent evolution of urine patch emissions (FU,age) was parameterized with an expo- nential decay function ﬁtted to the data points in Fig. 8: Table 3. Flux measurements using the FB technique (mean ± SD) of background and excreta patches averaged over 20 days follow- ing a grazing phase. Only near-simultaneous measurements in both systems were considered. Measurement location System M System G Background (µg N2O-N m−2 h−1) 8 ± 8 5 ± 8 Urine (µg N2O-N m−2 h−1) 121 ± 130 162 ± 190 Dung (µg N2O-N m−2 h−1) 16 ± 18 35 ± 60 Measurement location System M System G Background (µg N2O-N m−2 h−1) 8 ± 8 5 ± 8 Urine (µg N2O-N m−2 h−1) 121 ± 130 162 ± 190 Dung (µg N2O-N m−2 h−1) 16 ± 18 35 ± 60 FU,age = a1 · expb1·1tEOG. (3) (3) times the values of background areas. The relative variability within the different source classes (urine, dung, background) were very high and resulted in standard deviations larger than the associated mean values. 3.2.1 Comparison of pasture systems The excreta ﬂuxes measured on system G tended to be somewhat higher in magnitude com- pared to system M, but no signiﬁcant difference (p > 0.05) was found. Also, for the background ﬂuxes no signiﬁcant (p > 0.05) difference between the two pasture systems was observed. Therefore all FB ﬂuxes were combined for further processing without taking into account the different pasture systems. The coefﬁcients of Eqs. (3)–(8) are presented in Table 4 and apply to ﬂuxes in units of µg N2O-N m−2 h−1. Dung patch ﬂuxes also showed a relation to excreta age (Fig. 8), however less pronounced compared to urine patches, and the highest emissions were typically observed between 4 and 11 days after dung deposition. However they were still smaller on average than the urine patch emissions during the entire observed age period. Because the evolution of dung emissions FD,age after the observed 20-day age period is un- clear and a meaningful functional extrapolation was not pos- sible, we decided to use a simple second-order polynomial for parameterization purposes. This allowed us to reproduce the initial increase with age and a rapid decrease to zero be- yond the measured age range: 3.2.2 Dependence on excreta age The information on the temporal pattern of the excreta and background ﬂuxes after grazing is important for the time in- tegration of the individual sources and for the comparison with the EC measurements. In order to analyze and parame- terize the temporal evolution of the emissions, the measured FB ﬂuxes of each source class were averaged over 3-day pe- FD,age = a2 + b2 · 1tEOG + c2 · 1tEOG2. (4) (4) The ﬁtted polynomial function is only applicable up to 1tEOG ≈25 d, where it crosses the zero line. The ﬁtted polynomial function is only applicable up to 1tEOG ≈25 d, where it crosses the zero line. 3.2.1 Comparison of pasture systems FB chamber ﬂuxes of background and dung patches were considerably smaller compared to the ﬂuxes of urine patches (Table 3, Fig. 7). Freshly deposited urine patches under 3 days old could result in N2O emissions larger than 100 Biogeosciences, 16, 1685–1703, 2019 www.biogeosciences.net/16/1685/2019/ K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1695 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1695 Figure 7. Scatterplot shows the comparison of near-simultaneous ﬂuxes for different sources measured with the fast box on the two pasture systems. The black line indicates the 1 : 1 line. Figure 8. N2O ﬂux evolution with time for urine patches, dung pats, and background areas. The ﬂuxes were measured with the fast box and averaged over 3-day periods, and the error bars show the stan- dard error of the measurements. The standard errors for the back- ground ﬂuxes are smaller than the symbols. The dotted lines show the ﬁtted curves through the averaged values of urine and dung patch emissions (see also Eqs. 3 and 4). riods and were related to the excreta age 1tEOG (Fig. 8), de- ﬁned as days after EOG (Sect. 2.4.2). Background ﬂuxes were on average considerably smaller g g Figure 7. Scatterplot shows the comparison of near-simultaneous ﬂuxes for different sources measured with the fast box on the two pasture systems. The black line indicates the 1 : 1 line. Figure 8. N2O ﬂux evolution with time for urine patches, dung pats, and background areas. The ﬂuxes were measured with the fast box and averaged over 3-day periods, and the error bars show the stan- dard error of the measurements. The standard errors for the back- ground ﬂuxes are smaller than the symbols. The dotted lines show the ﬁtted curves through the averaged values of urine and dung patch emissions (see also Eqs. 3 and 4). Figure 8. N2O ﬂux evolution with time for urine patches, dung pats, and background areas. The ﬂuxes were measured with the fast box and averaged over 3-day periods, and the error bars show the stan- dard error of the measurements. The standard errors for the back- ground ﬂuxes are smaller than the symbols. 3.2.3 Dependence on environmental conditions Measured chamber ﬂuxes were analyzed in relation to driv- ing soil parameters (Sect. 2.6). For dung patch emissions, no relation to these parameters was found (thus FD = FD,age). For background ﬂuxes no signiﬁcant dependence on soil tem- perature (p < 0.05), but a clear dependence on the volumet- ric water content (VWC) at a depth of 5 cm, was found. The background ﬂuxes had a large variability and could roughly be separated by three different VWC sectors (< 0.27, 0.27– 0.33, > 0.33). In the sector below a VWC of 0.27, ﬂuxes typically ranged between −3 and 15 µg N2O-N m−2 h−1, whereas in the upper sector above a VWC of 0.33 the ﬂuxes showed typical values between 0 and 30 µg N2O-N m−2 h−1. Nevertheless, the variability was especially pronounced in the VWC range between 0.27 and 0.33 with ﬂuxes ranging between 0 and 40 µg N2O-N m−2 h−1. Thus this VWC range also comprised the overall highest background ﬂuxes. How- ever, averaging the ﬂuxes by VWC intervals of 0.05 resulted in very similar values of about 12 ± 3 µg N2O-N m−2 h−1 above a VWC of 0.3. Hence, the measured background ﬂuxes could be parametrized with the following functional relation- ship: a similar 1tEOG was measured at a low TS (1 ◦C) and a lower VWC (0.3). Maximum positive measured FB ﬂux de- viations (Sect. 2.7) from Eq. (3) were generally observed for wet (VWC > 0.45) and warm (> 17 ◦C) soil conditions while low TS and VWC resulted in negative ﬂux deviations. Thus, the ﬁnal regression model for urine patch emissions (Eq. 6) consists of multiple equations (Eqs. 3, 7, 8) which relate the measured ﬂuxes to the temporal decay (Eq. 3) and a deviation 1FU,env to this decay, where 1FU,env was parametrized as a function of environmental driving parameters TS and VWCU (Eqs. 7 and 8, Fig. 9). FU = FU,age + 1FU,env (6) 1FU,env = (a4 + b4 · VWCU + c4 · TS) · CorrU,env (1tEOG) (7) (6) (7) CorrU,env corrects 1FU,env for different urine patch ages as the deviation can be larger for relatively new patches com- pared to older ones. This correction factor was found to be a linear relationship (p < 0.01) between 1.35 for a 1tEOG of 0 days (after the patch deposition) and 0.35 after 20 days. VWCU (Eq. K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1696 Figure 9. Surface plot shows the estimated N2O ﬂux deviation (Eqs. 6, 7; CorrU,env = 0) from the exponential ﬁt (Eq. 3) for urine patches depending on soil moisture VWCU and temperature at a depth of 5 cm. The black dots indicate the conditions under which ﬂux measurements with the FB were obtained. Table 4. Coefﬁcients (a–c), corresponding indices i, and signiﬁ- cance levels for the equations presented in Sect. 3.2. The equation coefﬁcients were ﬁtted using FB chamber measurements and yield ﬂuxes in units of µg N2O-N m−2 h−1. The input quantities are the soil temperature TS (in units of degrees Celsius), time since end of grazing 1tEOG (in units of days), and volumetric water content VWC (as a dimensionless fraction). Equation I ai bi ci Eq. (3) 1 587∗∗∗ −0.082∗∗ Eq. (4) 2 23∗ 5.4∗ −0.25∗ Eq. (5) 3 12.6∗∗∗ 0.267∗∗∗ 0.012∗ Eq. (7) 4 −1490∗∗∗ 2900∗∗∗ 23.9∗∗ Eq. (8) 5 0.098∗∗∗ −0.086∗∗ ∗∗∗Signiﬁcant at level p < 0.001. ∗∗Signiﬁcant at level p < 0.01. ∗Signiﬁcant at level p < 0.05. Equation I ai bi ci Eq. (3) 1 587∗∗∗ −0.082∗∗ Eq. (4) 2 23∗ 5.4∗ −0.25∗ Eq. (5) 3 12.6∗∗∗ 0.267∗∗∗ 0.012∗ Eq. (7) 4 −1490∗∗∗ 2900∗∗∗ 23.9∗∗ Eq. (8) 5 0.098∗∗∗ −0.086∗∗ ∗∗∗Signiﬁcant at level p < 0.001. ∗∗Signiﬁcant at level p < 0.01. ∗Signiﬁcant at level p < 0.05. Figure 9. Surface plot shows the estimated N2O ﬂux deviation (Eqs. 6, 7; CorrU,env = 0) from the exponential ﬁt (Eq. 3) for urine patches depending on soil moisture VWCU and temperature at a depth of 5 cm. The black dots indicate the conditions under which ﬂux measurements with the FB were obtained. ∗∗∗Signiﬁcant at level p < 0.001. ∗∗Signiﬁcant at level p < 0.01. ∗Signiﬁcant at level p < 0.05. 3.2.3 Dependence on environmental conditions 8) accounts for different soil moisture condi- tions at the surface below a urine patch and nearby back- ground areas and was parametrized as a function of back- ground VWC and 1tEOG (Eq. 8). FBG = a3 1 + exp(b3−VWC)/c3 . (5) (5) This logistic regression curve has a strong effect below VWC values of 0.30 but stays fairly constant at higher VWC con- tents and converges to a ﬂux of 12.6 µg N2O-N m−2 h−1. Be- low a VWC of 0.2 the logistic regression converges to a back- ground ﬂux of 0 µg N2O-N m−2 h−1. VWCU = VWC + a5 · expb5·1tEOG (8) (8) Measured urine patch emissions showed a clear response not only to the excreta age as shown in Sect. 3.2.2 but also to changes in TS and VWC. On a speciﬁc 1tEOG, FU,age could vary signiﬁcantly and typically correlated with soil condi- tions. The highest ﬂux (5117 µg N2O-N m−2 h−1, 1tEOG = 6 d) was measured at a TS of 18 ◦C and a VWC of 0.42 while the lowest measured ﬂux (34 µg N2O-N m−2 h−1) on www.biogeosciences.net/16/1685/2019/ Biogeosciences, 16, 1685–1703, 2019 Biogeosciences, 16, 1685–1703, 2019 3.3.1 Comparison between upscaled chamber and EC ﬂuxes Generally the ﬁeld-scale ﬂuxes represent the area integral of management-related (excreta patches) and environmen- Biogeosciences, 16, 1685–1703, 2019 www.biogeosciences.net/16/1685/2019/ www.biogeosciences.net/16/1685/2019/ K. Voglmeier et al.: Grazing-related nitrous oxide emission Figure 10. Time series of (a) environmental parameters and (b) up- scaled FB ﬂuxes (Sect. 2.7) for different paddocks (M11–M14) in system M. The colored rectangles at the bottom show the graz- ing phases on the four considered paddocks (grey colors indicating grazing on the remaining paddocks). (c) N2O ﬂuxes by EC and up- scaled FB during a full rotation between 23 September and 10 Au- gust 2016 for system M. K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1697 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale meier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1697 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1697 Figure 10. Time series of (a) environmental parameters and (b) up- scaled FB ﬂuxes (Sect. 2.7) for different paddocks (M11–M14) in system M. The colored rectangles at the bottom show the graz- ing phases on the four considered paddocks (grey colors indicating grazing on the remaining paddocks). (c) N2O ﬂuxes by EC and up- scaled FB during a full rotation between 23 September and 10 Au- gust 2016 for system M. Figure 11. Cumulative emissions for both systems obtained with the FB and EC technique during GOP 2016. The grey shaded bars indicate time periods which were excluded due to signiﬁcant overlapping N2O emissions from fertilization/harvest and grazing (Sects. 2.5, 3.1). in environmental driving parameter could be observed, it was less pronounced for the upscaled FB ﬂuxes in comparison to the EC ﬂuxes. Gap ﬁlling of the EC ﬂuxes (Sect. 2.5.3) allowed the cal- culation of the cumulative N2O emissions during the GOP Figure 11. Cumulative emissions for both systems obtained with the FB and EC technique during GOP 2016. The grey shaded bars indicate time periods which were excluded due to signiﬁcant overlapping N2O emissions from fertilization/harvest and grazing (Sects. 2.5, 3.1). Figure 11. Cumulative emissions for both systems obtained with the FB and EC technique during GOP 2016. The grey shaded bars indicate time periods which were excluded due to signiﬁcant overlapping N2O emissions from fertilization/harvest and grazing (Sects. 2.5, 3.1). Figure 10. 3.3.1 Comparison between upscaled chamber and EC ﬂuxes Time series of (a) environmental parameters and (b) up- scaled FB ﬂuxes (Sect. 2.7) for different paddocks (M11–M14) in system M. The colored rectangles at the bottom show the graz- ing phases on the four considered paddocks (grey colors indicating grazing on the remaining paddocks). (c) N2O ﬂuxes by EC and up- scaled FB during a full rotation between 23 September and 10 Au- gust 2016 for system M. in environmental driving parameter could be observed, it was less pronounced for the upscaled FB ﬂuxes in comparison to the EC ﬂuxes. Gap ﬁlling of the EC ﬂuxes (Sect. 2.5.3) allowed the cal- culation of the cumulative N2O emissions during the GOP (solid lines in Fig. 11). These area-related emissions were very similar between the two systems throughout the GOP with seasonal sums close to 1500 g N2O-N ha−1. Cumulating the N2O emissions not only enabled a more quantitative com- parison between the systems, but also allowed a better com- parison between the two measurement approaches (Fig. 11). The emissions of the upscaled FB matched the EC emis- sions rather well with differences of the seasonal sums below 3 %. Distinct differences were mainly observed in May and June when FB-derived emissions were signiﬁcantly overes- timated compared to EC. At the end of the grazing period and averaged over both systems, slightly higher emissions were estimated from the upscaling routine compared to the measured EC emissions. Monthly absolute differences be- tween the cumulative EC and the upscaled cumulative FB sums were normally distributed (p < 0.05) with 1σ values of 26 % and 25 % for systems M and G, respectively. Within this uncertainty range no difference between the two mea- surement approaches was observable. Gap ﬁlling of the EC ﬂuxes (Sect. 2.5.3) allowed the cal- culation of the cumulative N2O emissions during the GOP (solid lines in Fig. 11). These area-related emissions were very similar between the two systems throughout the GOP with seasonal sums close to 1500 g N2O-N ha−1. Cumulating the N2O emissions not only enabled a more quantitative com- parison between the systems, but also allowed a better com- parison between the two measurement approaches (Fig. 11). tally driven small-scale ﬂuxes. Therefore the relationships presented in Sect. 3.2.2 (dependency on excreta age) and Sect. 3.2.3 (environmental driving parameter) were applied to upscale the FB measurements to the paddock size during the GOP. 3.3.1 Comparison between upscaled chamber and EC ﬂuxes As shown as an example in Fig. 10 for an 18-day period, the magnitude of the management-related upscaled paddock ﬂuxes depended mainly on the grazing duration on the single paddocks (similar slope for different paddocks M11–M14). The maximum of the emissions was typically calculated at the end of the grazing period on the respective paddocks. The lower limit of the ﬂuxes was given by the estimated background ﬂuxes, especially at the beginning of a new ro- tation, and stayed therefore rather constant for VWC values above 0.3 (Eq. 5, Sect. 3.2.3). Variations in environmental conditions (mainly important for soil moisture) led to rapid changes in the emission level as long as signiﬁcant urine patch emissions were present. These rapid variations typi- cally occurred after stronger precipitation events (as shown in Fig. 10a for on-site meteorological and soil measurements). www.biogeosciences.net/16/1685/2019/ 3.3.2 Emission breakdown into contribution sources The excellent match between the EC ﬂuxes and the upscaled chamber-based ﬂuxes showed that the applied relationships which used excreta age and environmental parameters as in- put (see Sect. 3.2) were reasonable and allowed the separa- tion into single emission sources (Fig. 12). Except for the be- Upscaling the paddock ﬂuxes to the EC footprint allowed a direct comparison with the EC ﬂuxes on a half-hourly basis (Fig. 10c). The upscaled FB ﬂuxes compared well in mag- nitude with the measured EC ﬂuxes and showed a similar temporal behavior. While generally a response to variations www.biogeosciences.net/16/1685/2019/ www.biogeosciences.net/16/1685/2019/ Biogeosciences, 16, 1685–1703, 2019 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld sca K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1698 Figure 12. (a) Time series of upscaled FB ﬂuxes averaged over all paddocks of system M for all three emission sources during the grazing season 2016, and (b) retrieved cumulative emission con- tribution of the emission sources to the overall ﬁeld emission. whole pasture system, although the contribution of the cen- tral paddocks X.11, X.12 and X.21, X.22 to the EC foot- print is generally higher than the contribution of the other more distant paddocks (Fig. 4). We found no indication of signiﬁcant differences between the paddocks concerning soil conditions, vegetation productivity, or other characteristics (data not shown). An alternative upscaling of the FB mea- surements to the entire pasture system (without taking the EC footprint into account) representing the average emission over all rotation paddocks (Table 5, FB emissions upscaled to pasture system) differed less than 4 % from the EC footprint- related emissions. For assessing the effect of the N-reduced diet on excreta- related N2O emissions, the emissions per cow and grazing hour were compared, taking into account the different pasture sizes for systems M and G (according to Sect. 2.7). The cor- responding results in Table 5 show about 25 % lower excreta- related N2O emissions per cow for the herd in system M than for the herd in system G during the GOP. The difference is not statistically signiﬁcant, probably due to the consider- able uncertainties resulting from the FB upscaling procedure. For comparing the two herds the parallel direct EC measure- ments are better suited as only random uncertainties have to be taken into account (Sect. 2.5.3), yet they also include the background emissions. The EC-based N2O emissions per cow were 0.20±0.03 and 0.27±0.05 g N2O-N cow−1 h−1 for system M and system G, respectively, and resulted in a signif- icant difference of 0.07 ± 0.02 g N2O-N cow−1 h−1 between the two herds. This indicates the ability of a N-adjusted for- age to reduce the excreta N content and related emissions of N2O. www.biogeosciences.net/16/1685/2019/ It has to be noted that this evaluation does not comprise the full N2O emission of the pasture ﬁelds or of the milk pro- duction system but only the emissions related to grazing exc- reta following the IPCC concept (EF3PRP,CPP; IPCC, 2006). Any further N2O emissions, e.g., related to fertilizer appli- cation on the pastures or the supplement maize production, were not taken into account here. A comparison of entire pro- duction systems would require many additional assumptions outside the speciﬁc scope of this study. It also has to be con- sidered that the N optimization of the diet is not necessarily linked to the supplemental feed of arable crops like maize but may also be achieved with different feed strategies (e.g., grass varieties with a high content of water-soluble carbohy- drates; Misselbrook et al., 2013). Figure 12. (a) Time series of upscaled FB ﬂuxes averaged over all paddocks of system M for all three emission sources during the grazing season 2016, and (b) retrieved cumulative emission con- tribution of the emission sources to the overall ﬁeld emission. ginning of the grazing season when grazing rate was very low (see Fig. 2), the urine patch emissions dominated the ﬁeld- scale ﬂuxes. In May, this effect was even more pronounced due to the wet soil conditions. Based on the upscaling, the averaged urine patch emissions of both systems were respon- sible for about 57 % of the pasture emissions. Background contributed to about 38 % and dung emissions to about 5 % to the overall ﬁeld emissions. Both systems had very similar contributions, with only 1 % difference in the dung contribu- tion as a result of a different N excretion ha−1 on the pasture by dung (Table 5). Background emissions were simulated to be constant for most of the GOP due to the weak sensitivity of Eq. (5) to VWC and the undetected sensitivity towards soil temperature. K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale Table 5. Summary of cumulated grazing-related emissions for both pasture systems during the GOP 2016. The table shows the emissions per area (ﬁrst part), pasture area, excreta N input (second part), and the emissions per cow and grazing hour as well as the calculated EFs (third part). The uncertainties are given as 1σ. Parameter System M System G EC emission (kg N2O-N ha−1) 1.50 ± 0.21 1.51 ± 0.27 FB emissions upscaled to EC footprint (kg N2O-N ha−1) 1.51 ± 0.39 1.50 ± 0.37 FB emissions upscaled to pasture system (kg N2O-N ha−1)a 1.48 ± 0.38 1.48 ± 0.37 Pasture system area (ha) 1.88 2.51 Excreta N total (g N cow−1 h−1) 16.5 ± 1.2 19.6 ± 1.5 Urine N (g N cow−1 h−1) 10.2 ± 1.1 13.0 ± 1.3 Dung N (g N cow−1 h−1) 6.3 ± 0.7 6.5 ± 0.7 FB excreta emissions (g N2O-N cow−1 h−1) 0.12 ± 0.04 0.16 ± 0.05 EF excreta total (%)b 0.74 ± 0.26 0.83 ± 0.29 EF urine (%)b 1.09 ± 0.43 1.15 ± 0.43 EF dung (%)b 0.16 ± 0.06 0.17 ± 0.06 a Average emissions over all paddocks. b Based on FB emissions upscaled to the pasture system. a Average emissions over all paddocks. b Based on FB emissions upscaled to the pasture system. tural systems, background emissions are usually determined as emissions from (managed) plots receiving no fertilization in the study year. Thus they still include N inputs from plant residues and atmospheric deposition. Background emissions are also often regarded as a late effect of fertilization events from previous years (Bouwman, 1996; Gu et al., 2009). On pastures, background emissions may additionally result from trampling of the cows that can further stimulate the N2O pro- duction via denitriﬁcation due to soil compaction (Bhandral et al., 2007). based on the combined, upscaled FB measurements of urine and dung patches (see Sect. 3.3.2) relative to the N excreted on the pastures during the GOP (Table 5). Their uncertainty is deﬁned by the combined uncertainty of the upscaling method (Sect. 3.3.1) and the N input estimation (7.5 %). The differ- ence in the EFs between the systems is therefore not statisti- cally signiﬁcant. The resulting EFs were signiﬁcantly smaller compared to the proposed default EF3PRP,CPP of the IPCC guidelines for cattle excreta (2 %; IPCC, 2006), which makes the use of the latter in the Swiss national inventory questionable. K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale The upscaled FB measurements also allowed us to sepa- rately calculate the EFs for urine and dung. We found EFs of 1.12±0.43 % and 0.16±0.06 % for urine and dung, respec- tively (average of both systems due to small difference; see Table 5). These EFs are comparable to the results of newer studies (0.59 % and 0.26 % for urine and dung patches com- bined from cattle and sheep; Cai and Akiyama, 2016; 1.18 % and 0.31 % for cattle urine and dung; Krol et al., 2016). The large difference of the EFs for urine and dung also supports the suggestion of Krol et al. (2016) to disaggregate the EF by excreta type in emission inventories. The implementation of excreta-speciﬁc EFs could allow for a more precise calcu- lation of the grazing-related N2O emissions, e.g., as dietary effects regarding the N intake predominantly affect the ex- creted urine N, which is the main source for the high N2O emission associated with excreta (Dijkstra et al., 2013). 4.1 Area-related and animal-related emissions The EC and upscaled FB emission results presented in Sect. 3.3.1 are normalized by area and showed the emissions for the EC footprint (see also summary in Table 5). The good agreement with a relative difference below 1.5 % for yearly sums (which is far below the uncertainty range; see Table 5) between the two independent approaches supports their qual- ity (including the upscaling procedure) in this study. We as- sume that the EC ﬂuxes are on average representative for the Area- or cow-related emissions as described in Sect. 4.1 enabled the comparison of the different measurement ap- proaches and the discussion of the diet effects on N2O emis- sion. However, results presented in literature or used in na- tional inventories typically relate emissions to the N inputs within a given time period using EFs. The annual excreta- related EF (Table 5) in the present study was 0.74 ± 0.26 % for system M and 0.83±0.29 % for system G. These EFs are Biogeosciences, 16, 1685–1703, 2019 www.biogeosciences.net/16/1685/2019/ K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1699 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 1700 and EC ﬂuxes were measured (July–October), the agreement between the systems was very good. 2013) on seasonal (e.g., due to variability in the N content of the fodder) and short-term scales (e.g., different urine vol- ume, different cows, difference between day and night), con- tinuous measurements throughout the grazing period for a representative number of cows would have been needed. This is only possible with measurement equipment directly placed on the cow. However, these measurements are often limited regarding animal welfare, are not well established and the as- sociated uncertainty is still considerable (Misselbrook et al., 2016). Thus, they were not used in this study. However, the parameterizations used for upscaling re- sulted in a poor performance for certain soil conditions. The limited sensitivity towards changes in VWC of the back- ground ﬂuxes is probably due to the fact that FB measure- ments were mainly performed during dry soil conditions. We have no explanation why we did not ﬁnd a signiﬁcant sensitivity of the background ﬂuxes towards changes in TS as reported by other studies (Butterbach-Bahl et al., 2013; Schindlbacher et al., 2004). Typically, increasing soil temper- ature leads to increased soil respiration, which subsequently can lead to a depletion of soil oxygen and further to higher denitriﬁcation rates. In contrast to background ﬂuxes, the urine patch emissions showed a clear response to changes in TS and VWC. This effect could be parametrized with a bilinear regression (Eqs. 7 and 8). This regression led to high upscaled emissions from urine patches, especially dur- ing wet soil conditions, and subsequently to an overestima- tion of the cumulative emissions in May and June compared to the EC systems. N2O emissions often have an emission maximum during moderately wet soil conditions (VWC be- tween 0.40 and 0.45) while completely anaerobic conditions at saturated VWC can lead to a complete denitriﬁcation with only marginal N2O emissions (Butterbach-Bahl et al., 2013). Such conditions have been very rare during the FB measure- ments (see Fig. 9) and therefore may not be adequately rep- resented in the derived parameterization. A general trend to- wards lower emissions during very wet soil conditions was also observed by the EC systems (not shown). However, in order to avoid mixing results of the different measurement systems and thus reducing the explanatory power of the sys- tem intercomparison, we decided to base the environmental regression analysis (Sect. 5 Concluding remarks The temporal dynamics of background areas and excreta patches were observed by fast-box (FB) chamber measure- ments on the pasture. We found no signiﬁcant temporal pat- tern of the background ﬂuxes. Urine patch emissions were parametrized by an exponential decay with time, whereas a less pronounced dependency on excreta age of dung emis- sions was observed. This relation was parametrized with a quadratic function and a maximum after about 10 days. On a ﬁeld-scale level, urine patch emissions dominated the pas- ture emissions during the grazing season. Nevertheless, back- ground ﬂuxes contributed signiﬁcantly to the pasture emis- sions as well. The origin of these background ﬂuxes is still uncertain and should be addressed in further studies. K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale 3.2.3) only on data measured by the FB. The combined approach of EC and FB measurements al- lowed the quantiﬁcation of the uncertainty of the upscaling routine, and the good match between the two measurement approaches also validates the resulting contributions of the different emission sources on the ﬁeld scale. The uncertainty associated with the upscaling mainly resulted from missing FB measurements during wet soil conditions (e.g., in spring), which prevented the use of a more complex parameteriza- tion of environmental driver effects on background and urine emission. In summary, the experimental setup resulted in ro- bust ﬁeld-scale emissions, allowed us to compare the two pasture systems, and yielded source-speciﬁc emission factors for dung and urine patches. 4.3 Upscaling of FB ﬂuxes Urine patch emissions were parametrized with an expo- nential decay and maximum initial emissions of about 600 µg N2O-N m−2 h−1 that is close to the maximum aver- aged emissions measured by Barneze et al. (2015) from man- ually applied urine in laboratory conditions and on a grass- land. A strong emission response to urine application was generally reported in the literature, however, with a large range of different emission dynamics and magnitudes (e.g., two emission peaks due to nitriﬁcation and denitriﬁcation, emission peak after a few days with near exponential decay afterwards, signiﬁcant emissions after weeks to a month; Bell et al., 2015; Cardenas et al., 2016; Chadwick et al., 2018). Similar to our study, reported dung patch emissions by those studies were much lower compared to urine-induced emis- sions. The background emissions measured by FB cannot be attributed to a speciﬁc N input in a quantitative way, but the annual sum of 1.03 kg N2O-N ha−1 yr−1 from this study (extrapolated using Eq. 5 and VWC data for the whole year) compares well with background emissions reported by a meta-study of Kim et al. (2013, median 0.7 and mean 1.52 kg N2O-N ha−1 yr−1) for agricultural lands. In agricul- We found that pasture emissions were dominated by excreta-related emissions during the GOP (about 60 %). On a seasonal basis, the upscaled aggregated ﬂuxes compared well with the gap-ﬁlled EC measurements, which also indicates the validity of the source attribution in the upscaled emis- sions. Especially during time periods when both FB ﬂuxes www.biogeosciences.net/16/1685/2019/ Biogeosciences, 16, 1685–1703, 2019 K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale Competing interests. The authors declare that they have no conﬂict of interest. Competing interests. The authors declare that they have no conﬂict of interest. Betteridge, K., Costall, D. A., Li, F. Y., Luo, D., and Ganesh, S.: Why we need to know what and where cows are urinating – a urine sensor to improve nitrogen models, Proc. N. Z. Grassl. As- soc, 75, 119–124, 2013. Acknowledgements. We gratefully acknowledge the funding from the Swiss National Science Foundation (project NICEGRAS, no. 155964) and from the Swiss Federal Ofﬁce for the Environment (contract no. 16.0030.KP/P031-1675). We wish to thank Lukas Eg- gerschwiler, Robin Giger, Walter Glauser, Andreas Münger, and Jens Leifeld for support in the ﬁeld and helpful discussions. We especially acknowledge the contribution of Harald Menzi in the de- sign and planning of the experiment and Arjan Hensen for lending us the fast box and advising how to use it. We are grateful to Al- brecht Neftel for the helpful discussions and advice concerning the measurements. We thank Daniel Bretscher for the support with the N balance computation of the cows and the discussions of these data. Karl Voglmeier was additionally supported by a MICMoR fel- lowship through KIT/IMK-IFU. Bhandral, R., Saggar, S., Bolan, N., and Hedley, M.: Transfor- mation of nitrogen and nitrous oxide emission from grassland soils as affected by compaction, Soil Tillage Res., 94, 482–492, https://doi.org/10.1016/j.still.2006.10.006, 2007. Biermann, T., Babel, W., Ma, W., Chen, X., Thiem, E., Ma, Y., and Foken, T.: Turbulent ﬂux observations and modelling over a shallow lake and a wet grassland in the Nam Co basin, Tibetan Plateau, Theor. Appl. Climatol., 116, 301–316, https://doi.org/10.1007/s00704-013-0953-6, 2014. Bouwman, A. F.: Direct emission of nitrous oxide from agricultural soils, Nutr. Cycl. Agroecosystems, 46, 53–70, https://doi.org/10.1007/BF00210224, 1996. Bracher, A., Schlegel, P., Münger, A., Stoll, W., and Menzi, H.: Möglichkeiten zur Reduktion von Ammoniakemissionen durch Fütterungsmassnahmen beim Rindvieh (Milchkuh), SHL Agro- scope, Zollikofen Posieux, 2011. Review statement. This paper was edited by Andreas Ibrom and re- viewed by two anonymous referees. Butterbach-Bahl, K., Baggs, E. M., Dannenmann, M., Kiese, R., and Zechmeister-Boltenstern, S.: Nitrous oxide emissions from soils: how well do we understand the processes and their con- trols?, Philos. Trans. R. Soc. B Biol. Sci., 368, 20130122, https://doi.org/10.1098/rstb.2013.0122, 2013. K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale K. Voglmeier et al.: Grazing-related nitrous oxide emissions: from patch scale to ﬁeld scale northern Spain, Agric. Ecosyst. Environ., 135, 132–139, https://doi.org/10.1016/j.agee.2009.09.007, 2010. northern Spain, Agric. Ecosyst. Environ., 135, 132–139, https://doi.org/10.1016/j.agee.2009.09.007, 2010. to the current default EF of 2 % for cattle excreta provided by the guidelines of the IPCC. The ﬁndings also exhibited clear differences in the individual EFs for urine and dung (1.12±0.43 % and 0.16±0.06 %, respectively, averaged over systems M and G), suggesting a corresponding disaggrega- tion in emission inventories. to the current default EF of 2 % for cattle excreta provided by the guidelines of the IPCC. The ﬁndings also exhibited clear differences in the individual EFs for urine and dung (1.12±0.43 % and 0.16±0.06 %, respectively, averaged over systems M and G), suggesting a corresponding disaggrega- tion in emission inventories. Barneze, A. S., Minet, E. P., Cerri, C. C., and Misselbrook, T.: The effect of nitriﬁcation inhibitors on nitrous oxide emissions from cattle urine depositions to grassland under summer conditions in the UK, Chemosphere, 119, 122–129, https://doi.org/10.1016/j.chemosphere.2014.06.002, 2015. Bates, G., Quin, B., and Bishop, P.: Low-cost detection and treat- ment of fresh cow urine patches, in Moving farm systems to im- proved attenuation, edited by: Currie, L. D. and Burkitt, L. L., Vol. 28, 12, Palmerston North, New Zealand, 2015. Data availability. Data obtained in this study are available online at https://doi.org/10.5281/zenodo.2601821 (Voglmeier et al., 2019). Bell, M. J., Rees, R. M., Cloy, J. M., Topp, C. F. E., Bagnall, A., and Chadwick, D. R.: Nitrous oxide emissions from cattle exc- reta applied to a Scottish grassland: Effects of soil and climatic conditions and a nitriﬁcation inhibitor, Sci. Total Environ., 508, 343–353, https://doi.org/10.1016/j.scitotenv.2014.12.008, 2015. 4.4 Advantages and problems of experimental setup The presented ﬁeld campaign was designed to estimate the N2O emissions of two parallel grazing systems and to com- pare different feeding diets of the herds. Field-scale emis- sions derived by the EC method resulted in a wide range of measured emissions, which were mainly driven by en- vironmental and management-related parameters. Neverthe- less, the setup with two towers allowed for a good compar- ison with a sufﬁcient number of measured ﬂuxes from both systems. Due to a delayed installation of the EC tower at sys- tem G, all ﬂuxes prior to the middle of April had to be gap ﬁlled, which resulted in a higher associated uncertainty. The combined approach with EC and FB measurements proved to be appropriate to observe and quantify the mag- nitude of the pasture emissions and to calculate the contri- bution of the single emission sources. The different diet of the cows resulted in an excreta-related N2O emission differ- ence of about 25 % between the two cow herds and revealed the large potential of a N-optimized feeding strategy to re- duce grazing-related N2O emissions. In this study, the N op- timization was achieved by adding maize silage to the fodder in system M. However, a reduction in excreted N can poten- tially be realized by other means as well (e.g., grass varieties with a high content of water-soluble carbohydrates). The excreta-related EFs derived from the upscaled FB measure- ments were 0.74 ± 0.26 % for system M and 0.83 ± 0.29 % for system G and were thus signiﬁcantly lower compared The excreta N input derived by the animal budget approach at a temporal resolution of 1 day was needed in order to quan- tify the EF of the two systems and to upscale FB chamber measurements to the ﬁeld scale. Nevertheless, direct mea- surements would have been preferable. However, as the N content in the excreta is highly variable (Betteridge et al., Biogeosciences, 16, 1685–1703, 2019 www.biogeosciences.net/16/1685/2019/ 1701 Review statement. 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https://openalex.org/W2995497531	https://roderic.uv.es/bitstream/10550/72527/1/135555.pdf	English	null	On Augmented Reality for the Learning of 3D-Geometric Contents: A Preliminary Exploratory Study with 6-Grade Primary Students	Education sciences	2,019	cc-by	4,917	Article Míriam Flores-Bascuñana 1, Pascual D. Diago 1,* , Rafael Villena-Taranilla 2 1 Míriam Flores-Bascuñana 1, Pascual D. Diago 1,* , Rafael Villena-Taranilla 2 and Dionisio F. Yáñez 1 1 Departament de Didàctica de la Matemàtica, Universitat de València, 46010 València, Spain; miﬂobas@alumni.uv.es (M.F.-B.); Dionisio.Yanez@uv.es (D.F.Y.) 2 LabinTIC (Laboratorio de Integración de las TIC en el aula), Universidad de Castilla-La Mancha 1 Departament de Didàctica de la Matemàtica, Universitat de València, 46010 València, Spain; miﬂobas@alumni.uv.es (M.F.-B.); Dionisio.Yanez@uv.es (D.F.Y.) 2 LabinTIC (Laboratorio de Integración de las TIC en el aula), Universidad de Castilla-La Mancha 02001 Albacete, Spain; rafa.villenataranilla@gmail.com * Correspondence: Pascual.Diago@uv.es; Tel.: +34-963-864-487 1 Departament de Didàctica de la Matemàtica, Universitat de València, 46010 València, Spain; miﬂobas@alumni.uv.es (M.F.-B.); Dionisio.Yanez@uv.es (D.F.Y.) 2 LabinTIC (Laboratorio de Integración de las TIC en el aula), Universidad de Castilla-La Mancha, 02001 Albacete, Spain; rafa.villenataranilla@gmail.com * Correspondence: Pascual.Diago@uv.es; Tel.: +34-963-864-487 Received: 19 November 2019; Accepted: 18 December 2019; Published: 21 December 2019 p g * Correspondence: Pascual.Diago@uv.es; Tel.: +34-963-864-487 Received: 19 November 2019; Accepted: 18 December 2019; Published: 21 December 2019 Abstract: Nowadays, Augmented Reality (AR) is one of the emerging technologies with a greater impact in the Education ﬁeld. Research has proved that AR-based activities improve the teaching and learning processes. Also, the use of this type of technology in classroom facilitates the understanding of contents from different areas as Arts, Mathematics or Science. In this work we propose an AR-based instruction in order to explore the beneﬁts in a 6th-grade Primary course related to 3D-geometry shapes. This ﬁrst experiment, designed from an exploratory approach, will shed light on new study variables to perform new implementations whose conclusions become more consistent. The results obtained allow us to envisage that AR-based proposals slightly improve the classical didactic methods. Keywords: Augmented Reality; geometry; 3D-geometric shapes; spatial vision; primary education education sciences education sciences education sciences 1. Introduction In the last decade the use of the technological tools linked to the Information and Communication Technologies (ICT) has acquired a big importance, both as a didactic tool and as well as a provider of new environments for promoting the learning. Different authors are reporting the learning effects of a signiﬁcant use of technological environments in the classroom, specially those regarding to the Augmented Reality (AR) [1–3]. Although, AR has been deﬁned from different perspectives [4], we understand an AR environment as a system that allows for combining real world objects with virtual objects or superimposed information. As a result virtual objects seem to coexist in the same space with the real world [5]. These AR-based learning environments reinforce the signiﬁcant learning through new possibilities and motivational practices [6,7]. The AR has been considered as a technological tool capable to permeate on the teaching and learning mechanisms providing signiﬁcant impact on both processes. Due to the affordability and the ease of use, AR has been reported as a mid-term technological adoption (2–3 years) for K-12 levels and High Education levels [8,9]. Possibilities for AR applications in research are also promising. Different studies had demonstrated the AR usefulness for increasing the student motivation, as well as for enhancing a wide variety of learning domains [3,7,10–16]. In particular, the meta-analysis provided by [4] among JCR publications on the state of AR applications in the Education ﬁeld, concludes that there is a increasing trend, specially during the last 4 years, on the Science and Humanities & Arts areas. The AR has been applied with different goals to medicine, engineering, psychological treatments, etc. [17]. Concerning the educational approach, the beneﬁts of AR applications had been told to Educ. Sci. 2020, 10, 4; doi:10.3390/educsci10010004 www.mdpi.com/journal/education 2 of 9 Educ. Sci. 2020, 10, 4 strengthen the comprehension of complex concepts, supporting the contextualisation [11]. In this line, the use of the AR-based technologies allows an individual learning for each student and the manipulation of both tangible and virtual objects in an enhanced and motivational scenery. In spite of the generalised though that AR is an expensive resource, the integration of AR devices in the classroom does not imply a high cost because of the mobile devices. Different educational studies are providing clues to implement AR-based proposals at different ages [10,18,19]. 2. AR for Geometric and Spatial Learning Geometry comprises those branches of mathematics that exploit visual intuition (the most dominant of our senses) to remember theorems, understand proofs, inspire conjectures, perceive realities, and give global insight (Zeeman, quoted in [24], p. 12). Historically, spatial abilities have been addressed from a psychological standpoint [25]. Nowadays, the recent research on teaching and learning of Geometry covers geometric and spatial thinking among other content domains, as the geometric measurement or the teacher development [26]. In one hand, the spatial reasoning, underlying the most geometric thought, is deﬁned as a vital capacity for human action and thought, not always supported in school curricula ([27], p. 3). On the other hand, the geometric reasoning consists on the inventions and the use of formal conceptual systems that allows us to investigate the space and the shape in a mathematical way ([28], p. 843). The learning of Geometry at Primary levels can be exciting and fun for those pupils who are engaged with Mathematics, but the same cannot be told for those pupils showing less interest in Mathematics [29]. Usually, the geometric ideas in the curriculum are reduced to the identiﬁcation of 2D- and 3D-geometric shapes, related to area and volume measurements, not taking to much time on developing spatial reasoning and visualisation contents [26]. This approach does not ﬁt the international guidelines, that claim the use of visualisation, spatial reasoning, and geometric modelling to solve problems [30]. In this line, Duval in 1998 stood up for a teaching of Geometry beyond the arithmetic operations, in which three cognitive processes are involved: visualisation, construction and reasoning [31]. This position has strong implications on the teaching of Geometry, in the sense of involving spatial reasoning tasks on the Geometry teaching proposals. Moreover, has been stated that the interaction with technological environments improves the visualisation and spatial reasoning on the students [30,32]. The use of manipulatives in the Mathematics classroom is not new. In the longstanding history different kinds of physical materials have been used in order to explore, acquire, or investigate mathematical concepts or processes and to perform problem-solving activities drawing on perceptual (visual, tactile, or, more generally, sensory) evidence [33]. Nowadays, the technological progress is providing a big amount of virtual materials that, combined with the physical ones, offer a wide variety of possibilities to explore teaching activities in new and motivational manners. 1. Introduction As we will describe in the next section, the use of AR can support the learning of speciﬁc domains regarding to geometric and spatial abilities. The key point is that AR enhances the students’ visual perception. An AR-based activity allows us to manipulate virtual objects as well as to physical objects in the real world [14]. Thus, students can move around a 3D-virtual ﬁgure and view it from any vantage point, just like a real object. Different researchers have delved into the geometric and spatial abilities developed by students when using AR-based activities from Primary to High Education levels [14,20–22]. In particular, the 3D-activities based on AR in which the students have to represent and manipulate spatial and virtual objects are depicting promising results for extending the resources used in the Mathematics classroom [23]. Although nowadays AR has been incorporated to many educational practices [11], different aspects have to be explored in order to complete the implementation of AR-based activities in real class situations. In particular, this work pretends to explore the applicability of AR-based activities for the learning of 3D-geometric contents in a 6-grade Primary classroom. 3. Aim With this research we aim to explore the potential of the AR-based activities through a short classroom intervention in a 6th-grade Primary class. Moreover, this experiment aims to test if AR-based activities enhances the acquisition of 3D-geometric contents, in particular those related with spatial abilities. 2. AR for Geometric and Spatial Learning In particular, the AR-based learning experiences produce as physical beneﬁts as virtual beneﬁts [23]. 3 of 9 Educ. Sci. 2020, 10, 4 Concerning the AR-based educational implementations related to Mathematics, these can be a powerful tool to strengthen the spatial visualisation. From a Mathematical Education approach, the spatial visualisation is determined as a set of mental skills that allow individuals to act in the context of mathematical graphical representations, taking this context in a broad sense that includes the usual representations in the different mathematical ﬁelds such as Geometry, Algebra, Arithmetic or Statistics [34]. In this line, the manipulation of AR 3D-geometric shapes enhances the spatial visualisation ability, in the terms described by Gutiérrez [35], by means of the use of visual or spatial elements, developed to solve problems or demonstrate properties. The learning of geometric contents through AR implementations is still a recent ﬁeld [23,36,37]. Modern dynamic geometric software (DGS) has stimulated research on students’ assessment regarding geometric content [38], through software like GeoGebra [39] or 3D computer-aided design (CAD) software [36]. However, the incorporation of AR into teaching situations still requires the exploration of several principles, such the design of the technological environment, the viability of the implementation, the curricular contents capable to address with the AR-based activities, the limitations posed by the context, the digital competences of the teachers and/or students, etc. [11]. Along these lines, this work tries to explore different variables related to the application of AR implementations on a real class context. 4. Materials and Methods This quasi-experimental study consists of an AR-based intervention of 5 sessions in a 6th-grade Primary classroom related to 3D-geometric contents. Although we have considered experimental and control groups to test our hypothesis, this is an exploratory study since we have not established an standard pre-post comparison. However, to determine the level of the students in geometric concepts, an initial test was developed. It was divided in ﬁve basic questions displayed in Table 1. The results presented in this manuscript have to be considered as a case study of an speciﬁc group from an speciﬁc school. Table 1. Questions of the initial test. Table 1. Questions of the initial test. Table 1. Questions of the initial test. 1 What is a polyhedron? 2 What types of polyhedrons you know? 3 Can you identify the notable elements of a polyhedron? 4 True of false: • Every revolution solid has a vertex. • A cylinder has two bases which are similar polygons. • A cone has a single vertex. 5 Draw the developments of a cone and of a prism. y • A cylinder has two bases which are similar polygo A h i l y • A cone has a single vertex. 4.2. Participants As mentioned above, the participants of this exploratory research were 30 students (56% male) of 6th Primary course (age 11 to 12 year-old) from an Spanish school. The sample was ethnically and socio-economically diverse. The intervention was conducted during the last semester of the academic year 2018–2019. The students were equally distributed into experimental group and control group, consisting each one of 15 students. Those groups were randomly selected. All participants were informed that we were carrying a research study and warned that the results obtained in the different assessment tests would not be considered in their grades. In addition, the parents were informed about this intervention with an ordinary communication. 4.1. The Intervention Design In order to test if AR-based activities enhances the geometric reasoning acquisition we have designed an intervention to be conducted in a Primary school. The intervention was intentionally designed to have a short duration (3.75 h), consisting in 5 sessions (described in Table 2). We opted for designing a 5-session duration intervention in order to be realistic according to the time usually devoted to technological activities (AR-based in our case) in real class situations. The participating students were separated into an experimental group and a control group. During the intervention, 4 of 9 Educ. Sci. 2020, 10, 4 both groups carried out the same 3D-geometric activities, but the experimental group uses AR-based materials and the control group worked with traditional manipulative materials. The schematic path of the experimental design of our research is provided in Figure 1 and will be described in detail later in the text. Figure 1. Scheme of the experimental design followed in this work. Figure 1. Scheme of the experimental design followed in this work. Table 2. Distribution of sessions and contents addressed in each of the designed sessions. Table 2. Distribution of sessions and contents addressed in each of the designed sessions. Session Content 1 3D-geometric shapes, deﬁnition of polyhedron 2 Identiﬁcation of 3D-geometric shapes through using videos 3 Review of 2D shapes and identiﬁcation. Identiﬁcation of a polyhedron using his development 4 Identiﬁcation 3D-geometric shapes in our everyday life 5 Characteristics and classiﬁcation of 3D-geometric shapes 5. Results and Discussion As a pre-test was not administered previously to the intervention, in the present study we use the results provided by the instrument to focus, exclusively, on the beneﬁts of implementing AR-based activities from an exploratory approach. In the following, we describe the results obtained by group in the initial test. These results prove that the group is homogeneous and that the students do not know the 3D-geometric concepts previous to the intervention abovementioned. 4.4. Instrument In order to measure the learning achieved by the students an instrument was speciﬁcally developed taking into account the contents that the students deal with during the intervention phase. The instrument (no validated) consisted of six items (showed in Table 3). All items were related to the 3D-geometric contents from the 6th Primary grade curriculum. Each item in the test was coded with a score between 0 (totally incorrect) and 10 (totally correct). The test was completed by the students two days after the intervention in a paper-and-pencil format. Table 3. Items selected to be completed by each student as ﬁnal assessment. Table 3. Items selected to be completed by each student as ﬁnal assessment. Final Assesment Items 1 Explain what 3D-geometric shapes are and in which two groups we can classify them 2 Put the parts of these 3D-geometric shapes (Shapes: hexagonal prism, cone and sphere. Parts: base, face, edge, vertex, body and radius) 3 Complete the table with the characteristics of the 3D-geometric shapes (name, number of faces, number of edges and number of vertices) 4 Circle with red the 3D-geometric shapes that are polyhedrons and circle with blue those that are round shapes 5 Write the name of the following developed 3D-geometric shapes 6 Match the 3D-geometric shape with arrows to its development and try to draw the missing one 4.3. Procedure and Materials The schematic procedure for this exploratory experiment is depicted in Figure 1. Once the class was separated in two groups both receive an intervention for the learning of 3D-geometric content according to their grade level. The academic content related to the intervention has not been presented prior in the academic course. Participants, in both the control and the experimental group, had to complete the same set of tasks during the intervention phase. The only difference was that the experimental group uses AR-based technological devices and the control group uses standard manipulative materials. It is important to mention that all students had been introduced to AR-based devices during the previous academic course. Specifically, they had been working with AR-devices on Science subject. Therefore, no instrumental factors due to the ability on the use of the AR-devices interfere in our experiment. The intervention took 5 sessions, 45 min each one, related to different aspects of 3D-geometric content, as shown in Table 2. Participants, divided into experimental and control group were encouraged to complete the tasks in each session. The control group made use of manipulative materials (Figure 2, right panel) while the experimental group worked with AR-based applications (Figure 2, left panel), in particular they work with the apps Geometry and Quiver. The manipulative materials of the control group were those typically present on an standard Primary classroom, i.e., 3D solids and developed nets. 5 of 9 5 of 9 Educ. Sci. 2020, 10, 4 Figure 2. (Left panel): AR-based task. (Right panel): standar manipulative task. Figure 2. (Left panel): AR-based task. (Right panel): standar manipulative task. 5.1. Mean Scores on the Initial Test In this section we show the results of the answers given by the complete group, i.e., 30 students. In the ﬁrst question (Table 1), only 4 students deﬁne correctly a polyhedron (13.33%). In terms of the typos of polyhedrons more than half of the students confuse the 3D-shapes with the 2D-shapes (53.33%). Only two are capable to explain the different typos of polyhedrons. Respect to the notable elements, any scholar made a satisfactory identiﬁcation on the parts of a polyhedron. It is interesting to note that only 26.66% of the students distinguish between 1 and 4 notable elements. The best results are provided in the 4th question, due to 42% of the pupils mark successfully 2 phrases. However, in Educ. Sci. 2020, 10, 4 6 of 9 this question a noise is introduced in the result because the multiple choice answer. Finally, 13% of the students are not capable to draw neither the development of a cone nor the development of a prism, 17% of the students answer correctly to this question and the rest of the students only draw one development. In conclusion, the results obtained allow us to divide the class in the two groups (experimental and control) using any probabilistic method. 5.2. Mean Scores on the Final Assessment Test Firstly, we address to the assessment of the items posed on the final test. Figure 3 shows the mean results obtained for each item, the mean score taking into account all students was a 8.38. It is important to point out that in the question 4 the mean score is bigger than 9. It indicates that the students are capable to distinguish between prisms and revolution solids. Therefore, using AR-based activities as well as using manipulative elements, the scholars improve their capacity to understand the principal properties of the solids. Also, in question 6, the mean achieves 9.23, therefore, the pupils draw correctly the development of the 3D solids. It is specially relevant in AR-based activities because of the apps used to teach it. In questions 1 and 2, the mean is between 8.5 and 8.9. The mistakes presented in the answers are due to little confusions in similar polyhedron and special notable elements as the radius. We can considerate that the concepts are satisfactory acquired. In the items 3 and 5 the mean is minor than the rest of items. Probably, the principal reason which explains these scores is that items are related with memorising abilities. In any case, the results evidence the improvement showed by the students in comparison with the initial prove. On the other hand, the exploratory study points out that teaching 3D-geometric solids with AR produces a more motivating learning. Also, the performance showed by the students points along these lines. But more rigorous experiments are necessary to obtain clearer conclusions. Figure 3. Mean score obtained for each item of the ﬁnal test. 5.3. Comparison between Control and Experimental Groups Figure 3. Mean score obtained for each item of the ﬁnal test. 5.3. Comparison between Control and Experimental Groups 5.3. Comparison between Control and Experimental Groups By the initial test, we suppose that the students of control and experimental groups obtain similar qualiﬁcations before the instruction (see Section 5.1). However, the mean score obtained for the experimental group, 8.7, is slightly higher than the score obtained for the control group, 8.1. It is important to remark that the manipulative didactic elements are an excellent way to introduce 3D-geometric solids and their characteristics. Thus, this explains that the differences between the 7 of 9 Educ. Sci. 2020, 10, 4 results of the two methods (manipulative vs. AR-based) are minor. 5.2. Mean Scores on the Final Assessment Test Despite of this, with the AR-based activities presented in this paper the mean score is improved. Also, the students present a high degree of satisfaction when learn with this new instruction based on AR. 6. Final Remarks The present study provides results on the effectiveness of AR-based instruction for the development of 3D-geometric contents in a 6th-grade Primary students. This study has underlined the potential of AR-based activities in promoting students’ acquisition of geometric and spatial abilities, according to previous works [11,15]. However, our results are weak due to the absence of an evaluation of the students’ knowledge previously to the intervention phase. This experimental approach has shown hints for future intervention designs, as the study of 3D-geometric development learning with AR-based technology. Moreover, it will be interesting to introduce new and more difﬁcult 3D shapes, as the semiregular polyhedrons, oblique cylinders or oblique cones, that are not habitual to ﬁnd as manipulative materials. Also, we plan to measure emotional factors in further interventions using AR-based activities. The goal will be to check the inﬂuence of these technological environments on the students’ motivation. Therefore, future studies are necessary in order to delve into the causes of the academic gains showed on this research. Author Contributions: Data curation, R.V.-T. and D.F.Y.; Investigation, R.V.-T. and P.D.D.; Methodology, M.F.-B.; Project administration, P.D.D.; Supervision, P.D.D. and D.F.Y.; Writing—original draft, M.F.-B.; Writing—review & editing, P.D.D., R.V.-T. and D.F.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Conselleria d’Innovació, Universitats, Ciència i Societat Digital de la Generalitat Valenciana, grant number GV/2019/146. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Author Contributions: Data curation, R.V.-T. and D.F.Y.; Investigation, R.V.-T. and P.D.D.; Methodology, M.F.-B.; Project administration, P.D.D.; Supervision, P.D.D. and D.F.Y.; Writing—original draft, M.F.-B.; Writing—review & editing, P.D.D., R.V.-T. and D.F.Y. All authors have read and agreed to the published version of the manuscript. 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https://openalex.org/W2001379032	http://file.scirp.org/pdf/AJPS_2013041710244261.pdf	English	null	Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo	American journal of plant sciences	2,013	cc-by	7,671	American Journal of Plant Sciences, 2013, 4, 824-837 http://dx.doi.org/10.4236/ajps.2013.44101 Published Online April 2013 (http://www.scirp.org/journal/ajps) American Journal of Plant Sciences, 2013, 4, 824-837 Received February 23rd, 2013; revised March 26th, 2013; accepted April 2nd, 2013 Received February 23rd, 2013; revised March 26th, 2013; accepted April 2nd, 2013 Received February 23rd, 2013; revised March 26th, 2013; accepted April 2nd, 2013 Copyright © 2013 Marcel Muengula-Manyi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT The responses of eight genetically improved and eight local cassava varieties to cassava mosaic disease (CMD) were evaluated under field conditions at two sites, in Eastern Kasaï, region of the DR-Congo). The varieties were planted using randomized complete block design with three replications. The rate of cuttings sprouted varied significantly from variety to variety and from location to location. Local varieties were severely infected than improved varieties through- out the trial period. In general, the level of CMD incidence for genetically improved varieties was below 15% while it reached 100% for the local cassava varieties six months after planting (MAP). This trend was also observed for the CMD severity and gravity. The mean scores for CMD severity were 2 and 3.6 for genetically improved and local varie- ties, respectively at 6 MAP. CMD gravity for improved varieties was below 21% for genetically improved varieties and exceeded 85% for local varieties at the end of trials. Area Under the Severity index Progress Curve (AUSiPC) and Area Under Disease Progress Curve (AUDPC) estimates confirmed that improved varieties were moderately infested com- paratively to local varieties. Molecular analysis is being conducted to determine the genetic variability and complexity of the cassava mosaic virus strains involved. Keywords: Cassava Mosaic Disease; Local Cassava Varieties; Genetically Improved Cassava Varieties; AUSiPC; AUDPC; Disease Index; DR-Congo Corresponding author. Copyright © 2013 SciRes. Marcel Muengula-Manyi1, Lyna Mukwa2, Kabwe K. Nkongolo3, Patrick Tshilenge-Djim1, Stephan Winter4, Claude Bragard5, Adrien Kalonji-Mbuyi1,6 1Unit of Phytopathology, Faculty of Agronomy, University of Kinshasa, Kinshasa, Democratic Republic of Congo; 2Plant Clinic of Kinshasa, Kinshasa, Democratic Republic of Congo; 3Department of Biology, Laurentian University, Sudbury, Canada; 4Plant Virus Collection, DSMZ Deutsche Sammlung von Mikroorganismen und Zellkuturen GmbH, Braunschweig, Germany; 5Earth and Life Institute, Applied Microbiology-Phytopathology, Université Catholique de Louvain, Louvain-la-Neuve, Belgium; 6Regional Nuclear Energy Center, Kinshasa, Kinshasa, Democratic Republic of Congo. Email: *knkongolo@laurentian.ca 1. Introduction nificant yield losses of up to 95%, especially in fields where susceptible cultivars are grown [8]. A key factor in the genesis and spread of the pandemic has been the re- combination between two distinct cassava mosaic gemi- niviruses to produce a novel and more virulent hybrid [9]. To manage this pandemic, several genetically improved varieties resistant to the CMD have been developed and released in diverse agricultural regions. Egesi et al. [10] reported that in most resistant varieties, the disease is usually confined only to a few leaves or branches. How- ever, several of these varieties have not been easily adopted because several farmers’ preferred characteris- tics related to environmental conditions, organoleptic traits and marketability have been ignored by several Cassava mosaic disease (CMD) is the principal constraint of cassava (Manihot esculenta Crantz) crop production in sub-Saharan African countries [1-4]. It is caused by sev- eral cassava mosaic geminiviruses and is the most im- portant disease of cassava in Africa and the Indian sub- continent [5,6]. In DR-Congo (DRC) where cassava is a source of food and income for about 70% of population, CMD represents an important threat to food security for many Congolese families who rely on this staple food [7]. In sub-Saharan African countries, CMD can cause sig- Copyright © 2013 SciRes. AJPS Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 825 ture averages 25˚C and annual rainfall is close to 1500 mm. cassava improved programmes [7]. In addition, newly introduced improved varieties have been abandoned by farmers in areas where there were grown at a large scale, because of the loss of resistance to CMD due in part to new strains and agronomic practices that are favourable to the virus spread [7,9,11]. 2.1. Field Trials Field experiments were conducted at two sites in Ganda- jika region (Eastern Kasai) in DR-Congo (Figure 1). Site 1 was located at INERA research station (23˚57'E, 06˚48'S and 754 m altitude) and site 2 in Mpiana (23˚56'E, 06˚36'S and 685 m altitude). Site 1 was chosen because it is considered to be a low CMD-spread site where the use of resistant varieties and phytosanitation practice are applied. The choice of the site 2 (Mpiana) was motivated by the fact that it has been used for over 10 years by farmers to grow cassava with limited phyto- sanitation practices. 2.3. Experimental Design The study was conducted in 2009 during the growing season B (from February to July). In each site, two sepa- rate trials were conducted. The cassava materials were planted in a randomized complete block design (RBCD) with three replications. Each variety represented a treat- ment. Hardwood stem cuttings of each variety were planted in a plot of 30 plants at spacing of 1 m × 1 m, resulting in a population of 10,000 plants·ha−1. Each plot measured 5 m × 4 m. Plots were weeded whenever nec- essary and no pesticide or fertilizer was applied through- out the period of the study. Gandajika region falls within the Aw4 climate type according to Köppen classification characterized with 4 months of dry season (from mid-may to august) coupled with 8 months of rainy season, sometimes interrupted by a short dry season in January/February. Daily tempera- 2.2. Cassava Varieties Used in the Study The plant materials used in the present study consisted of 16 different local and genetically improved cassava va- rieties. Eight local varieties including Kamana-mabanza, Kavuandula, Luenyi, Madame, Muana-mpiana, Mutom- bo-tshomba and Tshilombo were evaluated. The eight improved varieties that were also evaluated include Bu- tamu, Disanka, Gandajika, F 100, Mvuazi, Sadisa, TME 419 and Zizila. These new improved varieties were pro- vided by the National Cassava Programme (PRONAM) while local varieties were from farmers’ fields. Hard- wood stem cuttings used as planting material were se- lected from symptom-less plants. The main objective of the present study is to charac- terize field responses of new genetically improved and local cassava varieties from a savannah region of DR- Congo. Copyright © 2013 SciRes. 2.4. Data Recorded and Statistical Analysis Gandajika Figure 1. DR-Congo map. The arrow indicates the region (Gandajika) where the field trials were conducted. The selected parameters used to compare varieties in- clude rate of stem cuttings sprouted, cassava mosaic dis- ease (CMD) incidence, severity and gravity, and tuberous root yield. The rate of cuttings sprouted was recorded 2 weeks after planting and was assessed for each variety based on the percentage of cuttings sprouted compared to the total number of stem cuttings planted in the plot. CMD incidence, severity and gravity were recorded at 1, 3 and 6 months after planting (MAP). The CMD inci- dence represented the percentage of diseased plants compared to the total number of plants present in the plot. The CMD symptom severity was assessed for each plant in the plot using a scale of 1 to 5 described by Hahn et al., [12] and Terry and Hahn [13], where: 1 = no symptoms; 2 = mild chlorosis, mild distortions at bases of most leaves, while the remaining parts of the leaves and leaf- lets appear green and healthy; 3 = pronounced mosaic pattern on most leaves, narrowing and distortion of the lower one-third of the leaflets; 4 = severe mosaic distor- tion of two thirds of most leaves and general reduction of leaf size and stunting of shoots; and 5 = very severe mo- Figure 1. DR-Congo map. The arrow indicates the region (Gandajika) where the field trials were conducted. Copyright © 2013 SciRes. AJPS AJPS Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 826 saic symptoms on all leaves, distortion, twisting, mis- shapen and severe leaf reductions of most leaves accom- panied by severe stunting of plants. assessed per plant by the percentage of cassava leaves with typical symptoms of the disease. CMD incidence and gravity data were tested for ho- mogeneity of variance before analyses. 3. Results where SS1 is the CMD severity score at time t1 and SS2 is the CMD severity score at time t2. The CMD gravity was Morpho-agronomic data of all the varieties used in the present study are described in Table 1. The results for where SS1 is the CMD severity score at time t1 and SS2 is the CMD severity score at time t2. The CMD gravity was Table 1. Some agro-morphological characteristics of genetically improved and local cassava varieties used in the study. Principals morphological characters Principals agronomic characters Variety Varietal type Leaf colour Lobe shape Tuber root skin colour Flesh colour Number of tuberous root/plant Yield in research station Yield in farmer field Status to CMD Improved variety Butamu Sweet Light green Oblong Brown Yellow 4 - 5 25 - 40 t·ha−1 10 - 20 t·ha−1 Tolerant Disanka Bitter Purple to dark green Oblong Brown White 6 - 8 35 - 50 t·ha−1 20 - 25 t·ha−1 Resistant F100 - - - - - - - - Tolerant Gandajika Bitter - - - White 6 - 8 25 - 35 t·ha−1 15 - 20 t·ha−1 Resistant Mvuazi Bitter Purple to dark green Lanceolate Brown White 6 - 8 35 - 50 t·ha−1 20 - 25 t·ha−1 Resistant Sadisa Bitter Purple green to dark green Lanceolate Yellowish white Yellowish white 6 - 8 25 - 45 t·ha−1 7 - 10 t·ha−1 Sensible TME419 - - - - - - - - Resistant Zizila Sweet Purple to dark green Lanceolate Dark brown White 5 - 8 25 - 35 t·ha−1 10 - 20 t·ha−1 Resistant Local variety Kamana mabanza Sweet Dark green to dark green Oblong - White 6 - 9 - - Susceptible Kavuandula - - - - White 5 - 7 - - Susceptible Luenyi Sweet Dark green - - White 6 - 8 25 - 35 t·ha−1 10 - 20 t·ha−1 Susceptible Madame Sweet - - - White 5 - 7 - - Susceptible Muana mpiana - - - - White 5 - 7 - - Susceptible Mutombo tshomba Sweet - - - White 5 - 8 - - Susceptible Nzaza Sweet - - - White 4 - 6 - - Susceptible Tshilobo Sweet Light green Lanceolate - White 6 - 8 25 - 40 t·ha−1 10 - 20 t·ha−1 Susceptible - : Data not available. 2.4. Data Recorded and Statistical Analysis They were trans- formed using the formula arcsin y y  as described by Legendre and Legendre [15], where y represent the proportion of infected plants (level of incidence) or the proportion of leaves with CMD symptoms (level of grav- ity). Data were subjected to the analysis of variance (ANOVA) using the Statistix 8 (free version) and R softwares. Mean effects were separated by least signifi- cant differences (LSD) at 5% of probability level. The response of each variety to CMD was also evaluated by determining the disease index (DI) which was calculated using the formula described by Fargette and Vié (1994) where: DI = CMD incidence (%) × CMD severity/100. The Area Under Disease Progress Curve (AUDPC) was calculated using CMD incidence as described by Shaner and Finney [14] as follows:   1 1 AUDPC 2 n i i i x x t          , where xi is CMD incidence at the time i, n is the number of data taken and t is the number of days between the registration of xi and 1 ix . The mean severity score was used to calculate the adapted Area Under Severity Index Progress Curve (AUSiPC). This was calculated based on the following formula:   1 1 2 2 1 1 AUSiPC 2 n SS SS x t t            3.2. Cassava Mosaic Disease Incidence It was noted that all cassava varieties used in the present study expressed cassava mosaic disease symptoms over- time. In general, the local varieties showed higher CMD incidence values compared to genetically improved va- rieties. One month after planting (1 MAP), CMD symp- toms were observed only on local varieties. The highest incidence of CMD at 3 MAP was observed on Sadisa plants (6.7%) and F100 plants (9.5%) at sites 1 and 2 respectively, while the lowest incidence was observed on Gandajika plants (3%) at site 1 and Disanka and Ganda- jika plants (2.8%), at site 2 (Table 1). At 6 MAP, the highest incidence was recorded on Disanka (9.7%) and Gandajika (13.8%) plants at sites 1 and 2, respectively 3. Results orphological characteristics of genetically improved and local cassava varieties used in the study. Table 1. Some agro-morphological characteristics of genetically improved and local cassava vari AJPS Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 827 For local cassava varieties, the highest rate of cuttings sprouted was observed in Luenyi (94.4%) at sites 1 and 2, while the lowest rate was recorded in Kavuandula and Mutombo-tshomba (66.6%) at site 1 and Tshilobo (50%) at site 2 (Figure 2). For local cassava varieties, the highest rate of cuttings sprouted was observed in Luenyi (94.4%) at sites 1 and 2, while the lowest rate was recorded in Kavuandula and Mutombo-tshomba (66.6%) at site 1 and Tshilobo (50%) at site 2 (Figure 2). the rate of cuttings sprouted are illustrated in Figure 2. Data for CMD incidence and gravity are summarized in Tables 2 and 3, respectively. Results of rate cuttings and CMD severity are presented in Figures 2 and 3, respec- tively. Area Under Disease Progress Curve (AUDPC) and the total amount of disease severity expressed as the factor Area Under Severity index Progress Curve (AU- SiPC) are illustrated in Figures 4 and 5, respectively. Details on disease index curves overtimes for all cassava varieties are presented in Figures 6 and 7. Copyright © 2013 SciRes. Copyright © 2013 SciRes. AJPS 3.1. Rate of Stem Cuttings Sprouted AJPS Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 828 Table 3. Cassava mosaic disease gravity in field trials using genetically improved and local cassava varieties in Gandajika (DR-Congo). Site 1: INERA Site 2: MPIANA Type of variety 1 MAP 3 MAP 6 MAP 1 MAP 3 MAP 6 MAP Improved TME 419 0 0 2.0 (0.08) 0 0 4.6 (0.12) Mvuazi 0 6.2 (0.20) 4.7 (0.17) 0 5.8 (0.14) 10.1 (0.19) Sadisa 0 2.9 (0.09) 4.6 (0.12) 0 4.7 (0.12) 9.3 (0.18) Disanka 0 0 3.6 (0.18) 0 4.0 (0.11) 7.5 (0.16) Zizila 0 0 1.6 (0.07) 0 11.1 (0.28) 20.8 (0.39) F100 0 8.3 (0.17) 2.4 (0.12) 0 4.2 (0.12) 7.4 (0.16) Butamu 0 0 1.7 (0.07) 0 7.4 (0.16) 9.1 (0.18) Gandajika 0 3.1 (0.10) 5.1 (0.21) 0 8.2 (0.23) 12.4 (0.34) LSD(0.05) - - - - - - Local Madame 15.6 (0.33) 45.0 (0.73) 90.2 (1.26) 39.9 (0.68) 76.8 (1.15) 100 (1.57) Nzanza 7.7 (0.16) 34.7 (0.62) 94.7 (1.43) 28.8 (0.56) 71.7 (1.09) 98.2 (1.49) Muana mpiana 25.1 (0.52) 49.6 (0.78) 97.2 (1.47) 30.4 (0.58) 71.7 (1.09) 95.7 (1.40) Kamana mabanza 3.6 (0.11) 37.2 (0.65) 93.4 (1.35) 28.0 (0.55) 63.8 (1.01) 85.4 (1.24) Tshilobo 33.0 (0.60) 56.4 (0.85) 90.4 (1.25) 23.8 (0.50) 100 (1.57) 100 (1.57) Kavuandula 7.9 (0.16) 28.0 (0.55) 94.3 (1.37) 24.4 (0.51) 72.8 (1.10) 96.4 (1.41) Luenyi 11.7 (0.28) 45.1 (0.73) 100 (1.57) 19.5 (0.45) 48.8 (0.77) 90.3 (1.25) Mutombo tshomba 28.8 (0.56) 42.5 (0.70) 100 (1.57) 19.8 (0.46) 66.5 (1.04) 100 (1.57) LSD(0.05) 0.34 0.12 0.23 0.13 0.65 0.24 Data (in parentheses) were transformed using formula arcsin y  y (Legendre and Legendre, 1998); LSD: Least significant difference; MAP: months after planting. aic disease gravity in field trials using genetically improved and local cassava varieties in Gandajika Table 3. Cassava mosaic disease gravity in field trials using genetically improved and local cassava varieties in Gandajika (DR-Congo). while the lowest incidence was observed on Zizila plants (2.8%) at site 1 and Disanka plants (2.8%) at site 2 (Ta- ble 2). the maximum level of incidence was also observed on Madame plants (100%) while the lowest level of inci- dence was observed on Kamana-mabanza (75.5%). At 6 MAP, all the cassava varieties (100%) were infested at two sites (Table 2). 3.1. Rate of Stem Cuttings Sprouted In each site, analysis of variance revealed significant differences (P < 0.05) among varieties. In general, the rate of cuttings sprouted varied among variety and among sites. The highest rate of cuttings sprouted for genetically improved varieties was recorded in Mvuazi and Ganda- jika (100%), for sites 1 (INERA) and 2 (Mpiana), respec- tively (Figure 2). The lowest rate was recorded in Sadisa (33.3%) at site 1 and in F100 (72.2%) at site 2 (Figure 2). Table 2. Cassava mosaic disease incidence in field trials using genetically improved and local cassava varieties in Gandajika (DR-Congo). Site 1: INERA Site 2: MPIANA Type of variety 1 MAP 3 MAP 6 MAP 1 MAP 3 MAP 6 MAP Improved TME 419 0 0 3.7 (0.11) 0 0 3.3 (0.10) Mvuazi 0 5.5 (0.19) 5.5 (0.19) 0 3.3 (0.10) 3.3 (0.10) Sadisa 0 6.7 (0.15) 6.7 (0.15) 0 7.4 (0.16) 7.4 (0.16) Disanka 0 0 9.7 (0.31) 0 2.8 (0.09) 2.8 (0.09) Zizila 0 0 2.8 (0.09) 0 6.1 (0.20) 6.1 (0.20) F100 0 4.2 (0.12) 8.9 (0.24) 0 9.5 (0.18) 9.5 (0.18) Butamu 0 0 3 (0.10) 0 3 (0.10) 3 (0.10) Gandajika 0 3 (0.10) 8.5 (0.29) 0 2.8 (0.09) 13.8 (0.37) LSD(0.05) - - - - - - Local Madame 55.5 (0.84) 100 (1.57) 100 (1.57) 71.2 (1.00) 100 (1.57) 100 (1.57) Nzanza 33.3 (0.52) 100 (1.57) 100 (1.57) 52.1 (0.80) 88.9 (1.36) 100 (1.57) Muana mpiana 88.9 (1.36) 100 (1.57) 100 (1.57) 63.8 (0.92) 93.3 (1.41) 100 (1.57) Kamana mabanza 15.1 (0.24) 44.4 (0.72) 100 (1.57) 41.1 (0.69) 75.5 (1.13) 100 (1.57) Tshilobo 68.9 (1.07) 100 (1.57) 100 (1.57) 100 (1.57) 100 (1.57) 100 (1.57) Kavuandula 33.3 (0.52) 100 (1.57) 100 (1.57) 100 (1.57) 100 (1.57) 100 (1.57) Luenyi 66.7 (1.04) 100 (1.57) 100 (1.57) 75.7 (1.13) 87.9 (1.27) 100 (1.57) Mutombo tshomba 83.3 (1.30) 100 (1.57) 100 (1.57) 100 (1.57) 100 (1.57) 100 (1.57) LSD(0.05) - 0.10 - 0.26 0.41 - Table 2. Cassava mosaic disease incidence in field trials using genetically improved and local cassava varieties in Gandajika (DR-Congo) aic disease incidence in field trials using genetically improved and local cassava varieties in Gandajika Table 2. Cassava mosaic disease incidence in field trials using genetically improved and local cassava v (DR-Congo). able 2. Cassava mosaic disease incidence in field trials using genetically improved and local cassava vari DR C ) Copyright © 2013 SciRes. 3.1. Rate of Stem Cuttings Sprouted For local varieties, analysis of variance revealed that CMD incidence was not significantly different (P > 0.05) among varieties at 1 and 6 MAP at site 1 (INERA). Sta- tistical analysis of data from site 2 (Mpiana) revealed significant differences (P < 0.05) in CMD incidence among varieties at 1 and 3 MAP. The highest incidence was recorded on Muana-mpiana plants (88.9%) at site 1, and on plants from Kavuandula, Mutombo-tshomba and Tshilobo (100%) at site 2, while the lowest incidence was recorded on Kamana-mabanza plants (15.1%) and (41.1%), at sites 1 and 2, respectively at 1 MAP (Table 1). At 3 MAP, the highest level of incidence was ob- served on plants from Kavuandula, Luenyi, Madame, Muana-mpiana, Mutombo-tshomba, Nzaza and Tshilobo (100%) while the lowest level of incidence was recorded on Kamana-mabanza (44.4%) plants at site 1. For site 2, Copyright © 2013 SciRes. 3.3. Cassava Mosaic Disease Severity In general, in each site, high CMD severity values (mean severity ≥ 3) were recorded for local varieties compared to improved varieties. In fact, during the trials period, the CMD severity for each improved variety did not exceed the mean score of 2 (Figure 3). Improved cassava varie- ties expressed typical symptoms of CMD three MAP. At this stage, at site 1 (INERA), only four varieties (Ganda- jika, F 100, Mvuazi and Sadisa) expressed CMD symp- toms. However, at site 2 (Mpiana), all the varieties ex- cept TME 419 developed CMD symptoms. At 6 MAP, all the genetically improved arieties tested were infested v AJPS 829 Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 0 20 40 60 80 100 120 TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA Variety Rate of cuttings sprouted (%) INERA MPIANA (a) 0 20 40 60 80 100 120 MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA Variety Rate of cuttings sprouted (%) INERA MPIANA (b) Figure 2. Rate of cuttings sprouted (14 days after planting). (a) Genetically improved varieties and (b) Local varieties. 0 20 40 60 80 100 120 TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA Variety Rate of cuttings sprouted (%) INERA MPIANA (a) (a) 0 20 40 60 80 100 120 MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA Variety Rate of cuttings sprouted (%) INERA MPIANA (b) Figure 2. Rate of cuttings sprouted (14 days after planting). (a) Genetically improved varieties and (b) Local varieties. Variety (b) (b) (b) ate of cuttings sprouted (14 days after planting). (a) Genetically improved varieties and (b) Local varieties Copyright © 2013 SciRes. AJPS Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 830 0 0,5 1 1,5 2 2,5 TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA Variety CMD severity (scale 1 - 5) INERA MPIANA (a) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA Variety CMD severity (scale 1 - 5) INERA MPIANA (b) Figure 3. Cassava mosaic disease severity (scale 1-5) recorded at 6 months after planting (MAP) at two sites in Gandajika (DR-Congo). (a) Genetically improved varieties; (b) Local varieties. Copyright © 2013 SciRes. Copyright © 2013 SciRes. 3.3. Cassava Mosaic Disease Severity Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 830 0 0,5 1 1,5 2 2,5 TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA Variety CMD severity (scale 1 - 5) INERA MPIANA (a) INERA MPIANA (a) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA Variety CMD severity (scale 1 - 5) INERA MPIANA (b) Figure 3. Cassava mosaic disease severity (scale 1-5) recorded at 6 months after planting (MAP) at two sites in Gandajika (DR-Congo). (a) Genetically improved varieties; (b) Local varieties. Variety (b) Variety (b) (b) Figure 3. Cassava mosaic disease severity (scale 1-5) recorded at 6 months after planting (MAP) at two sites in Gandajika (DR-Congo). (a) Genetically improved varieties; (b) Local varieties. Copyright © 2013 SciRes. AJPS 831 Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 0 200 400 600 800 1000 1200 TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA Variety AUDPC INERA MPIANA (a) 0 200 400 600 800 1000 1200 TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA Variety AUDPC INERA MPIANA (a) 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA Variety AUDPC INERA MPIANA (b) Figure 4. Cassava mosaic disease incidence expressed as the Area Under Disease Progress Curve (AUDPC) recorded at 2 sites in Gandajika (DR-Congo). (a) Improved varieties and (b) Local varieties. AUDPC INERA MPIANA (a) 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA Variety AUDPC INERA MPIANA (b) Figure 4. Cassava mosaic disease incidence expressed as the Area Under Disease Progress Curve (AUDPC) recorded at 2 sites in Gandajika (DR-Congo). (a) Improved varieties and (b) Local varieties. Variety (b) (b) Figure 4. Cassava mosaic disease incidence expressed as the Area Under Disease Progress Curve (AUDPC) recorded at 2 sites in Gandajika (DR-Congo). (a) Improved varieties and (b) Local varieties. Copyright © 2013 SciRes. Copyright © 2013 SciRes. Copyright © 2013 SciRes. 3.3. Cassava Mosaic Disease Severity AJPS Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 832 0 50 100 150 200 250 300 TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA Variety AUSiPC INERA MPIANA (a) 0 50 100 150 200 250 300 350 400 450 500 MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA Variety AUSiPC INERA MPIANA (b) Figure 5. Cassava disease severity expressed as the Area Under Disease index Progress Curve (AUSiPC) recorded at 2 sites in Gandajika (DR-Congo). (a) Genetically improved varieties; (b) Local varieties. Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 832 0 50 100 150 200 250 300 TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA Variety AUSiPC INERA MPIANA (a) (a) 0 50 100 150 200 250 300 350 400 450 500 MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA Variety AUSiPC INERA MPIANA (b) Figure 5. Cassava disease severity expressed as the Area Under Disease index Progress Curve (AUSiPC) recorded at 2 sites in Gandajika (DR-Congo). (a) Genetically improved varieties; (b) Local varieties. Variety (b) Variety (b) (b) Figure 5. Cassava disease severity expressed as the Area Under Disease index Progress Curve (AUSiPC) recorded at 2 sites in Gandajika (DR-Congo). (a) Genetically improved varieties; (b) Local varieties. Copyright © 2013 SciRes. AJPS 833 Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 0 0,05 0,1 0,15 0,2 0,25 1 2 3 4 5 6 MAP Disease index TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA 0 0,05 0,1 0,15 0,2 0,25 1 2 3 4 5 6 MAP Disease index TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA (a) 0 0,05 0,1 0,15 0,2 0,25 0,3 1 2 3 4 5 6 MAP Disease index TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA (b) Figure 6. Disease index curves recorded on different genetically mproved varieties at (a) Site 1 (INERA) and at (b) Site 2 (MPIANA) in Gandajika (DR-Congo). MAP represents months after planting. Copyright © 2013 SciRes. 3.4. Cassava Mosaic Disease Gravity CMD gravity for improved varieties was less than 21% regardless of the site. Typical CMD symptoms were ob- served in plots with genetically improved varieties from 3 MAP. In each site, analysis of variance did not reveal significant differences (P > 0.05) among improved varie- ties during the experimentation period (6 months). At 3 MAP, the highest level of gravity was observed in F 100 (8.3%) and Zizila (11.1%) while the lowest level of grav- ity was recorded in Sadisa (2.9%) and Disanka (4%), at site 1 and site 2, respectively (Table 3). At 6 MAP, the highest CMD gravity was recorded in Gandajika (5.1%) and Zizila (20.8%), while the lowest level of gravity was observed in Zizila (1.6%) and TME 419 (4.6%), at sites 1 and 2, respectively (Table 3). 1 and 2, respectively (Table 3). 1 and 2, respectively (Table 3). with the severity score of 2 for each variety at both sites (Figure 3). 3.5. Area Under Disease Progress Curve (AUDPC) CMD symptoms were observed in local variety plants as early as 1 MAP at both sites. At 3 MAP, the highest score was observed on Luenyi and Mutombo-tshomba plants (2.7) at site 1 and on Muana-mpiana (3) at site 2. The lowest severity score was noted in Kavuandula (2) and Kamana-mabanza, Kavuandula, Mutombo-tshomba and Nzaza (3), at sites 1 and 2, respectively. At 6 MAP, the highest CMD severity was observed in Kamana-ma- banza, Muana-mpiana, Mutombo-tshomba and Tshilobo (mean score 3.7) while the lowest level of CMD severity was recorded on Kavuandula, Luenyi, Madame and Nzaza plants (mean score 3.3) in site 1. However, at site 2, the highest level of CMD severity was recorded on Tshilobo plants (mean score 4) while the lowest severity was recorded on Kamana-mabanza plants (mean score 3). In each trial, the total amount of disease that occurred was calculated and expressed as the Area Under Disease Progress Curve (AUDPC). AUDPC values of the differ- ent varieties were compared and the results are presented in Figure 3. Analysis of variance for AUDPC values revealed significant differences (P < 0.05) among local varieties only. In site 1 (INERA), the highest value of AUDPC was observed on plants from Sadisa (improved) and Muana-mpiana (local) varieties while the lowest value was recorded on plants from Butamu (improved) and Kamana-mabanza (local). For site 2 (Mpiana), the highest AUDPC value for genetically improved varieties was recorded in F 100 while the lowest value was ob- served in TME 419. For local varieties, the highest value of AUDPC was observed on Kavuandula, Mutombo- tshomba and Tshilobo, while the lowest value was in Kamana-mabanza (Figure 4). 3.6. Area Under the Severity Index Progress Curve (AUSiPC) In each site, the Area Under Severity index Progress Curve (AUSiPC) calculated for data covering the 6 month trial period did not show significant differences (P > 0.05) among improved and local varieties. At the end of the experimentation period, the highest AUSiPC value for improved varieties was recorded on plants from Gandajika, F 100, Mvuazi and Sadisa (255) varieties while the lowest value of AUSiPC was observed in Bu- tamu and Zizila (165) for site 1. However, at site 2 (Mpiana), the AUSiPC values were 255 for all varieties, except for TME 419 variety which expressed the lowest AUSiPC value of 195 (Figure 5). The AUSiPC value estimated for local varieties revealed that, the highest value was recorded on plants from Mutombo-tshomba (425) while the lowest value was observed on plants from Kavuandula and Nzaza (390) at site 1. For site 2, the highest value of AUSiPC was recorded in Tshilobo (410) while the lowest value was observed in Kavuan- dula (375) (Figure 5). It was noted that the level of CMD gravity for local varieties was higher compared to improved varieties values at both sites. Analysis of variance revealed sig- nificant differences (P < 0.05) among varieties for both site 1 (INERA) and site 2 (Mpiana). At 1 MAP, the highest level of gravity was observed on Tshilobo plants (33%) and Madame plants (39.9%), while the lowest CMD gravity was recorded on Kamana-mabanza plants (3.6%) and Luenyi plants (19.5%), at site 1 and site 2, respectively (Table 3). At 3 MAP, the highest level of CMD gravity was recorded on Tshilobo variety (56% at site 1) and (100% at site 2), while the lowest level of gravity was recorded on Nzaza (34.7% at site 1) and Lu- enyi (48.8% at site 2) varieties. At 6 MAP, the maximum level of gravity (100%) was recorded on Mutombo- tshomba and Luenyi varieties at site 1 and on Tshilobo, Madame and Mutombo-tshomba varieties at site 2, while the lowest level of gravity was observed on Madame (90.2%) and Kamana-mabanza (85.4%) varieties, at sites 3.3. Cassava Mosaic Disease Severity AJPS 0 0,05 0,1 0,15 1 2 3 4 5 6 MAP Disease index TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA (a) 0 0,05 0,1 0,15 0,2 0,25 0,3 1 2 3 4 5 6 MAP Disease index TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA (b) Figure 6. Disease index curves recorded on different genetically mproved varieties at (a) Site 1 (INERA) and at (b) Site 2 (MPIANA) in Gandajika (DR-Congo). MAP represents months after planting. Copyright © 2013 SciRes. AJPS MAP (a) 0 0,05 0,1 0,15 0,2 0,25 0,3 1 2 3 4 5 6 MAP Disease index TME 419 MVUAZI SADISA DISANKA ZIZILA F100 BUTAMU GANDAJIKA (b) Figure 6. Disease index curves recorded on different genetically mproved varieties at (a) Site 1 (INERA) and at (b) Site 2 (MPIANA) in Gandajika (DR-Congo). MAP represents months after planting. (b) Figure 6. Disease index curves recorded on different genetically mproved varieties at (a) Site 1 (INERA) and at (b) Site 2 (MPIANA) in Gandajika (DR-Congo). MAP represents months after planting. Copyright © 2013 SciRes. AJPS Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 834 Mosaic Disease (CMD) Infection in a Savannah Region of the DR Congo 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 1 2 3 4 5 6 MAP Disease index MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA (a) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 1 2 3 4 5 6 MAP Disease index MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA (b) Figure 7. Disease index curves recorded on different local cassava varieties at (a) Site 1 (INERA) and at (b) Site 2 (MPIANA) n Gandajika (DR-Congo). MAP represents months after planting. i Copyright © 2013 SciRes. AJPS 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 1 2 3 4 5 6 MAP Disease index MADAME NZAZA MUANAMPIANA KAMANA MABANZA TSHILOBO KAVUANDULA LUENYI MUTOMBO TSHOMBA Disease index Disease index (b) Figure 7. Disease index curves recorded on different local cassava varieties at (a) Site 1 (INERA) and at (b) Site 2 (MPIANA) n Gandajika (DR-Congo). MAP represents months after planting. i Copyright © 2013 SciRes. 3.3. Cassava Mosaic Disease Severity AJPS 835 Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo Copyright © 2013 SciRes. 3.7. Disease Index (DI) The DI calculated six months after planting (6 MAP) did not vary significantly (P > 0.05) among varieties, for both genetically improved and local varieties in each site. The highest DI for improved varieties was recorded in Disanka, F 100 and Gandajika (0.2) while the lowest value of DI was recorded in Butamu, TME 419 and Zizila (0.06) at site 1. At site 2, the highest DI was ob- Copyright © 2013 SciRes. AJPS AJPS Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo Assessing Reactions of Genetically Improved and Local Cassava Varieties to Cassava Mosaic Disease (CMD) Infection in a Savannah Region of the DR-Congo 836 served in Gandajika variety (0.26) while the lowest level of DI was observed in Butamu, Disanka, Mvuazi and TME 419 (0.06) varieties (Figure 6). For local varieties the highest DI value was recorded on plants from Ka- mana-mabanza, Muana-mpiana, Mutombo-tshomba and Tshilobo (3.66) at site 1 and on Tshilobo plants (4.00) at site 2, while the lowest DI was recorded on plants from Kavuandula, Luenyi, Madame and Nzaza (3.33) and on Kamana-mabanza plants (3.00) at site 2, respectively (Figure 7). The AUSiPC estimates in the present study indicated that the disease developed with a high degree of severity in local varieties compared to improved varieties. The same trend was observed for the factor AUDPC that was high for local varieties than improved varieties. In fact, data reported by Banito et al. [22] on expression of re- sistance and tolerance of cassava genotypes to bacterial blight revealed similar trend. These authors noted in general that the local genotypes were severely infected and expressed higher values of AUSiPC than improved genotypes. A study conducted on effect of mineral fer- tilization on CMD expression by Muengula-Manyi et al. [23] reported that values of AUDPC for local cassava varieties were higher than for improved varieties plants. In general, the disease index (DI) recorded on improved varieties did not exceeded value of 1 while it reached up to 4 for Tshilobo (a local variety) plants at the site 2. According to Adriko et al. [19], DI depended on the ini- tial health status of plots and on the variety used. 4. Discussion In the present study show that the rate of cuttings spouted varies from one variety to another. This variability may be related to interactions that exist between genotypic characters of each variety and soil conditions of each site where cassava was planted. p Genetically improved and local cassava varieties evaluated in the present study responded differently to cassava mosaic disease (CMD) infection. In general, at both sites, typical CMD symptoms were observed on all cassava varieties. However, the levels of different CMD symptoms and indices varied considerably among im- proved and local varieties. For example, the mean of CMD incidence remained below 15% for improved va- rieties, while it reached 100% for all the local varieties at 6 MAP. The level of CMD incidence varied from one site to another and among varieties. This is consistent with previous findings reported by Otim-Nape [16], Nweke [17], Zinga et al., [18], and Adriko et al., [19]. The results of the present study show that the disease severity was moderate for genetically improved varieties and severe for local varieties. In general, the disease se- verity recorded on local cassava varieties depended on the type of variety planted. Our observations corroborate with data reported by Fauquet and Fargette [20] who revealed that the severity of CMD symptoms varies with the cultivar and increases with the plant age until about 60 days after planting. These authors noted afterwards that the level of CMD severity depended on several fac- tors such as the cultivar, climatic conditions, and grow- ing season. According to Abdullahi et al. [21], the dif- ferences observed in CMD incidence and severity among cassava clones are due to genotypic factors. In the pre- sent study, the CMD gravity remained below 21% for improved varieties while reaching 100% for some local varieties at 6 MAP. This finding suggests that the leaves present in the cassava plant expressed CMD symptoms with different levels of severity. In Gandajika region, studies conducted earlier revealed that the gravity of CDM symptoms varied among leaves of a same cassava plant. This observation suggests that the virus is not dis- tributed uniformly in different plant tissues. In conclusion, differences in reaction of cassava varie- ties to CMD infection were observed in the targeted sa- vannah region in DR-Congo. 3.7. Disease Index (DI) In the present case, the high value of DI observed on local va- rieties plants suggest that cuttings used from these varie- ties were initially infested although they did not ex- pressed CMD symptoms. In this study, the low level of disease parameters recorded in improved varieties ap- pears to be related to a presence of CMD tolerance genes as suggested by Muengula-Manyi et al. [23]. served in Gandajika variety (0.26) while the lowest level of DI was observed in Butamu, Disanka, Mvuazi and TME 419 (0.06) varieties (Figure 6). For local varieties the highest DI value was recorded on plants from Ka- mana-mabanza, Muana-mpiana, Mutombo-tshomba and Tshilobo (3.66) at site 1 and on Tshilobo plants (4.00) at site 2, while the lowest DI was recorded on plants from Kavuandula, Luenyi, Madame and Nzaza (3.33) and on Kamana-mabanza plants (3.00) at site 2, respectively (Figure 7). 5. Acknowledgements Authors express their gratitude to the Canadian Interna- tional Development Agency (CIDA) for financial support and to the CUI-CUD through the Project KIN 01 (Mo- lecular Biology) for the Doctoral fellowship to MMM. Thanks to Mbuyamba and technicians of the “Program- me National Manioc (PRONAM)” of the Research Cen- ter of the “Institut National pour l’Etude et la Recherche Agronomiques (INERA/Gandajika)” for their technical assistance during the experimentation period. 4. Discussion The CMD parameters and indices analysed revealed that the disease was very pro- nounced in local varieties compared to genetically im- proved varieties. This indicates that improved cassava varieties assessed in the present study carry genes for resistance to CMD viral strains in the targeted areas. Molecular characterization of strains collected in the study areas is being conducted to determine the level of their genetic variability and complexity. Copyright © 2013 SciRes. doi:10.1556/APhyt.38.2003.1-2.14 [3] J. P. Legg and B. Owor, “Cassava Mosaic Disease in Africa: Where Are the Epidemics?” Africa Crop Science Conference Proceedings, Vol. 6, 2003, pp. 322-328. [15] P. Legendre and L. 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https://openalex.org/W4362553610	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_from_Single-Cell_Sequencing_Reveals_Trajectory_of_Tumor-Infiltrating_Lymphocyte_States_in_Pancreatic_Cancer/22541815/1/files/40005181.pdf	English	null	Supplementary Figure from Single-Cell Sequencing Reveals Trajectory of Tumor-Infiltrating Lymphocyte States in Pancreatic Cancer	null	2,023	cc-by	1,418	2. Supplementary Figure S1 caption.… SUPPLEMENTARY FIGURE S1 Single cell sequencing reveals trajectory of tumor-infiltrating lymphocyte states in pancreatic cancer 1. Supplementary Figure S1……………………………………………………………………………………………………………2 2. SUPPLEMENTARY FIGURE S1 The uniform manifold approximation and projection (UMAP) of 39,694 single T-cells from 57 pancreatic ductal adenocarcinoma (PDAC) samples and 22 uninvolved samples, color coded by (A) cohort and (B) patient. Each dot is a single cell and is colored according to dataset/patient and transcriptomic cell state respectively. (C) Violin plots showing relative expression of the defining gene for each tumor-infiltrating lymphocyte (TIL) cell state. Plots are color coded according to level of expression as indicated by the color key. (D) RNAscope of cells in the PDAC TME: (i) Field view of tumor cells (KRT19/MUC1+) and CD3/CD4/CD8-expressing T cells from a highly T cell-infiltrated PDAC sample, and examples of (ii) CD4+ cells and (iii) CD8+ cells expressing markers of the single-cell RNAseq-defined T- cell states (left label) present in the PDAC TME (top label). Labels on the right indicate all markers in the panel for the slide and their associated pseudo-coloring. Each cell state is depicted for one cell, in two patients. Supplementary Figure S1. The uniform manifold approximation and projection (UMAP) of 39,694 single T-cells from 57 pancreatic ductal adenocarcinoma (PDAC) samples and 22 uninvolved samples, color coded by (A) cohort and (B) patient. Each dot is a single cell and is colored according to dataset/patient and transcriptomic cell state respectively. (C) Violin plots showing relative expression of the defining gene for each tumor-infiltrating lymphocyte (TIL) cell state. Plots are color coded according to level of expression as indicated by the color key. (D) RNAscope of cells in the PDAC TME: (i) Field view of tumor cells (KRT19/MUC1+) and CD3/CD4/CD8-expressing T cells from a highly T cell-infiltrated PDAC sample, and examples of (ii) CD4+ cells and (iii) CD8+ cells expressing markers of the single-cell RNAseq-defined T- cell states (left label) present in the PDAC TME (top label). Labels on the right indicate all markers in the panel for the slide and their associated pseudo-coloring. Each cell state is depicted for one cell, in two patients. Supplementary Figure S1. The uniform manifold approximation and projection (UMAP) of 39,694 single T-cells from 57 pancreatic ductal adenocarcinoma (PDAC) samples and 22 uninvolved samples, color coded by (A) cohort and (B) patient. Each dot is a single cell and is colored according to dataset/patient and transcriptomic cell state respectively. (C) Violin plots showing relative expression of the defining gene for each tumor-infiltrating lymphocyte (TIL) cell state. SUPPLEMENTARY FIGURE S1 Supplementary Figure S1 caption.………………………………………………………………………………………………3 MDA1_T12 D A C B Supplementary Figure S1 −3 0 3 6 −4 0 4 UMAP 1 UMAP 2 PUMCH MDA2 MDA1 −3 0 3 6 −4 0 4 UMAP_1 UMAP_2 MDA1_01 MDA1_02 MDA1_03 MDA1_04 MDA1_05 MDA1_06 MDA1_07 MDA2_01 MDA2_02 MDA2_03 MDA2_04 MDA2_05 MDA2_06 MDA2_07 MDA2_08 MDA2_09 MDA2_10 MDA2_11 MDA2_12 MDA2_13 MDA2_14 MDA2_15 MDA2_16 MDA2_17 MDA2_18 MDA2_19 MDA2_20 MDA2_21 MDA2_22 MDA2_23 MDA2_24 MDA2_25 MDA2_26 PUMCH_01 PUMCH_02 PUMCH_03 PUMCH_04 PUMCH_05 PUMCH_06 PUMCH_07 PUMCH_08 PUMCH_09 PUMCH_10 PUMCH_11 PUMCH_12 PUMCH_13 PUMCH_14 PUMCH_15 PUMCH_16 PUMCH_17 PUMCH_18 PUMCH_19 PUMCH_20 PUMCH_21 PUMCH_22 PUMCH_23 PUMCH_24 PUMCH_25 PUMCH_26 PUMCH_27 PUMCH_28 PUMCH_29 PUMCH_30 PUMCH_31 PUMCH_32 PUMCH_33 PUMCH_34 PUMCH_35 patient.4 DAPI+ CXCR4+ IFIT1- CCR7- CD4+ DAPI+ CXCR4- IFIT1 DAPI+ CXCR4- IFIT1 DAPI+ CXCR4+ CXCL13- FOXP3- CD4+ DAPI+ GZMK- CD4 DAPI+ ZNF683+ IL7R DAPI+ GZMK+ CXCL13- PRF1- CD8A+ DAPI+ GZMK+ CXCL13- PRF1- CD8A+ CD4-CXCR4 CD8-GZMK NF683 CCR7 MDA1_T11 MDA1_T09 MDA1_T09 MDA1_T08 MDA1_T10 MDA1_T01 MDA1_T01 MDA1_T12 (i) (ii) (iii) Supplementary Figure S1 B Supplementary Figure S1 −3 0 3 6 −4 0 4 UMAP_1 UMAP_2 MDA1_01 MDA1_02 MDA1_03 MDA1_04 MDA1_05 MDA1_06 MDA1_07 MDA2_01 MDA2_02 MDA2_03 MDA2_04 MDA2_05 MDA2_06 MDA2_07 MDA2_08 MDA2_09 MDA2_10 MDA2_11 MDA2_12 MDA2_13 MDA2_14 MDA2_15 MDA2_16 MDA2_17 MDA2_18 MDA2_19 MDA2_20 MDA2_21 MDA2_22 MDA2_23 MDA2_24 MDA2_25 MDA2_26 PUMCH_01 PUMCH_02 PUMCH_03 PUMCH_04 PUMCH_05 PUMCH_06 PUMCH_07 PUMCH_08 PUMCH_09 PUMCH_10 PUMCH_11 PUMCH_12 PUMCH_13 PUMCH_14 PUMCH_15 PUMCH_16 PUMCH_17 PUMCH_18 PUMCH_19 PUMCH_20 PUMCH_21 PUMCH_22 PUMCH_23 PUMCH_24 PUMCH_25 PUMCH_26 PUMCH_27 PUMCH_28 PUMCH_29 PUMCH_30 PUMCH_31 PUMCH_32 PUMCH_33 PUMCH_34 PUMCH_35 patient.4 B Su −3 0 3 6 −4 0 4 UMAP_1 UMAP_2 M M M M M M M M M M M M M M M M M patient.4 A −3 0 3 6 −4 0 4 UMAP 1 UMAP 2 PUMCH MDA2 MDA1 B A MDA1_T12 MDA1_T12 MDA1_T08 MDA1_T08 MDA1_T12 D C −3 0 3 −4 0 4 UMAP 1 UMAP 2 PUMCH MDA2 MDA1 −3 0 3 6 −4 0 4 UMAP_1 UMAP_2 MDA1_02 MDA1_03 MDA1_04 MDA1_05 MDA1_06 MDA1_07 MDA2_01 MDA2_02 MDA2_03 MDA2_04 MDA2_05 MDA2_06 MDA2_07 MDA2_08 MDA2_09 MDA2_10 MDA2_12 MDA2_13 MDA2_14 MDA2_15 MDA2_16 MDA2_17 MDA2_18 MDA2_19 MDA2_20 MDA2_21 MDA2_22 MDA2_23 MDA2_24 MDA2_25 MDA2_26 PUMCH_01 PUMCH_03 PUMCH_04 PUMCH_05 PUMCH_06 PUMCH_07 PUMCH_08 PUMCH_09 PUMCH_10 PUMCH_11 PUMCH_12 PUMCH_13 PUMCH_14 PUMCH_15 PUMCH_16 PUMCH_17 PUMCH_18 PUMCH_20 PUMCH_21 PUMCH_22 PUMCH_23 PUMCH_24 PUMCH_25 PUMCH_26 PUMCH_27 PUMCH_28 PUMCH_29 PUMCH_30 PUMCH_31 PUMCH_32 PUMCH_33 PUMCH_34 PUMCH_35 DAPI+ CXCR4- IFIT1+ CCR7- CD4+ DAPI+ CXCR4- IFIT1+ CCR7- CD4+ DAPI+ CXCR4+ IFIT1- CCR7- CD4+ DAPI+ CXCR4- IFIT1- CD4+ CCR7+ DAPI+ CXCR4- IFIT1- CD4+ CCR7+ DAPI+ CXCR4+ CXCL13- FOXP3- CD4+ DAPI+ CXCR4- CXCL13- FOXP3+ CD4+ DAPI+ GZMK- CD4- ZNF683+ CD8A+ DAPI+ CXCR4- CXCL13+ CD4+ FOXP3- DAPI+ CXCR4- CXCL13- CD4+ FOXP3+ DA ZNF IL7 CXC CD8 DAPI+ GZMK- CXCL13- PRF1+ CD8A+ DA GZ CX PR CD DAP GZM CXC PRF CD8 DAPI+ GZMK- CXCL13+ PRF1- CD8A+ DAPI+ GZMK+ CXCL13- PRF1- CD8A+ DA GZM CXC PRF CD8 DAPI+ CCR7+ IL7R+ CXCR6- CD8A+ DA CC IL7 CXC CD8 CD4-FOXP3 CD4-CXCR4 CD8-GZMK CD8-ZNF683 CD8- GZMB/PRF1 CD8-CXCL13 CD8- CCR7/IL7R CD4-CCR7 CD4-CXCL13 CD4-MX1 MDA1_T11 MDA1_T09 MDA1_T09 MDA1_T09 MDA1_T09 MDA1_T08 MDA1_T08 MDA1_T08 MDA1_T10 MDA1_T10 MDA1_T05 MDA1_T01 MDA1_T01 MDA1_T12 8.8µM DAPI+ CXCR4- CXCL13+ FOXP3- CD4+ DAPI KRT19/MUC1 CD4 CD8A CD3E/CD3D/CD3G DAPI+ GZMK- CXCL13- DAP GZM CXC -MX1 MDA1_T05 50µM (i) (ii) (iii) MDA1_T05 MDA1_T01 MDA1_T01 UMAP 2 D C C D MDA1_T01 (i) MDA1_T12 MDA1_T08 MDA1_T08 MDA1_T12 DAPI+ CXCR4- IFIT1+ CCR7- CD4+ DAPI+ CXCR4- IFIT1+ CCR7- CD4+ DAPI+ CXCR4+ IFIT1- CCR7- CD4+ DAPI+ CXCR4- IFIT1- CD4+ CCR7+ DAPI+ CXCR4- IFIT1- CD4+ CCR7+ DAPI+ CXCR4+ CXCL13- FOXP3- CD4+ DAPI+ CXCR4- CXCL13- FOXP3+ CD4+ DAPI+ CXCR4- CXCL13+ CD4+ FOXP3- DAPI+ CXCR4- CXCL13- CD4+ FOXP3+ CD4-FOXP3 CD4-CXCR4 CD8-GZMK CD8-ZNF683 CD8- GZMB/PRF1 CD8-CXCL13 CD8- CCR7/IL7R CD4-CCR7 CD4-CXCL13 CD4-MX1 M M MDA1_T09 M MDA1_T08 MDA1_T10 MDA1_T10 MDA1_T05 A1_T01 MDA1_T12 8.8µM DAPI+ CXCR4- CXCL13+ FOXP3- CD4+ DAPI KRT19/MUC1 CD4 CD8A CD3E/CD3D/CD3G CD8-MX1 µM (ii) (iii) M M M MDA1_T12 MDA1_T08 MDA1_T08 MDA1_T12 DAPI+ CXCR4- IFIT1+ CCR7- CD4+ DAPI+ CXCR4- IFIT1+ CCR7- CD4+ DAPI+ CXCR4+ IFIT1- CCR7- CD4+ DAPI+ CXCR4- IFIT1- CD4+ CCR7+ DAPI+ CXCR4- IFIT1- CD4+ CCR7+ DAPI+ CXCR4+ CXCL13- FOXP3- CD4+ DAPI+ CXCR4- CXCL13- FOXP3+ CD4+ DAPI+ GZMK CD4- ZNF68 CD8A+ DAPI+ CXCR4- CXCL13+ CD4+ FOXP3- DAPI+ CXCR4- CXCL13- CD4+ FOXP3+ DAPI+ GZMK CXCL PRF1+ CD8A+ DAPI+ GZMK CXCL1 PRF1- CD8A+ DAPI+ GZMK CXCL1 PRF1- CD8A+ DAPI+ CCR7+ IL7R+ CXCR6 CD8A+ CD4-FOXP3 CD4-CXCR4 CD8-GZMK CD8-ZNF683 CD8- GZMB/PRF1 CD8-CXCL13 CD8- CCR7/IL7R CD4-CCR7 CD4-CXCL13 CD4-MX1 MDA1_T11 MDA1_T09 MDA1_T09 MDA1_T08 MDA1_T08 MDA1_T10 MDA1_T10 MDA1_T05 MDA1_T12 8.8µM DAPI+ CXCR4- CXCL13+ FOXP3- CD4+ CD3G DAPI+ GZMK CXCL1 IFIT1+ CD8A+ CD8-MX1 (ii) (iii) MDA1_T05 MDA1_T01 MDA1_T01 MDA1_T12 MDA1_T08 MDA1_T08 MDA1_T12 DAPI+ CXCR4- IFIT1+ CCR7- CD4+ DAPI+ CXCR4- IFIT1+ CCR7- CD4+ DAPI+ CXCR4+ IFIT1- CCR7- CD4+ DAPI+ CXCR4- IFIT1- CD4+ CCR7+ DAPI+ CXCR4- IFIT1- CD4+ CCR7+ DAPI+ CXCR4+ CXCL13- FOXP3- CD4+ DAPI+ CXCR4- CXCL13- FOXP3+ CD4+ DAPI+ CXCR4- CXCL13+ CD4+ FOXP3- DAPI+ CXCR4- CXCL13- CD4+ FOXP3+ CD4-FOXP3 CD4-CXCR4 CD4-CCR7 CD4-CXCL13 CD4-MX1 MDA1_T09 MDA1_T08 MDA1_T10 MDA1_T10 MDA1_T05 MDA1_T12 8 8 M DAPI+ CXCR4- CXCL13+ FOXP3- CD4+ (ii) (ii) MDA (i) (i) MDA1_T12 DAPI+ GZMK- CD4- ZNF683+ CD8A+ DAPI+ ZNF683+ IL7R- CXCR6- CD8A+ DAPI+ GZMK- CXCL13- PRF1+ CD8A+ DAPI+ GZMK- CXCL13- PRF1+ CD8A+ DAPI+ GZMK- CXCL13+ PRF1- CD8A+ DAPI+ GZMK- CXCL13+ PRF1- CD8A+ DAPI+ GZMK+ CXCL13- PRF1- CD8A+ DAPI+ GZMK+ CXCL13- PRF1- CD8A+ DAPI+ CCR7+ IL7R+ CXCR6- CD8A+ DAPI+ CCR7+ IL7R+ CXCR6- CD8A+ CD8-GZMK CD8-ZNF683 CD8- GZMB/PRF1 CD8-CXCL13 CD8- CCR7/IL7R MDA1_T11 MDA1_T09 MDA1_T09 MDA1_T09 MDA1_T08 MDA1_T08 MDA1_T01 + DAPI+ GZMK- CXCL13- IFIT1+ CD8A+ DAPI+ GZMK- CXCL13- IFIT1+ CD8A+ CD8-MX1 MDA1_T05 (iii) MDA1_T05 MDA1_T01 MDA1_T01 (iii) 50µM CD8- CCR7/IL7R 8.8µM Supplementary Figure S1. SUPPLEMENTARY FIGURE S1 Plots are color coded according to level of expression as indicated by the color key. (D) RNAscope of cells in the PDAC TME: (i) Field view of tumor cells (KRT19/MUC1+) and CD3/CD4/CD8-expressing T cells from a highly T cell-infiltrated PDAC sample, and examples of (ii) CD4+ cells and (iii) CD8+ cells expressing markers of the single-cell RNAseq-defined T- cell states (left label) present in the PDAC TME (top label). Labels on the right indicate all markers in the panel for the slide and their associated pseudo-coloring. Each cell state is depicted for one cell, in two patients.
https://openalex.org/W2018085232	https://espace.curtin.edu.au/bitstream/20.500.11937/7210/2/236320_236320.pdf	English	null	Improving Clinical Practice: What Dentists Need to Know about the Association between Dental Fear and a History of Sexual Violence Victimisation	International journal of dentistry	2,015	cc-by	10,640	Hindawi Publishing Corporation International Journal of Dentistry Volume 2015, Article ID 452814, 12 pages http://dx.doi.org/10.1155/2015/452814 Hindawi Publishing Corporation International Journal of Dentistry Volume 2015, Article ID 452814, 12 pages http://dx.doi.org/10.1155/2015/452814 Correspondence should be addressed to Marika Guggisberg; m.guggisberg@curtin.edu.au Correspondence should be addressed to Marika Guggisberg; m.guggisberg@curtin.edu.au Received 5 August 2014; Revised 23 November 2014; Accepted 2 December 2014 Academic Editor: Kee-Yeon Kum Copyright © 2015 H. H. Larijani and M. Guggisberg. 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. Anecdotal evidence suggests lack of dentist knowledge and uncertainty about how clinical practice can be improved when dealing with victims of sexual violence. This systematic review presents a synthesis of the available literature, which examined the association between dental fear and a history of sexual violence victimisation. All studies indicated, to various degrees, that dental fear is associated with a history of sexual violence victimisation. The analysis identified several common themes including a perception of lack of control, avoidance behaviours, experiences of flashbacks, feelings of embarrassment, difficulties with the physical proximity to the dentist, the sex of the dentist reminding patients of the perpetrator, being placed into a horizontal body position, the specific impact of fellatio, the smell of latex, experienced lack of knowledge of dental professionals leading to insensitive treatment as well as revictimisation experiences, and the occurrence of disproportionate dental problems among patients who had experienced event(s) of sexual violence. All these themes are discussed in detail. Specific strategies are offered to assist dental practitioners in providing sensitive treatment for patients with a history of sexual violence. Additionally, several suggestions are made that may assist both researchers and dental practitioners alike. Houman Hadad Larijani1 and Marika Guggisberg2 1Pacific Smile Group, Melbourne, VIC 3000, Australia 2Curtin University, Perth, WA 6845, Australia 1. Introduction Researchers found that visiting a dentist for dental treatment can elicit unwanted memories [11, 16]. This may result in experiences of disempowerment and anticipation of pain and/or discomfort [16]. Some studies indicated that the experience of sexual violence could potentially lead to the development of fear, anxiety, and/or dissociation in victims, resulting in poor relationship between the victims and health care practition- ers, particularly the dentist [16–19]. This issue may in turn result in misdiagnosing of existing dental problems as well as considering the victims as difficult dental patients [15]. Additionally, published evidence indicates that sexual vio- lence victims tend to have low self-worth and low self-esteem, which may force them to avoid attending the dental setting for enjoying the dental treatments [32]. Thus, it may not be surprising to observe high rate of oral and dental diseases among those who experienced sexual violence. Dougall and Fiske [12] argued that sexual violence victims are particularly likely to avoid visiting the dentist, which per se results in the deterioration of their oral health, and, as a consequence, problems including pain and discomfort. Perpetrators frequently groom vulnerable individuals by starting with activities that appear safe. They often attribute positive motives to their sexual behaviours, which suggest love or necessary education [11]. In a dental setting, patients are asked to trust the dentist, who frequently assures patients that even though they may experience painful or unpleasant situations during the treatment, it will be beneficial for them in the end [15]. This may be a similar experience to the vic- timisation event(s) and can lead to perceived revictimisation or fear thereof. Some characteristics of patients who had experienced sexual violence in dental surgery include dislike of being touched, fear of judgment and sensitivity to perceived criti- cism, sensitivity to particular smells, and especially sensitivity to having instruments in the mouth [16, 17, 19]. Since sexual violence may involve the victim’s mouth, oral and dental treatment may be particularly challenging, even threatening, given the requirement of the patient’s mouth being touched and intruded by the dentist’s fingers and instruments [11]. Consequently, destructive psychological impacts of sexual violence may negatively affect the ability to enjoy benefits of dental care [12]. 1. Introduction less frequent or irregular dental visits, cancelation, and/or deferrals of appointment and treatment [3, 4]. Dental fear has also been associated with increased oral ill health, impaired self-rated oral health, social withdrawal, and an eventually greater need for dental treatments [8]. Dental fear has been recognised as an important clinical problem hampering the provision of oral health care [1]. Despite improvement of the painless dentistry and increased awareness of dental practitioners that building trusting rela- tionships is crucial, dental fear still remains a challenging issue for both dentists and patients [2]. The concept of “dental fear” refers to patients’ feelings of anxiousness and worry about dentistry and receiving future dental care. This fear can range from mild anxiety to extreme fear [1]. The experience of dental fear was identified as an important barrier to dental health care, which can have long-term consequences [3, 4].f An estimated 8–19% Australians experience dental fear [9], in spite of changes in teaching and practice of dentistry. The majority of Australians experience various levels of dental fear, but a small significant number of patients suffer from a severe form with a significant impact [10]. High levels of dental fear are associated with poorer oral health outcomes such as decayed and missing teeth [1]. Adults with a history of sexual violence victimisation may experience particular difficulties visiting the dentist due to high levels of fear [11]. Generally, sexual violence is experienced by both genders in all communities, cultures, religious and ethnic backgrounds, and economic and social Dental patients suffering from dental fear experience higher levels of psychological distress related to dental treatment than those with no fear [5]. Extreme dental fear is associated with poor oral health [1, 6, 7], as a result of International Journal of Dentistry 2 classes [12]. However, sexual violence is a gendered crime with females usually being victims, while males almost always are perpetrators [13, 14].i requesting dental care might have experienced sexual vio- lence in the previous stages of their lives. This suggests that dentists may work with the victims of sexual violence on a regular basis without any awareness of their history. There- fore, the clinical implications of managing dental patients with sexual violence victimisation have to be considered. Victims of sexual violence may find it especially prob- lematic to tolerate dental treatments and frequently cancel scheduled dental appointments or avoid dental treatment [15]. 1. Introduction Although the association between experiencing sexual violence and medical care has been established for some domains of health care such as internal medicine or gynecol- ogy and obstetrics [11], knowledge of the association between a history of sexual violence, dental fear, and oral health is in its infancy [16]. Limited publications in this field with gaps in knowledge suggest a need for a systematic review to better establish the association between dental fear and a history of sexual violence victimisation. This paper fills this gap in the literature. 1.1. General Health Effects of Experiencing Sexual Violence. Sexual violence is identified as a serious public health problem [13, 25]. A significant body of research found a strong association between sexual violence victimisation and poor health outcomes [13, 14, 25, 26]. It can affect victims’ mental and physical health across the life course [27, 28]. Victims of sexual violence, for instance, have been shown to be at an increased risk for anxiety disorders, depression, posttraumatic stress disorder [13, 28], obesity and eating disorders, suicide and self-harm behaviours [29], and health risk behaviours such as alcohol/other drugs use [14, 30]. Victimisation by sexual violence can significantly influence nearly all aspects of victims’ lives including the ability to participate in the workforce and social relationships [13, 31].tt International Journal of Dentistry (i) investigation of the association between dental fear and sexual violence (either as an independent event or as a part of a series of variables under investigation); (ii) studies written in English language; f g Wade and Tavris [38] reported an association between individuals’ wellbeing and locus of control, arguing that people with external LOC are more likely to develop psy- chological disorders than those with higher internal LOC who were found to be less likely to experience physical and psychological symptoms. Similarly, people with high internal LOC were found to have a higher capacity to cope with daily stressors and psychiatric symptoms, which frequently occur in victims of sexual violence [13, 39]. (iii) studies published between 1995 and 2011. 2.2. Data Extraction. The initial search located 18 articles (published between 2000 and 2011). After screening the titles and reviewing the abstracts, 10 studies were excluded, as they did not meet the inclusion criteria. The remaining eight studies were reviewed and critically appraised. Given the limited number of obtained articles it was decided to relax the inclusion criteria and repeat the procedure with a timeframe between 1995 and 2011. Six additional articles were located of which two examined the association between dental fear and a history of sexual violence victimisation. Consequently, 10 articles were included in the final analysis. 2.2. Data Extraction. The initial search located 18 articles (published between 2000 and 2011). After screening the titles and reviewing the abstracts, 10 studies were excluded, as they did not meet the inclusion criteria. The remaining eight studies were reviewed and critically appraised. Given the limited number of obtained articles it was decided to relax the inclusion criteria and repeat the procedure with a timeframe between 1995 and 2011. Six additional articles were located of which two examined the association between dental fear and a history of sexual violence victimisation. Consequently, 10 articles were included in the final analysis. Abramson and Seligman [40] argued that when stress is uncontrollable, individuals experience heightened arousal, which was found to be a key feature of anxiety. In this regard, Hood and Carter [41] reported that individuals with a history of CSA were more likely to develop external LOC given their lack of control over future events. 2.3. Data Synthesis. Obtained information from the stud- ies included in this research was coded. Each publication was allocated a study identification number. International Journal of Dentistry 3 International Journal of Dentistry Table 1: Terms used for literature search. Number Terms 1 Dental fear, dental phobia, dental anxiety, and dental phobic patients. 2 Sexual abuse, sexual violence, sexual assault, child sexual abuse, rape, incest, fellatio, and traumatic experience. Table 1: Terms used for literature search. Number Terms 1 Dental fear, dental phobia, dental anxiety, and dental phobic patients. 2 Sexual abuse, sexual violence, sexual assault, child sexual abuse, rape, incest, fellatio, and traumatic experience. publications will be discussed below. First, a brief summary of the included studies will be presented. 3.1. Overview of the Included Publications. The following section presents the results of this analysis discussing the main themes that emerged (see Table 3). It provides the findings extracted from the studies included in this review. consensus method was applied to make the final decision on whether or not to include a study [33]. After all the studies were located and after deciding which ones to include in the review, data were collated and summarized with a descriptive analysis. Key elements of the studies are provided in Table 2 which provides details on study and participant characteristics as well as intervention and results. 3.2. Lack of Control (Theme 1). Lack of control was reported by all the included studies, making it the most common theme. The concept of control, as a main predictor of health behaviour in psychological research [34], is defined as the perception that an individual has the necessary skill, talent, resources, or opportunities to acquire desirable outcomes or avoid harmful situations through his or her own actions [34]. 3.2. Lack of Control (Theme 1). Lack of control was reported by all the included studies, making it the most common theme. The concept of control, as a main predictor of health behaviour in psychological research [34], is defined as the perception that an individual has the necessary skill, talent, resources, or opportunities to acquire desirable outcomes or avoid harmful situations through his or her own actions [34]. 2.1. Inclusion Criteria. The following three criteria needed to be fulfilled for studies to be included: 3.2.1. Sexual Victimisation and General Perception of Control. Locus of control (LOC) theory [35] provides a framework in which the impacts of sexual violence victimisation on generalised perception of control can be assessed [36]. It refers to the extent to which individuals believe they can control subsequent events affecting them [35, 37]. International Journal of Dentistry The coding scheme collected data on participants’ characteristics (e.g., age, gender), year of publication, setting (e.g., community or clinical), location (e.g., country and city), research design (e.g., quantitative, qualitative, or mixed method), variables of interest (e.g., childhood sexual abuse), prevalence (number and percentage) of participants with dental fear, and a history of sexual violence and specific findings (e.g., presence or absence of an association between dental fear and a history of sexual violence victimisation). 3.2.2. Concept of Control and Dental Fear. Control as a crucial issue in the development of dental fear was recently examined by Armfield and colleagues [10, 42]. Both studies found that the perception of lack of control was a main concern among dental phobic patients and strongly associated with dental fear. Armfield et al. [42] assessed the association between dental fear and perceived lack of control among 3,937 participants as part of the National Survey of Adult Oral Health (NSAOH), in Australia between 2004 and 2006. The study found that the perceptions of uncontrollability and dangerousness were significantly associated with high levels of dental fear. 2. Methods A comprehensive literature search identified publications that investigated the association between dental fear and a history of sexual violence victimisation. Different databases including Pubmed, Medline, Google scholar, ProQuest, Sci- enceDirect, and Willey online were searched using a variety of search strategies. Furthermore, a manual search of key journals was conducted including hand search of hard copies of the Australian Dental Association Journal. A strict protocol was followed. Original articles included in the systematic review were appraised and synthesized in a rigorous way. The review protocol is outlined below. The primary author screened the abstracts of many studies and was able to reject those papers identified as not fulfilling the inclusion criteria. A long list of papers was created of papers that required to be read in more detail and further studies were located through the reference lists of obtained articles. Data extraction yielded a relatively small number of studies. A record was created for search terms used (Table 1) either individually or in combination and the decision-making process of including or rejecting the paper in the analysis. The critical appraisal of studies was undertaken to assess whether the studies met the inclusion criteria. The second author reviewed and checked the information provided, and In the aftermath, both genders often feel out of control and disempowered as they may realise that their wills and desires do not count [12]. Furthermore, victims may learn not to care about themselves and ignore or disregard generated signals by their bodies such as pain, injury due to the effects of sexual violence [12, 31]. Long-term coping skills may include compulsive behaviours masking their emotions that originated from experiencing sexual violence [12, 13]. 1.2. Oral Health Effects of Experiencing Sexual Violence. Considering the prevalence of sexual violence, Leeners and colleagues [11] anticipated that nearly 20% of female patients 3. Result and Discussion In a National Dental Telephone Interview Survey (NDTIS) using 1,084 Australian adults, Armfield [10] examined whether dental fear can be explained by negative dental experiences and/or by cognitive perceptions of going to the dentist. The study found that a perception of uncontrollability was a superior predictor of dental fear compared with negative dental experiences. This finding Analysed data are presented narratively. It was found that the studies were heterogeneous and it was decided that results should be summarised and presented most appropriately in narrative form. No attempt was made to undertake a statistical synthesis. Themes that emerged from the included 4 International Journal of Dentistry Table 2: Publications included in the analysis. ID Reference Year Participants number and age Setting and location Research design Variable Prevalence Specific findings 1 [17] 1996 462 F, 18–45 y Clinical, Seattle, USA Mixed method Dental fear, CSA, CPA, CN, ASA, and APA High dental fear = 25.5% High levels of dental fear in women are significantly associated with CSPA, CN, and ASPA. 2 [16] 2001 99 F, 17–67 y Clinical, Norway Quantitative Dental fear, CSPA, and ASPA Extreme dental fear: ST group = 23.1%, IC group = 29.3%, and OP group = 52.3% OP during SV was found to be predictive of the development of dental fear. 3 [18] 2004 108 F, 20–60 y Clinical, Norway Quantitative Dental fear, CSA Women with CSA = 53.7% Participants in fear and abuse group reported higher levels of dental fear in comparison with other groups. Concerning dental problems, fear and abuse and fear only group indicated more problems than the control group. 4 [19] 1997 181 F Clinical and community, USA Quantitative CSA Women with CSA = 72.9% (i) History of CSA is associated with current women’s dental experiences. (ii) Dental fear is associated with a history of sexual violence victimisation. 5 [15] 2005 58 M and 19 F, 24–62 y Clinical, Canada Qualitative CSA All participants were sexually abused People who have experienced sexual violence often dissociate as a way of coping with overwhelming stimuli. 6 [11] 2007 255 F, mean 38.65 Clinical and community, Germany Quantitative CSA Combination of SA and PA in women with CSA = 66% PA in women with CSA = 92.9% (i) History of CSA is associated with increased dental fear. (ii) CSA experiences may increase psychological strain during dental treatment. 3. Result and Discussion 7 [20] 2006 240 M and F, 19–79 y Clinical, Netherland Quantitative Dental anxiety, SV HDA = 58.75% Participants with HDA were more likely to have experienced SV as those with LDA. 8 [21] 2009 1462 M and F, 18–85 y Community, Germany Quantitative Dental anxiety, SV SV = 4.6% The rates of high dental fear related to the SV experience among those participants were 11.9% and 4.5%, respectively. 9 [22] 2011 1024 students, 16–≥60 y Community, UK Quantitative Dental anxiety, SV HDA = 11.2% Significant prevalence of high dental anxiety was found among participants who experienced SV. 10 [23] 2004 16 M and 14 F, 20–65 y Clinical, Denmark Qualitative Dental anxiety, SV, and embarrassment SV = 10% Severe dental fear is associated with remarkable psychological impairment due to avoidance of routine dental checkups and treatments and subsequently deteriorating individuals’ oral hygiene. APA: adult physical abuse; ASA: adult sexual abuse; CN: child neglect; ASPA: adult sexual and physical abuse; CPA: child physical abuse; CSA: child sexual abuse; CSPA: child sexual and physical abuse; F: female; HDA: high dental anxiety; IC: intercourse; LDA: low dental anxiety; M: male; OP: oral penetration; PA: physical abuse; SV: sexual violence; ST: sexual touching. International Journal of Dentistry 5 Table 3: Themes identified. 3. Result and Discussion Themes/study Walker et al.’s study, 1996 [17] Hays and Stanley’s study, 1997 [19] Willumsen’s study, 2001 [16] Willumsen’s study, 2004 [18] Moore et al.’s study, 2004 [23] Stalker et al.’s study, 2005 [15] De Jongh et al.’s study, 2006 [20] Leeners et al.’s study, 2007 [24] Oosterink et al.’s study, 2009 [21] Humphris and King’s study, 2011 [22] Lack of control/feeling of being helpless and powerless √ √ √ √ √ √ √ √ √ √ Avoidance/never go to the dentist √ √ √ √ √ √ √ — √ — Lack of awareness and knowledge of relationship between SV and DF — — √ √ — √ — √ — — Flashbacks — √ √ — — √ √ √ — — Embarrassment/fear of judgment and criticism — — — — √ √ — — √ √ Physical proximity/threatening — √ — √ — — — — — — Sex of dentist on SV — √ — √ — — — √ — — Problem with horizontal dental chair position √ √ — √ — √ — √ — — Fellatio specific impact √ √ √ — — — — — — — Disproportionate dental problems — √ — — — — — — — — Smell of latex — — — — — √ — — — — International Journal of Dentistry 6 corroborated previous results indicating a strong relationship between lack of control and dental fear. to appreciate provision of explanations about the treatment plan. The dentist can easily keep them informed regarding each step of dental treatment in advance. 3.2.3. Dental Fear, Sexual Violence Experience, and Lack of Control. All studies reported that participants experienced various degrees of helplessness, powerlessness, and incidents of lack of control during dental treatments. The experience of such feelings was believed to be the underlying problem in developing fears of feeling claustrophobic, experiencing chocking, severe gagging or being unable to breathe, and being trapped in the dental chair which interferes with dental treatments [17]. The following statement by a dentist provides evidence of this view: “She reported extreme fear of being trapped in the dental chair—by contrast her fear of needles, drills and painful treatment was low” [17, page 485]. Treatment Breaks and Use of “Stop” Signal. Allowing breaks during treatment, checking patients’ comfort levels, and using a previously agreed stop signal were strategies presented [12, 15, 17, 19]. 3. Result and Discussion The feeling of control can be increased by introducing choices including asking dental patients to raise their hands as a stop signal or to pause for a rest, requiring dentists to respond quickly to the patients’ signals [44]. This strategy was appreciated as the following statements in Stalker et al.’s study [15] suggest: “If I [put] my hands across like this, or I was blinking continually that was, “stop,” you know, and I was in control” (page 1280). Another participant stated, “Most of the time [with this dentist] it’s like, “You know the signals, right?” And I go “yeah.” And he’d always review the signals . . . “this is what you can do for yes, this is no, this is stop”” (page 1280). Feelings of helplessness and powerlessness in dental patients with sexual violence histories can potentially result in revictimisation if dental treatment is perceived to be fear pro- voking [15, 17, 19]. It stands to reason that experiencing such feelings stems from the similarity between sexual violence and perceived lack of control when visiting the dentist. In both situations a perception of victimisation may be evoked through power imbalance and a sense of helplessness and fear. However, anecdotal evidence suggests that some den- tists experience difficulties with this method when patients disclose CSA. Dentists reported that these patients may have difficulty using appropriate stop signals, which may be perceived as additional stress factor given the responsibility of having to provide the stop signal. In this regard, it will be important for the dentist to be sensitive to body language and developing a sense of the patient’s comfort level, assisting in the determination of when a break will be necessary. 3.2.4. Strategies to Increase Patient Control. Control is an issue of paramount importance and feeling safe in medical and dental environments is a critical factor for patients with a history of sexual violence [31]. Strategies addressing this issue can significantly help such dental patients to feel safer in the dental environment. Some of the studies reviewed proposed strategies to increase patients’ sense of control in dental settings [11, 16, 17]. They are discussed below. Providing Written Information. Another strategy to manage patients’ perceived lack of control is through providing writ- ten information [12]. This method appears to be particularly appreciated among patients with a history of CSA. 3. Result and Discussion “What I hear one day I won’t hear the next due to dissociation when I shut down. I find it an awful help if people write things down when I am nervous, as I won’t be listening” (page 303). As can be observed by the quote above, providing information in writing may assist patients, particularly those who tend to dissociate during dental treatment. Collaboration. Collaboration with patients was identified as one of the most important strategies. Walker et al. [17] argued that particularly people having experienced sexual violence require transparent communication and involve- ment in treatment decisions to share control. The utilisation of obtaining informed consent before each step of treatment was suggested by several studies [15, 16, 31, 43], involving patients in the treatment procedures. Asking permission before performing dental treatments will give simply back the control to the dental patients with history of sexual violence [12]. 3.3. Avoidance (Theme 2). Avoidance as a defence mechanism was the second major theme referred to by eight studies [15–21, 23]. Avoidance may comprise refusal to encounter activities, objects, and situations as they represent uncon- scious aggressive or sexual impulses or punishment for those impulses [45]. Dental patients who experienced sexual violence appeared to use avoidance behaviours as a coping mechanism to prevent any reminder of the past experiences. “Inform before You Perform.” The dentist-patient communi- cation was addressed with the following slogan by Stalker et al. [15]: “inform before you perform” (page 1280). The researchers found that pretreatment communication and detailed explanation of the treatment plan were appreciated by the participants and reduced their levels of distress and fear. For example, one participant stated, “I find her [the dentist] good because she does explain everything that she is going to do and why she’s doing it at the time and sort of checks, “Is that O.K.?”” (page 1280). Another participant noted, “He [the dentist] tells me what he’s going to do next. So, long before I can anticipate, he’s already told me . . .” (page 1280). As can be seen from these quotes, dental patients seem 3.3.1. Sexual Violence and Avoidance. Often, individuals who have experienced sexual violence try to avoid any reminders of the experiences consciously or unconsciously [45–52]. International Journal of Dentistry Feelings of guilt, shame, and inferiority Deterioration of dentition Avoidance of dental care Fear, anxiety Figure 1: Vicious circle of dental anxiety [8, 54]. Although avoidance can provide a short-term relief, addi- tional psychological problems have been reported including increased level of fear and anxiety, depressive symptoms, and PTSD [45, 52]. For instance, Wright et al. [52] found an association between the adoption of avoidance behaviour and severity of psychological problems. Feelings of guilt, shame, and inferiority 3.3.2. Dental Fear, Sexual Violence, and Avoidance. Frequent cancellations of the dental appointments were identified as a means of avoidance among dental patients who experienced sexual violence [15–21, 23]. The following statement quoted by a male participant in Stalker et al.’s study [15] illustrates this issue: “I put it off for about five or six times . . . my wife has been bugging me for a while now: “The dentist has been calling you, you’ve got to go now.” [I say] “OK, I’ll call her back” and I do not call her back” (page 1280). 3.3.2. Dental Fear, Sexual Violence, and Avoidance. Frequent cancellations of the dental appointments were identified as a means of avoidance among dental patients who experienced sexual violence [15–21, 23]. The following statement quoted by a male participant in Stalker et al.’s study [15] illustrates this issue: “I put it off for about five or six times . . . my wife has been bugging me for a while now: “The dentist has been calling you, you’ve got to go now.” [I say] “OK, I’ll call her back” and I do not call her back” (page 1280). Avoidance of dental care Figure 1: Vicious circle of dental anxiety [8, 54]. g Stalker and colleagues [15] reported an association between dental fear and heightened sensitivity to dental procedures and visiting patterns. Invasion of personal space through an uninvited touch (either head and neck or arm) was found to be a main factor for avoidance behaviour [31]. It can be more difficult for individuals with a history of sexual violence to cope with reminders that may be perceived as continues victimisation. The imagination of such a feeling before visiting the dentist may increase the level of stress in these individuals and result in the cancellation of the appointment [12], which may lead to feelings of shame on behalf of patients and frustration for the dental practitioners [15]. International Journal of Dentistry The statement below reported by Dougall and Fiske [12] demonstrates the distress and dilemma that can be experienced: “I lost it when the nurse put her arm round me to comfort me, and she was only trying to help. I could not bear her to touch me and I could not tell her why” (page 303). for neglecting his teeth: “When they [the dentists] do my teeth, they are going to say, “Oh, you have not been taking care of them, you should have come in before”” [15, page 1280]. Additionally, experiences of childhood sexual violence by dental patients may cause a belief that they are undeserving and may expect to be harshly judged [12]. Havig [32] examined experiences of adults with a history of CSA in a health care setting. Findings suggested that exposure to sexual violence was associated with feelings of shame. This may result in neglecting personal hygiene and oral health [31]. Consequently, some individuals with a history of sexual violence victimisation may require a reminder regarding self-care and instead of reprimanding them, in fact, there is an opportunity for dental practitioners to assist sexual violence victims in maintaining a good oral hygiene [12]. 3.4. Embarrassment, Fear of Judgment, and Criticism (Theme 3). Researchers developed a “vicious circle of dental fear” framework to explain the association among avoidance, dental fear, and embarrassment. This cycle indicates how psychological variables including embarrassment in addition to dental fear result in avoidance, dental problem, and feeling of shame (Figure 1). Considering the vicious circle model, it can be observed that a high level of dental fear in patients is associated with increased avoidance behaviours resulting in extensive dental problems, which lead to feelings of guilt, shame and inferiority in the patients, and, eventually, feedback into the maintenance or exacerbation of presented dental fear [8, 54].i 3.4.1. Strategies to Overcome Avoidance Behaviour. Strategies to assist dental patients to overcome the problem of frequent cancellations of dental appointment are necessary. They may include included motivation strategy and a same-day appointment strategy, which are further outlined below. Motivation Strategy. Validation of dental patients’ courage and energy may motivate them to maintain a good oral health. Schachter et al. [31] recommended the utilisation of simple method for improving the oral hygiene of sexual violence victims. 3. Result and Discussion Various forms of avoidance were identified as a result of expe- riencing sexual violence including dissociation as a form of emotional numbing, alterations in body perception, amnesia for painful memories, and multiple personality disorder [53]. Other types of avoidant behaviour included suicidal attempts, problematic substance use, and tension-reducing activities such as self-harming behaviours [53]. 7 International Journal of Dentistry For example, an electric toothbrush because of its novelty effect might help these patients take a better care of their teeth. It was further argued that sexual violence vic- tims may improve their oral health and avoid experiences of shame and embarrassment, which would counteract previous hesitations of attending a dentist appointment. Four studies identified the issue of embarrassment and fear of judgment and criticism, as a prominent theme [15, 21– 23], which is associated with avoidance behavior in indi- viduals who have experienced sexual violence. For example, Stalker et al. [15] argued that feelings of shame due to fear of dental treatment among patients with a history of sexual violence prevented them from visiting the dentist. It was further reported that those patients who were constantly afraid of being reprimanded for neglecting their teeth have a tendency to avoid going to the dentist. As can be seen in the statement below, a male participant expected to be criticised Same-Day Appointment. Stalker and colleagues [15] pro- moted a same-day appointment strategy. This approach is beneficial as it allows taking advantage of a patient’s readiness to attend a dentist appointment while at the same time avoiding the inconvenience and financial implications of International Journal of Dentistry 8 repeated rescheduling. “If the patient feels “ready” on a particular day, he or she may call the office seeking to take a slot left open by a short-notice cancellation, if one is available, for that same day” (page 1280). This simple intervention may assist vulnerable individuals to attend to dental appointments more readily and resist the impulse of “backing out.” sexual violence and that patients feel safer and less distressed in this position.i Despite the benefits of semisupine position for dental patients who experienced sexual violence, it is not always possible to carry out the dental treatments in the semisupine position particularly during the procedures that need to be performed on upper jaw [12]. In such a case, providing a very detailed explanation for the patients that outlines why a supine position is required for a particular intervention to achieve the best possible quality of treatment was recom- mended. If patients are given detailed information why the supine position affords better visibility and, therefore, should not be avoided, they are likely to agree [12, 15]. 3.5. Flashbacks (Theme 4). International Journal of Dentistry [15] found that placing patients in the supine position triggered the memories of original sexual violence. Hays and Stanley [19] also noted that their participants experienced most discomfort with the placing of the dental chair in a horizontal position. 3.6. Patient Body Position (Theme 5). Five studies reported that reclining the dental chair could be experienced as particularly threatening to patients with a history of sexual violence [11, 15, 17–19]. For example, Stalker et al. [15] found that placing patients in the supine position triggered the memories of original sexual violence. Hays and Stanley [19] also noted that their participants experienced most discomfort with the placing of the dental chair in a horizontal position. g By contrast, Leeners et al. [11] reported that participants had no preference towards the sex of their dentist. They argued that sometimes the relationship with a neglectful mother might have had a negative impact on those who experienced sexual violence, which may counteract the rela- tionship with a perpetrator. Furthermore, given that the vast majority of perpetrators are men, there are still some individuals who are sexually victimised by women [11]. Consequently, it is fair to argue that being a male dentist may not necessarily constitute a trigger for victims of sexual violence. Patients with a history of sexual violence may feel vulnerable visiting a male or female dentist [11] and the sex of the dentist may need to be considered if there is a specific preference [12]. A patient who feels more comfortable to be treated by a female dentist should be encouraged and educated about the importance to have dental treatment by a woman as opposed to a man, without feeling guilty, given that this simple approach may prevent triggering flashbacks because of the dentist’s sex. A sense of power imbalance due to the horizontal body position may result in heightened vulnerability to revictim- isation and increased states of anxiety, particularly when dental practitioners are above the patients [11, 12, 15, 16, 19]. A male participant in Stalker et al.’s [15] study indicated feeling unsafe when being placed in a horizontal position in the dental chair. “You are . . . supine . . . with your head lowered . . . so you are really vulnerable physically, and many of us have been violated orally” (page 1280). 3.6.1. Strategies for Making Supine Position Acceptable to Vic- tims of Sexual Violence. International Journal of Dentistry An issue identified in five studies [11, 15, 16, 19, 20] was the experience of flashbacks during the dental care. A flashback, or involuntary recurrent memory, has been defined as “highly persistent memories of traumatic experiences that are activated automatically by features of the current environment and are accompanied by much reliving” [55, page 649]. Individuals with a history of sexual violence may reexperience the abusive situation in their imagination or thought processes as if it is occurring [11]. Another strategy when the supine position cannot be avoided is the provision of cover such as blanket, coat, or X-ray bib. Schachter et al. [31] and Williams [43] suggested that this method helps the patients feel more secure and less exposed within the dental environment. As a result, they tend to experience less anxiety and panic during the procedure. Perceived similarities between events of sexual violence and visiting the dentist may act as a trigger eliciting memories of victimisation experience [16, 19]. Studies demonstrated that individuals who experienced sexual violence intention- ally avoid visiting healthcare facilities because of flashbacks, rapid emergence of fear, anxiety, grief, or anger [11, 15, 16, 19, 20]. Conditions that may elicit flashbacks in dental surgery include being placed in horizontal position, the sex of the dentist, physical proximity of the dentist, oral manipulation, and the smell of latex gloves and even aftershave. An additional experience of safety may be achieved by providing instruments that assist patients to monitor the dental procedures (e.g., intraoral camera, a mirror) [15, 43]. Such an easy strategy is likely to remove uncertainties of dental procedures [43] and allows patients to feel less vulnerable. 3.7. Sex of the Dentist (Theme 6). Three studies investigated the association between the sex of the dentist and dental fear among participants with a history of sexual violence [11, 16, 19]. Hays and Stanley [19] found an association between distress symptoms and the dentist’s sex. The researchers reported that the rate of nausea was significantly lower among participants who visited a female dentist. This finding was corroborated by Willumsen [16] who indicated that women with history of CSA reported male dentist to be more fear provoking. 3.6. Patient Body Position (Theme 5). Five studies reported that reclining the dental chair could be experienced as particularly threatening to patients with a history of sexual violence [11, 15, 17–19]. For example, Stalker et al. International Journal of Dentistry Dental practitioners may use specific strategies to manage particularly those dental patients who experienced sexual violence and complain about the supine position. They include placing patients in semisupine posi- tion whenever possible, explaining the reasons for the supine position, covering the patients, and allowing monitoring the treatment procedures by the patients [12, 15, 31, 43]. Dougall and Fiske [12] reported that semisupine position of the patient in the dental chair with one foot on the ground appears to be tolerable and more favorable for individuals who experienced 3.7.1. Strategies to Decrease the Likelihood of Revictimisation. Dougall and Fiske [12] recommended having a chaperone to 9 International Journal of Dentistry 3.9.1. Strategies to Assist Victims to Cope with the Effects of Fellatio. Dentists need to control the gag reflex among the dental patients who experienced forced oral sex [12]. A tool kit comprising several components including (a) relaxation, distraction, and desensitization techniques, (b) complementary therapies including transcutaneous electrical nerve stimulation (TENS) and hypnosis, and (c) psychologi- cal and behavioural therapies may enable dentists to choose the most appropriate strategy to providing dental treatments for patients suffering from anxiety and gag reflex [56]. help dental patients cope with the situation. The chaperone can be a member of dental team or preferably a trusted friend of the patients who can assist in coping with the distress of visiting the dentist. Another method they proposed is to leave the door of surgery open, as this may reduce the level of stress in the patients as they feel more safety. 3.8. Physical Proximity to the Dentist (Theme 7). The physical closeness of the dentist, which could act as a trigger eliciting negative memories of the original act(s) of sexual violence, was mentioned by two studies [18, 19]. Hays and Stanley [19] stated that their participants experienced the most discom- fort when the dentist was too close to them or touching their body. Similarly, Willumsen [18] indicated significant negative impacts from physical intimacy. Participants in both studies reported that the physical proximity to the dentist increased their level of fear and anxiety as a result of flashbacks and intrusive thoughts. fl Dougall and Fiske [12] strongly advised against pharma- cological control therapies or chemical restraint as strategies to control the gag reflex among dental patients with a history of sexual violence involving fellatio. International Journal of Dentistry Such an application per se may result in flashbacks and subsequently heighten their state of fear and anxiety. 3.10. Smell of Latex Gloves and Aftershave (Theme 9). Some victims of sexual violence may be sensitive to the smell of latex gloves. Victims felt reminded about the assault and could not tolerate the smell of latex gloves as it considerably increased the levels of fear and anxiety: “[Visiting] dentists, for me is, even on a good day, a total, absolute nightmare” [15, page 1281]. Stalker and colleagues’ study [15] found that the smell of latex gloves triggered memories of the smell of a condom used in unwanted sexual encounters, which increased the patients’ levels of fear.ht 3.8.1. Making the Position of the Dentist Acceptable to Vic- tims of Sexual Violence. The dentist’s body position and provision of ongoing information were found to be helpful strategies. Hays and Stanley [19] reported that the most helpful strategy was to be mindful of body position during treatment and not to lean in towards or touching the patient’s body. Such considerations can improve patients’ feelings of safety and prevent negative memories of victimisation events. 3.8.1. Making the Position of the Dentist Acceptable to Vic- tims of Sexual Violence. The dentist’s body position and provision of ongoing information were found to be helpful strategies. Hays and Stanley [19] reported that the most helpful strategy was to be mindful of body position during treatment and not to lean in towards or touching the patient’s body. Such considerations can improve patients’ feelings of safety and prevent negative memories of victimisation events. The smell of aftershave might also trigger memories of victimisation events making the dental visit a frightening ordeal [12]. The following statement indicates that some patients with sexual violence histories behave in an uncon- trolled way when they smell aftershave scent due to the smell triggering a flashback: “When the dentists smelt of that after- shave—I really freaked out” [12, page 299]. Providing ongoing information about the treatment was reported to be a useful strategy [31]. Informing patients in advance regarding the possible unintentional physical contacts can help dental patients who have been victimised by sexual violence to feel more secure and comfortable in the dental settings and also assist the dentist to render high quality treatments [12]. 3.10.1. Strategies to Address the Problem with Latex Gloves and Aftershave. 4. Conclusion This review examined the relationship between dental fear and a history of sexual violence victimization using a sys- tematic review methodology. The articles analysed yielded 10 prominent themes associated with dental fear in dental patients with a history of sexual violence victimisation. Data synthesis indicated that the issue of control was the most significant theme that emerged. The most important issue for dental phobic patients seems to be their perceived lack of control and the fear of negative dental experiences. Patients with a history of sexual violence victimisation reported feeling revictimised given the fear-provoking experience of dental treatment. Consequently, patient control is a critically important issue to increase patient perception of safety in dental environments. A collaborative approach using shared control by obtaining informed consent (asking permission for dental treatment), using the “inform before you perform” slogan, allowing breaks during treatment, and using a stop signal and checking comfort levels during treatment were suggested approaches. 3.11.1. Strategies to Enhance Dentists’ Knowledge and Pub- lic Awareness. There is a need to further educate dental practitioners about dental fear and its possible association with sexual victimisation. Willumsen [18] indicated that patients with a history of sexual violence have particular difficulties establishing a trusting relationship. As a result, all members of the dental team, particularly dentists, should learn how to identify the nonverbal sign of the patients’ fear and anxiety including tenseness, unusual request, and unfounded fears [12]. This analysis revealed that although dental patients with a history of sexual violence were aware that voicing their discomfort is difficult, they could speak loudly using their actions and body language [15, 17, 19], for instance, gaging when their mouths are intruded, cringing when the dentist touches them, and feeling terrified when being placed in the horizontal position. As a result, all dentists should be prepared to identify and manage such a situation. Furthermore, it is essential for dentists to know how to ask patients about a possible history of sexual violence. Stalker and colleagues [15] stated that asking a direct question regarding sexual victimisation may result in patients’ unwill- ingness to answer such a question honestly. Instead, they recommended asking less specific questions. For example, “Are there any parts of dental treatment that are particularly difficult for you? Is there anything we can do to help you feel more comfortable?” (page 1280). International Journal of Dentistry Two simple but useful strategies were rec- ommended by to help the dental patients with a history of sexual violence cope with the discussed situations, namely, the use of vinyl gloves and avoiding wearing aftershave [12, 15]. Wearing gloves is an essential part of infection control in the dental settings [57], which cannot be avoided. However, if dental patients with a history of sexual violence are anxious about the use of latex gloves, the dentist may consider the use of vinyl gloves as an alternative [15]. Dougall and Fiske [12] recommended avoiding use of aftershave in the dental surgery during the treatment of the patients because aftershave may heighten the levels of fear and discomfort experienced by patients, particularly if they have a history of sexual violence. 3.9. Oral Manipulation and Fellatio Special Effects (Theme 8). The especially negative effect of fellatio was referred to by three studies [16, 17, 19]. These studies found an association between a history of forced oral sex and high levels of dental fear. Willumsen [16] reported that women who experienced sexual violence including fellatio have higher levels of dental fear when compared to women with sexual violence victim- isation not involving fellatio. Negative impacts of forced oral sex might present a particular difficulty with oral health care in the later stages of life due to a symbolic recreation of the experience [17]. Similar findings were reported by Hays and Stanley [19]. Consequently, considering the impact of fellatio is imperative. It is undeniable that dental procedures require the intru- sion of the mouth and touching of the lips with instruments and/or the dentist’s fingers. Therefore, there is a particularly high possibility that patients who experienced sexual violence involving fellatio are particularly affected [11]. This experience can result in an especially heightened state of anxiety and activation of a gag reflex preventing those patients from enjoying the benefits of dental care [12]. 3.11. Public Awareness and Dentists’ Knowledge of Association between Dental Fear and Sexual Violence (Theme 10). Four studies [11, 15, 16, 18] referred to the general lack of public awareness and dentists’ knowledge of association between dental fear and sexual violence, as a prominent theme. These studies demonstrated that women who were exposed to sexual violence might have greater difficulty in establishing International Journal of Dentistry 10 a trusting relationship when compared to patients without sexual victimisation. International Journal of Dentistry Stalker and colleagues [15] found that the relationship between sexual violence victimisation and dental treatments along with its negative impacts on patients’ current dental conditions is not clearly understood by both affected patients and their dentists. That is why only a few patients in their study informed the dentists about their previous experience of sexual violence. 3.12.1. Strategies to Reduce the Rate of Dental Problems of Patients Who Are Victims of Sexual Violence. No specific strategy could be located within the analysis of the included studies to address the higher incidence of oral and dental diseases in individuals who were exposed to sexual violence within this analysis. However, all strategies addressing the underlying causes of dental fear among patients, particularly those experiencing sexual violence, appear to be useful in assisting dental patients to reduce the rate of dental problems and maintain a good oral condition. For example, recommending using an electric toothbrush may assist these patients maintain a good oral hygiene [31]. Leeners et al. [11] reported that only one-third of women with a history of CSA believed that disclosing information to the dentist would be potentially useful and result in improved dental treatment. Other participants expressed that the history of CSA was not taken into account by their dentist in a serious way. References [1] J. M. Armfield, G. D. Slade, and A. J. Spencer, “Dental fear and adult oral health in Australia,” Community Dentistry and Oral Epidemiology, vol. 37, no. 3, pp. 220–230, 2009. [18] T. 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Hausen, “Association between dental fear and dental attendance among adults in Finland,” Acta Odontologica Scandinavica, vol. 65, no. 4, pp. 224–230, 2007. [21] F. M. D. Oosterink, A. de Jongh, and I. H. A. Aartman, “Negative events and their potential risk of precipitating pathological forms of dental anxiety,” Journal of Anxiety Disorders, vol. 23, no. 4, pp. 451–457, 2009. [5] G. Klingberg and A. G. Broberg, “Dental fear/anxiety and dental behaviour management problems in children and adolescents: a review of prevalence and concomitant psychological factors,” International Journal of Paediatric Dentistry, vol. 17, no. 6, pp. 391–406, 2007. [22] G. Humphris and K. King, “The prevalence of dental anxiety across previous distressing experiences,” Journal of Anxiety Disorders, vol. 25, no. 2, pp. 232–236, 2011. [23] R. Moore, I. Brødsgaard, and N. Rosenberg, “The contribution of embarassment to phobic dental anxiety: a qualitative research study,” BMC Psychiatry, vol. 4, article 10, 2004. [6] S. Eitner, M. Wichmann, A. Paulsen, and S. Holst, “Dental anxiety—an epidemiological study on its clinical correlation and effects on oral health,” Journal of Oral Rehabilitation, vol. 33, no. 8, pp. 588–593, 2006. [24] B. Leeners, H. Richter-Appelt, B. Imthurn, and W. 4. Conclusion Utilising these questions by dentists will provide a situation that allows dental patients who experienced sexual violence to respond in an appropriate manner matching their level of comfort [15]. Asking these questions may also lead to the disclosure of a history of sexual violence, which can set the stage for a collaborative working relationship between dentists and dental patients who have a history of sexual violence [15]. A number of conclusions can be drawn from this analysis. Overall, it demonstrated a clear association between dental fear and a history of sexual violence victimisation. All studies indicated that victims of sexual violence are more likely to suffer from dental fear when compared to patients without a history of sexual victimisation. It was also found that an experience of sexual violence is likely to result in the development of dental fear or at least exacerbates the presented dental fear in dental patients.f Current knowledge on oral health effects of experienced sexual violence is emerging with more or less consistent practical applications of this knowledge, particularly in the United State of America. Importantly, this study revealed that overall knowledge of the consequences of sexual violence on dental fear and oral health is only in its infancy. This became evident during the data extraction phase that only a small number of international studies investigated the association between dental fear and a history of sexual violence vic- timisation. Consequently, we recommend specific research in this area to generate further empirical evidence on the issues discussed. It is of concern that the current lack of knowledge and insight of some dentists may restrict patients the opportunity to receive high quality and sensitive dental treatments, particularly if they have been subjected to sexual violence. Clearly, the importance of managing these patients 3.12. Disproportionate Dental Problems. Hays and Stanley [19] investigated the association between type and inci- dence of dental problems and a history of sexual violence. The researchers reported that the problems with temporo- mandibular joint (TMJ), bruxism, gingivitis, and number of extracted teeth were significantly higher in the participants with a history of sexual violence when compared with the general population. 11 International Journal of Dentistry hypothesis,” International Journal of Arts & Sciences, vol. 5, pp. 723–736, 2012. has not been stressed sufficiently yet and it appears imperative to improve understanding of this relationship among dental practitioners. [15] C. A. Stalker, B. D. C. Russell, E. References Rath, “Influence of childhood sexual abuse on pregnancy, delivery, and the early postpartum period in adult women,” Journal of Psychosomatic Research, vol. 61, no. 2, pp. 139–151, 2006. [7] A. A. Schuller, T. Willumsen, and D. Holst, “Are there differ- ences in oral health and oral health behavior between individ- uals with high and low dental fear?” Community Dentistry and Oral Epidemiology, vol. 31, no. 2, pp. 116–121, 2003. 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[52] M. O. Wright, E. Crawford, and K. Sebastian, “Positive resolu- tion of childhood sexual abuse experiences: the role of coping, benefit-finding and meaning-making,” Journal of Family Vio- lence, vol. 22, no. 7, pp. 597–608, 2007. [35] J. B. Rotter, “Generalized expectancies for internal versus external control of reinforcement,” Psychological Monographs, vol. 80, no. 1, pp. 1–28, 1966. [53] R. Lev-Wiesel, “Child sexual abuse: a critical review of inter- vention and treatment modalities,” Children and Youth Services Review, vol. 30, no. 6, pp. 665–673, 2008. [36] B. R. Bolstad and R. E. Zinbarg, “Sexual victimization, gener- alized perception of control, and posttraumatic stress disorder symptom severity,” Journal of Anxiety Disorders, vol. 11, no. 5, pp. 523–540, 1997. [54] U. Berggren and G. Meynert, “Dental fear and avoidance: causes, symptoms, and consequences,” The Journal of the Amer- ican Dental Association, vol. 109, no. 2, pp. 247–251, 1984. [37] H. 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https://openalex.org/W2567028502	https://europepmc.org/articles/pmc6130591?pdf=render	English	null	<i>Sempervivum davisii</i>: phytochemical composition, antioxidant and lipase-inhibitory activities	Pharmaceutical biology	2,016	cc-by	8,283	VOL. 55, NO. 1, 532–540 http://dx.doi.org/10.1080/13880209.2016.1255979 PHARMACEUTICAL BIOLOGY, 2017 VOL. 55, NO. 1, 532–540 http://dx.doi.org/10.1080/13880209.2016.1255979 PHARMACEUTICAL BIOLOGY, 2017 VOL. 55, NO. 1, 532–540 http://dx.doi.org/10.1080/13880209.2016.1255979 PHARMACEUTICAL BIOLOGY, 2017 ABSTRACT Context: Sempervivum davisii Muirhead (Crassulaceae) is a traditional medicinal herb from Eastern Anatolia. To date the composition of phytochemicals and physiological properties of this herb were not subjected to any research. j y Objective: This study identifies compounds in S. davisii hydrophilic extracts and evaluates their potential biological properties j y Objective: This study identifies compounds in S. davisii hydrophilic extracts and evaluates their potential biological properties. KEYWORDS Kaempferol; quercetin; volatile compounds; enzyme inhibition; FRAP; ORAC Materials and methods: Ethanol-based lyophilized extracts were obtained from aerial parts of plant (10 g of ground dry plant material in 200 mL of acidified aqueous ethanol, shaken for 2 h at 22 C with super- natant collected and freeze-dried under vacuum). Phytochemical composition was investigated by liquid chromatography mass spectrometry (LC-MS/MS, phenolics) and gas chromatography mass spectrometry (GC-MS, volatiles). Phenolic compounds were quantified by high-performance liquid chromatography (HPLC) and the Folin–Ciocalteu assay. Subsequently, antioxidant capacity [ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) assays] and enzyme inhibitory properties (iso- lated porcine pancreatic lipase) of the extracts were determined. p p p Results: Polyphenolic compounds were the main constituents of lyophilized extracts, among which kaempferol glycosides and quercetin hexoside dominated. The extracts exhibited potent antioxidant (FRAP values of 1925.2–5973.3 lM Fe2þ/g DW; ORAC values of 1858.5–4208.7 lM Trolox Eq./g DW) and moderate lipase inhibitory (IC50: 11.6–2.96 mg/mL) activities. Volatile compounds (nonanal, dehydroxylina- lool oxide isomers, 2-decenal, 2-undecenal, 2,6-di-tetr-butylphenol) were also found. y p Conclusions: Phenolic compounds with the dominating kaempferol and quercetin derivatives are the sources of potent antioxidant properties of S. davisii hydrophilic extracts. The extracts exhibit moderate inhibitory properties towards isolated pancreatic lipase. Introduction in a rosette. Young rosette leaves have scattered glandular hairs (Davis 1972). This species closely resemble S. armenum with the exception that S. armenum rosette leaves are glabrous when mature. Distinguishing these two Sempervivum species in the wild is difficult and therefore both species are equally utilized by the local population of Eastern Anatolia as a traditional herbal medicine. The genus Sempervivum (Crassulaceae) comprises approximately 50 species of wild-growing succulent perennial plants, spread in arid areas of Mediterranean countries from Spain to Minor Asia. Numerous Sempervivum species are utilized in traditional medi- cine to cure common ailments. In Turkey fresh juice from squeezed leaves of various Sempervivum plants is extensively used to treat earache and dermatophyte infections (Yes¸ilada et al. 1999). Among botanicals, S. tectorum L. is frequently used in Europe to relieve pain and sore ailments, particularly earache and ear inflammation, warts, ulcers, skin rash, insect bites and headache (Bremness & Norman 1995; Alberti et al. 2012). Extract of the same plant exhibited antioxidant properties in vitro (Kerry et al. 1992) and in vivo (Muselin et al. 2014), significant antinoci- ceptive (Alberti et al. 2012) and lipid-lowering activities in rats (Blazovics et al. 1993, 1994). Another Sempervivum species, S. armenum Boiss. Et Huet. was reported to be used by traditional healers in Turkey to treat cancer (Novaretti & Lemordant 1990). d h d f h h To date the phytochemical composition of S. davisii extracts and their physiological activities were not reported. Therefore, the present study investigates the composition and potential bio- logical properties of lyophilized hydrophilic extracts obtained from S. davisii stems, leaves and flowers. Yusuf Uzuna, Abdullah Dalara and Izabela Konczakb aDepartment of Pharmaceutical Botany, Faculty of Pharmacy, Yuzuncu Yil University, Van, Turkey; bFood Science & Technology, School of Chemical Engineering, The University of New South Wales, Kensington, New South Wales, Australia ARTICLE HISTORY Received 5 November 2015 Accepted 29 October 2016 Revised 3 September 2016 KEYWORDS Kaempferol; quercetin; volatile compounds; enzyme inhibition; FRAP; ORAC CONTACT Yusuf Uzun, PhD yusufuzun.pharm@gmail.com Yuzuncu Yil University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Zeve Campus, Van 65090, Turkey; Izabela Konczak, PhD i.konczak@unsw.edu.au Food Science & Technology, School of Chemical Sciences & Engineering, University of New South Wales, Sydney, NSW 2052, Australia 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri- bution, and reproduction in any medium, provided the original work is properly cited. ARTICLE HISTORY Received 5 November 2015 Accepted 29 October 2016 Revised 3 September 2016 mail.com Yuzuncu Yil University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Zeve Campus, zak@unsw.edu.au Food Science & Technology, School of Chemical Sciences & Engineering, University of New Quantification of phenolic compounds by high-performance liquid chromatography–diode array detector (HPLC–DAD) Quantification of phenolic compounds by the HPLC was conducted as described previously (Dalar & Konczak 2013) on HPLC system, which consisted of two LC-10ADVP pumps, SPD-M10ADVP diode array detector, CTO-1-ADVP column oven, DGU-12A degasser, SIL-10ADVP auto injector and SCL-10A system controller (Shimadzu Corporation, Kyoto, Japan) equipped with an Atlantis column (dC18, 4.6mm i.d 100mm length, 5lm particle size, Waters Associates, Chippendale, New South Wales, Australia). Analytical HPLC was run at 30 C and monitored at 280nm, 320nm, 370 and 520nm. Injection volume was 10lL. The follow- ing solvents in purified water with a flow rate of 1.0mL/min were used: A, 0.5% trifluoroacetic acid (TFA) and B, 0.5% TFA þ95% acetonitrile. The elution profile was a linear gradient elution for B of 0% to 60% over 20min followed by 100% B for 10min. The identity of compounds was confirmed by co-chromatography and comparison of their spectral characteristics with those of authentic standards (Sigma-Aldrich, Castle-Hill, New South Wales, Australia). The levels of phenolic compounds detected at 280nm were quanti- fied as mg gallic acid equivalents per gram dry weight of lyophilized extract (mg GA Eq./g DW) based on gallic acid calibration curve (concentration range: 0.0125–0.5mg/mL; r2¼1). Phenolic com- pounds detected at 320nm were calculated as mg of chlorogenic acid equivalents per gram dry weight (mg CHA Eq./g DW) based on chlorogenic acid calibration curve (concentration range: 0.0125–0.5mg/mL; r2¼1). Compounds detected at 370nm were quantified as mg rutin equivalent per gram dry weight (mg Rutin Eq./g DW) based on the rutin calibration curve (concentration range: 0.0125–0.5mg/mL; r2¼0.999). Total phenolic content Total phenolic content was determined using the Folin Ciocalteu assay (Singleton & Rossi 1965; Ainsworth & Gillespie 2007), with minor modifications as described previously (Konczak et al. 2010). Briefly, 25 lL of plant extracts (1mg/mL) were mixed with 125 lL Folin–Ciocalteu reagent earlier diluted with MilliQ water (1:10, v/v) in 96-well microplates and shaken for 3 min. In order to avoid the additive effects of ascorbic acid which can readily reacts with Folin–Ciocalteu reagent (Ainsworth & Gillespie 2007), the absorbance was measured at 600 nm for ascorbic acid correction. Subsequently, 125 lL of 6% Na2CO3 were added and the microplate was shaken for 12 min. The absorbance was then measured again at 600 nm. The results (F-C values) were expressed as mg gallic acid equivalents per gram of dry weight of the lyophilized extract (mg GAE/g DW), based on gallic acid standard curve and against a blank control. The analyses were conducted in triplicate. Identification of phenolic compounds by liquid chromatography–diode array–mass spectrometry (LC-DAD- MS/MS) Reagents Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich Inc. (St. Louis, MO) and were of analytical or HPLC grade. Porcine pancreatic lipase (type II) and orlistat were purchased from Sigma-Aldrich, Inc. (Sydney, Australia). Folin–Ciocalteu reagent was purchased from Merck (Darmstadt, Germany). Plant materials Sempervivum davisii plants (approximately 2 kg fresh weight) were harvested in G€orecek Village, S¸evkii Huseyin Place, Muradiye, Van City, in the Eastern Anatolia Region of Turkey, (GPS coordinates 39 05' 03.94" N; 4341' 33.36" E) in August, 2014. Their identity was confirmed by Muzaffer Mukemre at the Sempervivum davisii Muirhead from the north-eastern Anatolia region of Turkey is a perennial herb. Flowers are ovate to lanceolata, with yellowish petals. The leaves are thick, growing ading as Taylor & Francis Group. of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri- inal work is properly cited. PHARMACEUTICAL BIOLOGY 533 An aliquot (3 lL) of each ethanolic extract was chromatographed on a 150 2.1 mm i.d., 5 lm Luna Synergy Hydro column (Phenomenex, Torrance, CA) which was heated to 30 C. Analytes were separated using 0.5% of formic acid in purified water (solvent A) and 0.5% of formic acid in acetonitrile (solvent B) under a flow rate of 200lL/min. The gradient employed was 0% B for 2 min, and then increased to 100% B over 18 min, held for 1 min. The photodiode array detector was used to acquire data from 190–520 nm. Ions for mass spectrometry were generated using an electrospray source in either positive or negative mode (depending on analyte) under conditions set following optimiza- tion using chlorogenic acid (negative mode) or quercetin-3-gluco- side (positive mode). MS experiments in the full scan (parent and product-specific) and the selected reaction monitoring (SRM) mode were conducted. Ellagic acid, gallic acid, chlorogenic acid, quercetin-3-glucoside, kaempferol, luteolin, quercetin, rutin and cyanidin 3-glucoside were used as standard phenolic compounds. The composition of phenolic compounds was characterized based on their UV spectrum, retention time, co-chromatography with commercial standards, when available, and MS fragmentation patterns. Biological Sciences Department, Science Faculty, Yuzuncu Yil University, Turkey, according to the Flora of Turkey, volume 4 (Davis 1972) and a voucher specimen was deposited at the uni- versity’s herbarium (Herbarium code: VANF-164637; Collector code: MM336). The stems, leaves and flowers of fresh plants were divided, cleaned and dried at room temperature in the dark until dry. Subsequently, the samples were ground and stored at 20 C until analyzed. Preparation of lyophilized extracts The lyophilized extracts were prepared as described previously (Dalar & Konczak 2013). Briefly, the ground plant material was mixed with a 20-fold volume of acidified ethanol (80% ethanol, 19.9% H2O and 0.1% trifluoroacetic acid, v/v/v), shaken for 2 h at room temperature (22 C) and centrifuged for 20 min (15,320g) at 4 C (Sorvall RC-5B; DuPont, Wilmington, DE; rotor Beckman JA14 (137 mm) serial No. 02U8152; Thermo Fisher Scientific, Waltham, MA) with the supernatant collected. The supernatants from the consecutive extractions were combined. Solvent was evaporated under reduced pressure at 40 C. The derived fraction was dissolved in purified water and freeze-dried under a vacuum at 51 C to obtain a fine lyophilized powder used for analysis. Oxygen radical absorbance capacity (ORAC) assay Oxygen radical scavenging capacity was determined using the ORAC assay according to Prior et al. (2005) with some modifica- tions (Konczak et al. 2010). Preparations of fluorescein (120 nM) and AAPH (360 mM) were dissolved in phosphate-buffered saline (PBS; 75 mM, pH 7.0). Blank solution and diluted samples were placed in 96-well microplate, inserted into a fluorescence spectro- photometer (POLARstar Omega, BMG Labtech, Germany) and allowed to equilibrate at 37 C. Subsequently, AAPH was added to each well and fluorescence measurements initiated immedi- ately. Fluorescence (excitation wavelength 495 nm and emission wavelength 515 nm) was recorded every minute until the fluores- cence reached zero and a kinetic curve generated. The area under the curve (AUC) was integrated and standardized against a blank control. The measurements were carried out in triplicate. The antioxidant capacity of the samples was expressed as lM of Trolox equivalent per gram of dry weight of lyophilized extract (lM T Eq./g DW) based on a trolox standard curve. Identification of phenolic compounds by liquid chromatography–diode array–mass spectrometry (LC-DAD- MS/MS) To assist the spectrophotometric analysis of polymeric fraction, plant extract was hydrolyzed according to Pinto et al. (2008). Briefly, lyophilized plant extracts (10 mg) were added to 2 mL of 2N trifluoroacetic acid (TFA) in pyrex glass tubes. The tubes were placed into a heating instrument (Reacti-Therm Heating/stirring module, Selby Biolab, Pierce, Rockford, IL) and maintained at 120 C for 2 h. Subsequently, the solutions were transferred into LC-DAD-MS/MS analysis was conducted as described previously (Dalar & Konczak 2013) on a Quantum triple stage quadrupole (TSQ) mass spectrometer (Thermo Fisher Scientific, Waltham, MA) equipped with a quaternary solvent-delivery system, a col- umn oven, a photo-diode array detector and an auto sampler. Y. UZUN ET AL. 534 dilutions were mixed with 0.5 mL of copper tartrate reagent. The solution was heated (100 C for 15 min) and 0.5mL of arsenomoly- bolic acid reagent was added. Following mixing, absorbance was measured at 520 nm. The results were expressed as mg of glucose per gram lyophilized extract. 5 mL volumetric flasks and 80% methanol was added to adjust the volumes. The extracts were directly analyzed by HPLC-MS/MS as described earlier. Pancreatic lipase inhibitory activity Lipase inhibitory activity of the lyophilized plant extracts were assessed according to Sakulnarmrat and Konczak (2012), using 4- methylumbelliferyl oleate (0.1 mM) as a substrate, with the excep- tion of porcine pancreatic lipase (Sigma type II), which was pre- pared using a concentration of 0.085 g/mL. Relative lipase inhibition activity was calculated using the following formula: % Inhibition ¼ [1 – (FS – FSB)/(FC – FCB)] 100, where FS and FC were the values of samples and negative control measured fluorometrically at an emission wavelength of 460nm and excita- tion of 320 nm with slit widths of 5 nm (POLARstar Omega, BMG Labtech, Germany), and where FSB and FCB were, respectively the fluorescence readings of sample blank and control blank. Gas chromatography–mass spectrometry (GC–MS) analysis Gas chromatography–mass spectrometry (GC–MS) analysis Volatile compounds present in S. davisii were analyzed by gas chromatography–mass spectrometry (GC/MS) using a head space solid phase micro extraction and identified by the fragment ions and relative retention indices of their peaks with those of the MS library standards. Volatile compounds prepared by HS-SPME procedure were analyzed on Varian 3800 gas chromatograph dir- ectly interfaced with a Varian 2000 ion trap mass spectrometer (Varian Spa, Milan, Italy) equipped with a flame ionization detector (FID), at the injector temperature of 260 C in injection mode, splitless. The lyophilized extracts were chromatographed on a 60 0.25 mm i.d., 0.2 lm HP 88 column (Agilent Technologies, Santa Clara, CA). Oven temperature was pro- grammed as follows: 80 C at the start, followed by an increase to 190 C at a rate of 5 C/min, held at 190 C for 3min, followed by an increase at a rate of 5 C/min to 260 C and held for 3min. The subsequent temperature increase was at a rate of 2 C/min to 275 C and held for 12.5min. The carrier gas was helium, used at a constant pressure of 10psi; the transfer line temperature was 250 C; the ionization 8 mode, electron impact (EI); acquisition ion range, 40–200 m/z; scan rate, 1us-1. The compounds were identi- fied using the National Institute of Standards and Technology (NIST) library (NIST/WILEY/EPA/NIH), mass spectral library and verified by the retention indices, calculated as described by Van den Dool & Kratz (1963). Results were expressed as FID response area in relative percentages. Ferric reducing antioxidant power (FRAP) assay Ferric reducing antioxidant power (FRAP) assay Lyophilized plant extracts (2 g) were weighed into 40 mL vials and treated by divinyl benzene/carboxen/polydimethylsiloxane fibre with 50/30 mm film thickness. The fibre was preconditioned in the injection port of the gas chromatography (GC) as indicated by the producer before the analysis. The samples were placed in mininert push-button valves (Sigma-Aldrich) and kept at 35 C with continuous internal stirring and equilibrated for 30 min. Subsequently, the solid phase micro extraction (SPME) fibre was exposed for 40 min to the headspace at 35 C. SPME fibre was introduced into the GC injector after sampling and was left for 3 min to allow the analysis of thermal desorption. The headspace volume, heating temperature and time of the extraction were opti- mized according to Verzera et al. (2004). Total reducing capacity was determined using the FRAP assay (Benzie & Strain 1996) with minor modifications as described previously (Konczak et al. 2010). Briefly, the FRAP reagent consisted of 10 mL of 300 mM acetate buffer, 10 mL of 20 mM FeCl3 and 1 mL of 10 mM TPTZ solution. The acetate buffer (pH 3.6) consisted of 3.1 g of sodium acetate and 16mL acetic acid (glacial) per litre of water. The TPTZ solution consisted of 31.2mg of TPTZ in 10 mL of 40 mM hydrochloric acid. Briefly, 10lL of extracts were placed in 96-well microplate (Sarstedt Australia Pty Ltd., Technology Park, South Australia, Australia) and 200lL of FRAP reagent were added, followed by 15 s shak- ing. After incubation for 8 min, the absorbance was measured at 595 nm using a spectrophotometer (Wallac, Labsystems Multiskan MS; Thermo Fisher Scientific, Waltham, MA). The reducing capacity of the extracts were expressed as micromoles of iron (Fe2þ) per gram of dry weight of lyophilized extract (lM Fe þ 2/g DW) based on an iron sulphate standard (Fe2SO4) curve against a blank control. Phenolic compounds and sugars It was present at the highest level in flower extract (4.0%), followed by leaf (2.3%) and stem extract (3.1%). Other phenolic compounds detected in S. davisii stem and leaf extracts were apigenin, isorhamnetin, chlorogenic acid, hes- peritin pentoside, apigenin glucuronide, kaempferol glucuronide, quercetin rutinoside (Table 2). Flower extract contained p-coumaric acid, caffeic acid, apigenin, gallocatechin, isorhamnetin, hydroxy- benzoic-O-hexoside, chlorogenic acid, hesperitin pentoside, apige- nin glucuronide, kaempferol glucuronide and quercetin rutinoside (Table 2). Table 1. The yield of lyophilized extracts obtained from S. davisii Muirhead aer- ial plant parts, their phenolics and sugar contents. Table 1. The yield of lyophilized extracts obtained from S. davisii Muirhead aer- ial plant parts, their phenolics and sugar contents. Stem Leaf Flower Yield (%)a 9.68 17.87 16.70 Total phenolics at 280 nmb 45.9 ± 1.2c 82.7 ± 4.8b 223.4 ± 1.4a at 326 nmc 32.2 ± 0.7c 56.6 ± 0.1b 82.1 ± 2.3a at 370 nmd 40.0 ± 4.1c 62.0 ± 1.9b 113.7 ± 3.2a F-Ce 105.1 ± 2.7c 133.6 ± 3.3b 291.3 ± 8.6a Sugar content Total sugarsf 448.1 ± 8.3a 322.9 ± 15.5c 377.3 ± 11.2b Reducing sugarsf 273.9 ± 2.9a 206.8 ± 5.2c 232.6 ± 1.5b Means with different letters in the same row were significantly different at the level (p < 0.001); n ¼ 3. aPercentage original sample dry weight. bSum of all peaks detected by HPLC and quantified as mg Gallic acid Eq./g DW of lyophilized extract. cSum of all peaks detected by HPLC and quantified as mg CHA Eq./g DW of lyophilized extract. dSum of all peaks detected by HPLC and quantified as mg Rutin Eq./g DW of lyophilized extract. eTP: Folin–Ciocalteu values (mg Gallic acid Eq./g DW of lyophilized extract). fmg Glucose Eq./g DW of lyophilized extract. ial plant parts, their phenolics and sugar contents. Stem Leaf Flower Yield (%)a 9.68 17.87 16.70 Total phenolics at 280 nmb 45.9 ± 1.2c 82.7 ± 4.8b 223.4 ± 1.4a at 326 nmc 32.2 ± 0.7c 56.6 ± 0.1b 82.1 ± 2.3a at 370 nmd 40.0 ± 4.1c 62.0 ± 1.9b 113.7 ± 3.2a F-Ce 105.1 ± 2.7c 133.6 ± 3.3b 291.3 ± 8.6a Sugar content Total sugarsf 448.1 ± 8.3a 322.9 ± 15.5c 377.3 ± 11.2b Reducing sugarsf 273.9 ± 2.9a 206.8 ± 5.2c 232.6 ± 1.5b The composition of polymeric fraction of the extract was eval- uated following acid hydrolysis as described by Pinto et al. (2008). Phenolic compounds and sugars The yield of lyophilized extracts obtained from dry aerial parts of S. davisii plant (stems, leaves and flowers) is shown in Table 1. Leaf and flower tissues yielded the largest amount of lyophilized extracts, which represented over 15% of the dry weight. p y g The HPLC analysis revealed a presence of phenolic com- pounds, detected predominantly at 280, 326, and 370 nm. No compounds were detected at 520 nm, which indicates that the mixture did not contain anthocyanins. Flower extract contained the highest level of phenolics, approximately threefold that of leaf and fourfold that of stem extracts (Table 1). In agreement, the Folin–Ciocalteu (F-C) assay confirmed that flower was the richest source of phenolic compounds, followed by leaf and stem. The total phenolic contents of the evaluated extracts (105.1–291.3 mg GA Eq./g extract) were comparable to several common industrial medicinal herbs such as Cosmo sulphureus Cav. (Asteraceae) (102.5 ± 10.2 mg GA Eq./g extract), Tagetes erecta L. (Asteraceae) (212.9 ± 6.0 mg GA Eq./g extract), Cistus ladaniferus L. (Cistaceae) (103.2 ± 0.4 mg GA Eq./g extract), Eucalyptus globulus Labill. (Myrtaceae) (113.7 ± 0.3 mg GA Eq./g extract), Rosa dam- ascena Mill. (Rosaceae) (124.9 ± 1.5 mg GA Eq./g extract), Coffea arabica L. (Rubiaceae) (173.5 ± 1.9 mg GA Eq./g extract) and Ilex paraguariensis A. St. Hill. (Aquifoliaceae) (202.6 ± 5.1 mg GA Eq./g extract) (Dudonnne et al. 2009; Kaisoon et al. 2012). All extracts were rich in sugars, which contributed over 30% of the The third major phenolic compound represented by peak 3 (Figure 1) showed a negatively charged molecular ion ([M–1]) at m/z 623 and produced a MS/MS fragment of 285 m/z. A hexo- side moiety (162 amu) and a glucuronide moiety (176 amu) of neutral losses were also detected (Table 2). On the basis of molecular weight, fragmentation pattern and absorbance spec- trum, this compound was tentatively identified as kaempferol hexoside þ glucuronide. This compound was present in all plant parts. In stem, leaf and flower extract it contributed, respectively, 9.0%, 6.7% and 3.7% of total phenolics (Table 2). Phenolic compound represented by peak 1 (Figure 1) had a positively charged molecular ion ([M þ 1]þ) at m/z 465 and a 303/m/z MS/MS fragment, indicating a neutral loss of hexoside moiety (162 amu). Based on molecular weight, fragmentation pat- tern and absorbance spectrum, this compound was tentatively identified as quercetin (or hesperitin) hexoside (Table 2). Sugar content Total sugars and reducing sugars were assayed as described by Cardozo et al. (2011). The phenol–sulphuric acid method was used to determine total neutral sugars. Aliquots (0.8 mL) of dif- ferent extract dilutions were mixed with 0.04 mL of 80% phenol and 2 mL of H2SO4 and incubated for 20 min at 100 C. Absorbance was measured at 490 nm (Shimadzu 1601 spectrophotometer; Tokyo, Japan). The results were expressed as mg of glucose per gram dry weight (mg Glucose Eq./g DW) of lyophilized extract. Reducing sugars were measured using the Somogyi–Nelson method. Aliquots (0.1mL) of different extract PHARMACEUTICAL BIOLOGY 535 extracts dry weight with 322.9 ± 15.5 mg Glucose Eq./g DW in leaf extract and 448.1 ± 8.3 mg Glucose Eq./g DW in stem extract. Figure 1 represents a typical HPLC chromatogram of hydro- philic extract obtained from aerial parts of S. davisii plant. Oligomeric and/or polymeric compounds, potentially proantho- cyanidins, represented by a characteristic hump on the HPLC chromatogram dominated (Figure 1(A)). The LC/MS analysis revealed the presence of a rich mixture of phenolic compounds (Table 2). The major phenolic compound of S. davisii was tenta- tively identified as kaempferol hexoside (Figure 1, peak 4) by the presence of negatively charged molecular ions ([M þ 1]þ)/ ([M 1]) at m/z 449/447 and MS/MS fragments 287/285 m/z (Table 2). The loss of 162 amu indicated a hexoside unit. This compound constituted 17.5% of total phenolics in leaf extract, 21.0% in flower extract phenolics and 27.7% in stem extract (Table 2). Statistical analysis extracts dry weight with 322.9 ± 15.5 mg Glucose Eq./g DW in leaf extract and 448.1 ± 8.3 mg Glucose Eq./g DW in stem extract. leaf extract and 448.1 ± 8.3 mg Glucose Eq./g DW in stem extract. Figure 1 represents a typical HPLC chromatogram of hydro- philic extract obtained from aerial parts of S. davisii plant. Oligomeric and/or polymeric compounds, potentially proantho- cyanidins, represented by a characteristic hump on the HPLC chromatogram dominated (Figure 1(A)). The LC/MS analysis revealed the presence of a rich mixture of phenolic compounds (Table 2). The major phenolic compound of S. davisii was tenta- tively identified as kaempferol hexoside (Figure 1, peak 4) by the presence of negatively charged molecular ions ([M þ 1]þ)/ ([M 1]) at m/z 449/447 and MS/MS fragments 287/285 m/z (Table 2). The loss of 162 amu indicated a hexoside unit. This compound constituted 17.5% of total phenolics in leaf extract, 21.0% in flower extract phenolics and 27.7% in stem extract (Table 2). The mean values were calculated based on at least three determi- nations (n ¼ 3). One-way ANOVA followed by the Bonferroni post hoc test were performed to assess differences between the samples at the level of p < 0.05. Statistical correlation analyses were performed using Graphpad Prism 5 (Graphpad Software, CA). Results from correlation analysis were considered statistic- ally significant when p < 0.05. All half maximal inhibitory con- centration (IC50) values were calculated from the corresponding dose inhibition curves according to their best-fıt shapes based on at least four reaction points using Graphpad Prism. Results and discussion Phytochemical composition Phytochemical composition The second major phenolic compound of S. davisii (Figure 1, peak 2) gave a negatively charged molecular ion ([M 1]) at m/z 593 and displayed a MS/MS fragment of 285 m/z. It also exhibited a loss of rutinose moiety (308 amu) (Table 2). On the basis of molecular weight, fragmentation pattern and absorbance spectrum, this compound was tentatively identified as kaempferol rutinoside. Kaempferol rutinoside contributed a high 17.3% of total phenolics in flower extract, 15.1% in leaf and 10.1% in stem extract (Table 2; Figure 1). Phenolic compounds and sugars Phenolic compounds and sugars High-performance liquid chromatography profile of ethanol-based lyophilized extract obtained from S. davisii Muirhead aerial parts at (A) 280nm and (B) 370nm; (1) quercetin/hesperidin hexoside, (2) kaempferol rutinoside, (3) kaempferol hexoside þ glucuronide, (4) kaempferol hexoside. gure 1. High-performance liquid chromatography profile of ethanol-based lyophilized extract obtained from S. davisii Muirh 70nm; (1) quercetin/hesperidin hexoside, (2) kaempferol rutinoside, (3) kaempferol hexoside þ glucuronide, (4) kaempferol hexo oxide isomers with major contribution of cymene and nonanal. Flower extract contained a mixture of nonanal, 2-decenal, 2- undecenal, 2,6-di-tert-butylphenol, palmitic acid ethylester, 2.4- decadienal and octanal. kaempferol and its glycosides as the major phenolic compounds of S. tectorum. Our findings are in agreement with these earlier results and support the opinion that kaempferol and its glycosides are the characteristic key phenolic compounds of Sempervivum species. Compound 1 produced m/z 55, 67, 82, 96, 110, 123 and 137 mass spectra (Figure 2, Table 3). On the basis of molecular weight, fragment ions and MS library standards, this compound was tentatively identified as dehydroxylinalool oxide isomers. The isomers contributed a high 32.9% and 45.4% toward the volatile compounds mixture of, respectively, stem and leaf; only their traces (<2%) were found in the flower extract (Table 3). Phenolic compounds and sugars The major aglycone yielded was kaempferol. In agree- ment, kaempferol was the unique aglycone of the hydrolyzed oli- gomeric polyphenolic fraction of S. tectorum leaf extract (Abram & Donko 1999). Similarly, Stevens et al. (1996) identified kaemp- ferol as the principal flavonol obtained after acid hydrolysis of extracts of 34 Sempervivum species, including S. armenum, S. marmoreum Griseb. and S. tectorum. Alberti et al. (2012) ana- lyzed phenolic composition of S. tectorum. According to their study, kaempferol glycosides were the major bioactive com- pounds of S. tectorum extracts. Sentjurc et al. (2003) reported p aPercentage original sample dry weight. b bSum of all peaks detected by HPLC and quantified as mg Gallic acid Eq./g DW of lyophilized extract. cSum of all peaks detected by HPLC and quantified as mg CHA Eq./g DW of lyophilized extract. d dSum of all peaks detected by HPLC and quantified as mg Rutin Eq./g DW of lyophilized extract. y eTP: Folin–Ciocalteu values (mg Gallic acid Eq./g DW of lyophilized extract). fmg Glucose Eq./g DW of lyophilized extract. 536 Y. UZUN ET AL. Y. UZUN ET AL. and its glycosides as the major phenolic compounds oxide isomers with major contribution of cymene and Retention time (minutes) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Retention time (minutes) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 mAU mAU 0 0 10 20 30 40 50 60 20 40 60 80 100 280 nm (A) (B) 1 3 2 4 1 4 3 2 370 nm h-performance liquid chromatography profile of ethanol-based lyophilized extract obtained from S. davisii Muirhead aerial parts at (A) 280 uercetin/hesperidin hexoside, (2) kaempferol rutinoside, (3) kaempferol hexoside þ glucuronide, (4) kaempferol hexoside. UZUN ET AL. Retention time (minutes) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 mAU 0 20 40 60 80 100 280 nm (A) 1 3 2 4 Retention time (minutes) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 mAU 0 10 20 30 40 50 60 (B) 1 4 3 2 370 nm H h f l d h h f l f h l b d l h l d b d f S d M h d l (A) Figure 1. Volatile composition Mass spectrometric analysis revealed the presence of 11 volatile compounds in the lyophilized S. davisii extracts evaluated in this study. Figure 2 shows representative total ion chromatogram of volatile compounds detected in the aerial parts. Stem and leaf extracts exhibited a similar composition of volatile compounds, which was different to that of flower (Table 3). The major vola- tile compounds of stem and leaf extracts were dehydroxylinalool The major volatile compound (compound 4; 22.0% of the mixture) of flower extract produced fragment ions of 57, 70, 82, 98, 114 and 124 m/z (Figure 2, Table 3) and was tentatively iden- tified as nonanal. PHARMACEUTICAL BIOLOGY 537 Table 2. Mass spectrometric details (LC-MS/MS) and concentration (HPLC-DAD) of phenolic compounds in ethanol-based lyophilized extracts obtained from S. davisii Muirhead aerial plant parts. Table 2. Mass spectrometric details (LC-MS/MS) and concentration (HPLC-DAD) of phenolic compounds in ethanol-based lyophilized extracts obtained from S. davisii Muirhead aerial plant parts. MS/MS Stem Leaf Flower Individual phenolic compounds [M þ 1]þ/ [M 1] Fragments (m/z) (þ/) Concentration (mg/g DW)d Contribution (%) Concentration (mg/g DW)d Contribution (%) Concentration (mg/g DW)d Contribution (%) p-Coumaric acid1 -/163 -/119 ND – ND – T < 2% Caffeic acid1 -/179 -/135 ND – ND – T < 2% Apigenin1 -/269 -/117 T < 2% T < 2% T < 2% Gallocatechin1 -/305 -/225 ND – ND – T < 2% Isorhamnetin1 -/315 -/300 T < 2% T < 2% T < 2% Hydroxybenzoic acid-O- hexoside2 -/299 -/137 ND – ND – T < 2% Chlorogenic acid1,3 -/353 -/191 T < 2% T < 2% T < 2% Hesperitin pentoside2 435/- 303/- T < 2% T < 2% T < 2% Apigenin glucuronide2 -/445 -/269 T < 2% T < 2% T < 2% Kaempferol hexoside2,3 449/447 287/285 5.6 ± 0.01 17.5% 15.7 ± 0.3b 27.7% 17.2 ± 0.7a 21.0% Kaempferol glucuronide2 -/461 -/285 T < 2% T < 2% T < 2% Quercetin/hesperitin hexoside2,3 465/- 303/- 1.01 ± 0.0c 3.1% 1.3 ± 0.0b 2.3% 3.3 ± 0.0a 4.0% Kaempferol rutinoside2,3 -/593 -/285 3.3 ± 0.0c 10.1% 8.6 ± 0.1b 15.1% 14.2 ± 0.1a 17.3% Quercetin rutinoside (Rutin)1 611/609 303/301 T < 2% T < 2% T < 2% Kaempferol hexoside þ glucuronide2,3 -/623 -/285 2.9 ± 0.0c 9.0% 3.8 ± 0.0a 6.7% 3.1 ± 0.0b 3.7% T: traces; ND: not detected. Tentative assignment based on ms data only. 3Value is expressed as mg chlorogenic acid Eq/g DW lyophilized extract. 4 / d i h f l hili d T: traces; ND: not detected. Means with different superscript letters in the same row were significantly different at the level (p < 0.001); n ¼ 3. 1Assignment confirmed with reference standard. 2T i i b d d l Volatile composition 4lmol of drug equivalent per g dry weight of lyophilized extract (lM/g DW of lyophilized extract) Stem Leaf Flower Antioxidant activity ORAC1 1858.5 ± 105.7b 2021.8 ± 133.3b 4208.7 ± 240.8a FRAP2 1925.2 ± 44.4c 2372.2 ± 93.3b 5973.3 ± 96.8a Lipase-inhibitory activity IC50 3 11.16 ± 0.13c 7.83 ± 0.12b 2.96 ± 0.06a Orlistat Eq4 0.25 ± 0.00c 0.35 ± 0.01b 0.93 ± 0.02a Means with different superscript letters in the same raw were significantly differ- ent at the level (p < 0.001). p 1Oxygen radical absorbance capacity (lM Trolox Eq./g DW of lyophilized extract). 2Ferric reducing capacity (lM Fe2þ/g DW of lyophilized extract). 3H lf i l i hibit t ti ( l hili d t t/ L Antioxidant capacity 4lmol of drug equivalent per g dry weight of lyophilized extract (lM/g DW of lyophilized extract). The antioxidant capacity of hydrophilic extracts obtained from stem, leaf and flower of S. davisii is shown in Table 4. Flower extract exhibited the highest capacity demonstrated by both, the highest oxygen radical absorbance capacity (ORAC) and the fer- ric reducing antioxidant power (FRAP) values. Volatile composition Means with different superscript letters in the same row were significantly different at the level (p < 0.001); n ¼ 3. 1Assignment confirmed with reference standard. 2Tentative assignment based on ms data only. 3Value is expressed as mg chlorogenic acid Eq/g DW lyophilized extract. 4mg/g dry weight of lyophilized extract. Figure 2. Total ion chromatogram of volatile compounds profile of ethanol-based lyophilized extract obtained from S. davisii Muirhead aerial parts: (1) Dehydroxylinalool oxide isomers, (2) Octanal, (3) Cymene, (4) Nonanal, (5) 2-Decenal, (6) 2,4-Decadienal, (7) 2-Undecenal, (8) 2,6-Di-tert-butylphenol, (9) Palmitic acid ethylester. Figure 2. Total ion chromatogram of volatile compounds profile of ethanol-based lyophilized extract obtained from S. davisii Muirhead aerial parts: (1) Dehydroxylinalool oxide isomers, (2) Octanal, (3) Cymene, (4) Nonanal, (5) 2-Decenal, (6) 2,4-Decadienal, (7) 2-Undecenal, (8) 2,6-Di-tert-butylphenol, (9) Palmitic acid ethylester. Figure 2. Total ion chromatogram of volatile compounds profile of ethanol-based lyophilized extract obtained from S. davisii Muirhead aerial parts: (1) Dehydroxylinalool oxide isomers, (2) Octanal, (3) Cymene, (4) Nonanal, (5) 2-Decenal, (6) 2,4-Decadienal, (7) 2-Undecenal, (8) 2,6-Di-tert-butylphenol, (9) Palmitic acid ethylester. 538 Y. UZUN ET AL. Y. UZUN ET AL. 538 Table 3. Volatile compounds in lyophilized hydrophilic extracts prepared from S. davisii Muirhead aerial plant parts. Table 3. Volatile compounds in lyophilized hydrophilic extracts prepared from S. davisii Muirhead aerial plant parts. Relative concentration (%) Number Retention time Name of compound Molecular formula Molecular mass Fragment ions Stem Leaf Flower 1 15.4 Dehydroxylinalool oxide isomers C10H16O 152 55, 67 82, 96, 110, 123, 137 32.88 45.41 T 2 16.3 Octanal C8H16O 128 57, 69 81, 84, 100, 110 T T 3.88 3 16.5 Cymene C10H14 134 51, 77 91, 119, 134 3.76 4.75 ND 4 18.3 Cyclohexene C10H18O 154 69, 81 93, 121 T T ND 5 19.4 Nonanal C9H18O 142 57, 70,82 98, 114, 124 2.00 2.30 22.01 6 21.7 Cylodecane C10H20 140 55, 70 83, 97, 111, 140 T T ND 7 24.6 2-Decenal C10H18O 154 55, 70 83, 98, 110, 136 T T 16.39 8 26.9 2,4-Decadienal C10H16O 152 67, 81 95, 123, 152 T T 4.04 9 27.7 2-Undecenal C11H20O 168 55, 70 83, 97, 121, 166 T T 18.58 10 31.6 2,6-Di-tert-butylphenol C14H22O 206 57, 74 91, 163, 191, 206 ND ND 7.61 11 43.6 Palmitic acid ethylester C18H36O2 284 55, 88 101, 115, 157, 199, 241, 284 T T 4.08 T: traces; ND: not detected. Lipase inhibitory activity In the present study, S. davisii plant extracts exhibited a dose- dependent lipase inhibitory activity (Figure 3). Among them, flower extract exhibited the highest activity (IC50: 2.96mg/mL), and was followed by leaf and stem extracts. The lipase inhibitory activity of flower extract was higher than that of edible flowers from Thailand (IC50 values of 4.60–7.87mg/mL) (Kaisoon et al. 2012). The ORAC values of S. davisii varied from 1858.5 ± 105.7 (stem) to 4208.7 ± 240.8 (flower) lmol T Eq./g DW in the follow- ing order: stem leaf < flower (Table 4). The ORAC value of S. davisii flower extract was superior to that of pomegranate poly- phenol-rich extract (3210 lmol TE/g DW) used as a traditional medicine and a food supplement (Madrigal-Carballo et al. 2009). Leaf and stem extracts exhibited similar ORAC values to that of Hibiscus sabdariffa L. (2307 ± 10 lmol TE/g DW), a tropical plant commonly used for the preparation of soft drink (Fernandez- Arroyo et al. 2011). Volatile composition Relative concentration (%) Table 4. Antioxidant and lipase inhibitory activities of lyophilized hydrophilic extracts prepared from S. davisii Muirhead aerial plant parts. The second major volatile compound of flower extract had fragment ions of 55, 70, 83, 97, 121 and 166 m/z and was identified as 2-undecenal (Table 3). The compound 7 con- tributed 16.4% of the volatile mixture of flower extract and produced mass spectra m/z of 55, 70, 83, 98, 110 and 136. According to molecular weight, fragment ions and MS library standards this compound was tentatively identified as 2-decenal (Table 3). Minor volatile compounds present at trace level (< 2%) were octanal, cyclohexene, cylodecane, 2- decenal, 2,4-decadienal, 2-undecenal and palmitic acid ethyl- ester (Table 3). Table 4. Antioxidant and lipase inhibitory activities of lyophilized hydrophilic extracts prepared from S. davisii Muirhead aerial plant parts. Stem Leaf Flower Antioxidant activity ORAC1 1858.5 ± 105.7b 2021.8 ± 133.3b 4208.7 ± 240.8a FRAP2 1925.2 ± 44.4c 2372.2 ± 93.3b 5973.3 ± 96.8a Lipase-inhibitory activity IC50 3 11.16 ± 0.13c 7.83 ± 0.12b 2.96 ± 0.06a Orlistat Eq4 0.25 ± 0.00c 0.35 ± 0.01b 0.93 ± 0.02a Means with different superscript letters in the same raw were significantly differ- ent at the level (p < 0.001). 1O di l b b it ( M T l E / DW f l hili d Table 4. Antioxidant and lipase inhibitory activities of lyophilized hydrophilic extracts prepared from S. davisii Muirhead aerial plant parts. Stem Leaf Flower Antioxidant activity ORAC1 1858.5 ± 105.7b 2021.8 ± 133.3b 4208.7 ± 240.8a FRAP2 1925.2 ± 44.4c 2372.2 ± 93.3b 5973.3 ± 96.8a Lipase-inhibitory activity IC50 3 11.16 ± 0.13c 7.83 ± 0.12b 2.96 ± 0.06a Orlistat Eq4 0.25 ± 0.00c 0.35 ± 0.01b 0.93 ± 0.02a Means with different superscript letters in the same raw were significantly differ- ent at the level (p < 0.001). 1Oxygen radical absorbance capacity (lM Trolox Eq./g DW of lyophilized extract). 2Ferric reducing capacity (lM Fe2þ/g DW of lyophilized extract). 3Half maximal inhibitory concentration (mg lyophilized extract/mL). References Abram V, Donko M. 1999. Tentative identification of polyphenols in Sempervivum tectorum and assessment of the antimicrobial activity of Sempervivum L. J Agric Food Chem.47:485–489. g Ainsworth EA, Gillespie KM. 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nat Protoc. 2:875–877. and active oxygen species. Tatsimo et al. (2012) reported a pro- nounced DPPH radical scavenging activity of kaempferol glyco- sides, while Rice-Evans et al. (1997) reported its superior in vitro antioxidant capacity to that of vitamin C and vitamin E. Therefore, quercetin hexoside and kaempferol glycosides could be one of the main contributors to antioxidant properties of S. davisii extracts. In agreement, kaempferol and kaempferol-3-glu- coside were suggested as the major sources of antioxidant cap- acity of S. tectorum juice extract (Sentjurc et al. 2003). and active oxygen species. Tatsimo et al. (2012) reported a pro- nounced DPPH radical scavenging activity of kaempferol glyco- sides, while Rice-Evans et al. (1997) reported its superior in vitro antioxidant capacity to that of vitamin C and vitamin E. Therefore, quercetin hexoside and kaempferol glycosides could be one of the main contributors to antioxidant properties of S. davisii extracts. In agreement, kaempferol and kaempferol-3-glu- coside were suggested as the major sources of antioxidant cap- acity of S. tectorum juice extract (Sentjurc et al. 2003). Alberti A, Beni S, Lacko E, Riba P, Al-Khrasani M, Kery A. 2012. Characterization of phenolic compounds and antinociceptive activity of Sempervivum tectorum L. leaf juice. J Pharm Biomed Anal. 70:143–150. Artz WE, Bishop PD, Dunker AK, Schanus EG, Swanson BG. 1987. Interaction of synthetic proanthocyanidin dimer and trimer with bovine serum albumin and purified bean globulin fraction G-1. J Agr Food Chem. 35:417–421. Benzie IFF, Strain JJ. 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem. 239:70–76. Volatile compounds present in the extracts may be a second- ary source of antioxidant activities. Weak antioxidant activities of volatile compounds including nonanal, decenal, undecenal and linalool oxide and cymene were reported by others (Ruberto & Baratta 2000). Blazovics A, Feher J, Feher E, Kery A, Petri G. 1993. Liver protecting and lipid lowering effects of Sempervivum tectorum extract in the rat. Phytother Res. 7:98–100. Blazovics A, Gonzales-Cabello R, Barta I, Gergely P, Feher J, Kery A, Petri G. 1994. References Effect of liver-protecting Sempervivum tectorum extract on the immune reactivity of spleen cells in hyperlipidaemic rats. Phytother Res. 8:33–37. Bremness L, Norman J. 1995. The complete book of herbs & spices. NY: Viking Penguin. Phenolic compounds and antioxidant capacity davisii may play a role in the in vitro suppres- sion of pancreatic lipase activity. Conclusions Kaempferol glycosides (hexoside and rutinoside) were identified as the dominating phenolic compounds of lyophilized hydrophilic extracts prepared from stems, leaves and flowers of S. davisii and were accompanied by quercetin hexoside. The extracts contained a rich mixture of volatile compounds comprising nonanal, dehy- droxylinalool oxide isomers, 2-decenal, 2-undecenal and 2,6-di- tert-butylphenol, octanal, cymene, 2,4-decadienal and palmitic acid ethylester. The antioxidant activity of the evaluated extracts was equivalent or superior to that of commercially used industrial medicinal herbs. The extracts also exhibited moderate lipase- inhibitory activities. Figure 3. Dose-dependent antilipase activity of S. davisii Muirhead extracts. Table 5. Correlation analysis between phenolic compounds and antioxidant and lipase inhibitory activities of plant extracts obtained from S. davisii Muirhead aerial plant parts. Table 5. Correlation analysis between phenolic compounds and antioxidant and lipase inhibitory activities of plant extracts obtained from S. davisii Muirhead aerial plant parts. Table 5. Correlation analysis between phenolic compounds and antioxidant and lipase inhibitory activities of plant extracts obtained from S. davisii Muirhead aerial plant parts. Acknowledgements aerial plant parts. F-C FRAP ORAC Lipase Phenolics (280 nm) (mg Gallic acid Eq./g DWa) 0.9970 0.9908 0.9819 0.9971 Phenolics (326 nm) (mg Chlorogenic acid Eq./g DW) 0.8704 0.8401 0.8708 0.8708 Phenolics (370 nm) (mg Rutin Eq./g DW) 0.9760 0.9620 0.9460 0.9770 F-C (mg Gallic acid Eq./g DW) 1 0.9983 0.9936 1 FRAP (lM Fe2þ/g DW) 0.9983 1 0.9985 0.9849 ORAC (lM Trolox Eq./g DW) 0.9936 0.9985 1 0.9935 Lipase (lM Orlistat Eq./g DW) 1 0.9849 0.9935 1 Quercetin hexoside 0.9999 0.9999 0.9970 0.9999 Kaempferol hexoside 0.5000 0.4058 0.4200 0.5000 Kaempferol rutinoside 0.8710 0.8420 0.8130 0.8720 Kaempferol hexoside þ glucuronide 0.0025 0.0390 0.0560 0.0240 F-C: Folin–Ciocalteu method; FRAP: ferric reducing capacity; ORAC: oxygen rad- ical absorbance capacity. aDry weight of lyophilized extract. The authors thank Mr Muzaffer M€ukemre of the Biology Department in Yuzunci Yil University for help in plant identification and Dr A. S. Bengu in GC/MS analysis. Phenolic compounds and antioxidant capacity A strong positive relationship has been identified between total phenolic levels (F-C values) and antioxidant capacities of S. davi- sii extracts as evaluated using FRAP assay (r2¼0.9983) and ORAC assay (r2¼0.9936). The values of correlation coefficients between total phenolic content analyzed spectrophotometrically at 280 nm and antioxidant capacities (r20.9918) are similar to those between total phenolics (Folin–Ciocalteu values) and anti- oxidant capacities (Table 5). This suggests that phenolic com- pounds present in the extracts might be one of the main agents contributing to antioxidant capacities. y The total reducing capacity (FRAP values) of S. davisii extracts ranged from 1925.2 ± 44.4 (stem) to 5973 ± 96.8 (flower) lM Fe þ 2/g DW and were in the same order as the ORAC values (stem leaf < flower). The FRAP value of flower extract was approximately 2.5- to 3-fold that of leaf and stem extracts (Table 4). The FRAP value of S. davisii flower was sixfold that of allspice (1015 lM Fe2þ/g DW), cinnamon (984 lM Fe2þ/g DW) and approximately ninefold that of rosemary (669 lM Fe2þ/g DW) and thyme (637 lM Fe2þ/g DW), important commercial spices promoted for their health benefits (Dragland et al. 2003). The total reducing capacity of leaf was approximately fivefold that of saffron (478 lM Fe2þ/g DW), oregano (450 lM Fe2þ/g DW) and tarragon (433 lM Fe2þ/g DW) (Dragland et al. 2003). The FRAP value of S. davisii stem was fourfold that of a Chinese herbal medicinal plant, Sargentodoxa cuneata Rehd. Et Wils (Sargentodoxaceae) (453.53 ± 10.3 lM Fe2þ/g DW), which dis- played the highest FRAP value among 45 Chinese herbal medi- cinal plants (Li et al. 2008). The correlation analysis for the relationship between individ- ual phenolic compounds and antioxidant capacity showed that quercetin hexoside (r20.99) and kaempferol rutinoside (r20.8130) correlated very well with antioxidant activities with quercetin showing superior correlation. A moderate positive cor- relation was found for kaempferol hexoside (r20.4) (Table 5). Kaempferol and its glycosides are well-known for their potential antioxidant activities due to their ability to scavenge free radicals 539 PHARMACEUTICAL BIOLOGY Root Stem Leaf Flower 0 20 40 60 80 100 10 mg/mL 7.5 mg/mL 5 mg/mL 2.5 mg/mL Inhibition (%) Figure 3. Dose-dependent antilipase activity of S. davisii Muirhead extracts. compounds of S. davisii may play a role in the in vitro suppres- sion of pancreatic lipase activity. compounds of S. Disclosure statement The authors report no conflict of interest. The authors alone are responsible for the content of the paper. Phenolic compounds and lipase inhibitory activity Cardozo ML, Ordonez RM, Alberto MR, Zampini IC, Isla MI. 2011. Antioxidant and antiinflammatory activity characterization and genotoxic- ity evaluation of Ziziphus mistol ripe berries, exotic Argentinian fruit. Food Res Int. 44:2063–2071. Sempervivum davisii extracts evaluated in this study exhibited moderate lipase inhibitory activities. Strong positive correlations were identified between lipase inhibitory activities and total phe- nolics, antioxidant capacities, quercetin hexoside and kaempferol rutinoside (r20.8720). Relatively lower correlations were found for kaempferol hexoside (r2¼0.5000) and kaempferol hexosi- de þ glucuronide (r2¼0.0240) (Table 5). Artz et al. (1987) reported that proanthocyanidins or condensed tannins are prone to complexation with proteins, and dimers and monomers are less effective. Therefore, it can be speculated that polymeric Dalar A, Konczak I. 2013. 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Quantification of the polyphenolic fraction and in vitro antioxidant and in vivo anti-hyperlipemic activities of Hibiscus sabdariffa aqueous extract. Food Res Int. 44:1490–1495. p Ruberto J, Baratta MT. 2000. Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem. 69:167–174. y Sakulnarmrat K, Konczak I. 2012. Composition of native Australian herbs polyphenolic-rich fractions and in vitro inhibitory activities against key enzymes relevant to metabolic syndrome. Food Chem. 134:1011–1019. Muselin F, Trif A, Stana LG, Cristina TR, Gravila C, Macinic I, Dumitrescu E. 2014. Protective effects of aqueous extract of Sempervivum tectorum L (Crassulaceae) on aluminium-induced oxidative stress in rat blood. Trop J Pharm Res. 13:179–184. Sentjurc M, Nemec M, Connor HD, Abram V. 2003. Antioxidant activity of Sempervivum tectorum and its components. J Agr Food Chem. 51: 2766–2771. 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https://openalex.org/W2890644408	https://europepmc.org/articles/pmc6163900?pdf=render	English	null	Damage Detection in Active Suspension Bridges: An Experimental Investigation	Sensors	2,018	cc-by	17,535	Received: 8 August 2018; Accepted: 4 September 2018; Published: 7 September 2018 Abstract: This paper considers a Hilbert marginal spectrum-based approach to health monitoring of active suspension bridge hangers. The paper proposes to takes advantage of the presence of active cables and use them as an excitation mean of the bridge, while they are used for active damping. The Hilbert–Huang transform is used to calculate the Hilbert marginal spectrum and establish a damage index for each hanger of the suspension bridge. The paper aims to investigate the method experimentally, through a series of damage scenarios, on a laboratory suspension bridge mock-up equipped with four active cables; each active cable is made of a displacement actuator collocated with a force sensor. Different locations and levels of damage severity are implemented. For the ﬁrst time, the investigation demonstrates experimentally the effectiveness of the technique, as well as its limitations, to detect and locate the damage in hangers of a suspension bridge. Keywords: damage detection; active cable; Hilbert–Huang transform; suspension bridges, health monitoring Sensors 2018, 18, 3002; doi:10.3390/s18093002 sensors sensors Damage Detection in Active Suspension Bridges: An Experimental Investigation Fanhao Meng 1,2,* , Bilal Mokrani 3 , David Alaluf 2,4, Jingjun Yu 1,* and André Preumont 2 1 Robotics Institute, Beihang University, 100191 Beijing, China 2 Active Structures Laboratory, Université Libre de Bruxelles, 1050 Brussels, Belgium; David.Alaluf@esa.int (D.A.) andre.preumont@ulb.ac.be (A.P.) 3 Department of Mechanical, Materials and Aerospace Engineering, University of Liverpool, Liverpool L69 3BX, UK; Bilal.Mokrani@liverpool.ac.uk 4 Optoelectronics Section, ESA-ESTEC, 2201 AZ Noordwijk, The Netherlands * Correspondence: Fanhao.Meng@ulb.ac.be (F.M.); jjyu@buaa.edu.cn (J.Y.) 1 Robotics Institute, Beihang University, 100191 Beijing, China 2 Active Structures Laboratory, Université Libre de Bruxelles, 1050 Brussels, Belgium; David.Alaluf@esa.int (D.A.) andre.preumont@ulb.ac.be (A.P.) 3 Department of Mechanical, Materials and Aerospace Engineering, University of Liverpool, Liverpool L69 3BX, UK; Bilal.Mokrani@liverpool.ac.uk 4 Optoelectronics Section, ESA-ESTEC, 2201 AZ Noordwijk, The Netherlands * Correspondence: Fanhao.Meng@ulb.ac.be (F.M.); jjyu@buaa.edu.cn (J.Y.) 1. Introduction Over recent years, the improvement in construction materials, construction technology, computing ability, and above all the understanding of the physics of the complex phenomena which control the external loads acting on structures have revolutionized the civil engineering community, enabling the construction of large and elegant civil infrastructures. With the rising of large-scale civil engineering structures, structural health monitoring (SHM) problems have become a crucial scientiﬁc issue over the last two decades [1–3]. Suspension bridges are among civil structures vulnerable to damages, such as corrosion in the hangers; this study is concerned with this problem. The goal of health monitoring techniques is to be able to detect, locate and assess the extent of a damage to a structure to predict its safety and lifetime. As an alternative to existing local detection methods, vibration-based methods have been widely applied over the last years [4–8], where most of the damage information could be obtained by analyzing vibration data [9–13]. In general, the vibration data contains two parts, one is the measurable input applied to the bridge system and the other one is the output. From these vibration measurements, the real model of the structure can be obtained by a variety of parameter estimation methods [14]. However, in practice, applying an artiﬁcial force as the excitation for the bridge require extensive instrumentation and disruption of trafﬁc which make frequent tests less favorable [15]. For online health monitoring in real suspension bridge, the principal method to excite the bridge is relying upon available ambient excitation sources. It is practically Sensors 2018, 18, 3002; doi:10.3390/s18093002 www.mdpi.com/journal/sensors 2 of 20 Sensors 2018, 18, 3002 impossible to measure this ambient excitation and the outputs are the only information that can be used [16,17]. In addition, some novel integrated technologies have developed quickly and achieved positive results [18], for example, the unsupervised deep learning model [19] which incorporates synchrosqueezed wavelet transform, Fast Fourier Transform, and unsupervised deep Boltzmann machine; synchrosqueezed wavelet transform-fractality model [20]; and Fast Fourier Transform based wavelet ﬁnite element method [21]. In this study, we aim to combine active cable drive modes and Hilbert–Huang transform (HHT) method to monitor the health of suspension bridges’ hangers. We consider a suspension bridge equipped with a set of active cables/tendons which are used for vibration damping [22,23]. 1. Introduction We propose to take advantage of the active cables drive modes to excite the suspension bridge, and use the HHT of the outputs to establish a damage detection index, based on the Hilbert marginal spectrum. Indeed, the choice of HHT owes to its several merits demonstrated in many applications such as fatigue test of automobile gearbox [24,25], fault diagnosis in machinery [26,27] and locating combined damage in truss-type structures [28]. The HHT was proposed by Huang [29,30]. It has adaptive characteristics without any need to select a basic function, which may be advantageous for several applications. It mainly includes two aspects. First, the primary signals are decomposed into a series of time-dependent intrinsic mode functions (IMFs). Then, instantaneous frequency and amplitude are obtained and presented in the three-dimensional spectrum for every IMF, named as the Hilbert–Huang spectrum. The Hilbert marginal spectrum of the signal can be obtained by integrating the Hilbert–Huang spectrum along the time axis; it can accurately reﬂect the trend of the signal amplitude and the instantaneous frequency. Without any energy leakage, it has higher resolution and accuracy than the traditional Fourier spectrum [29]. The aim of this paper is to explore the potential of HHT as a method to establish a damage index for health monitoring of suspension bridges’ hangers. Since the method performs perfectly numerically, and for the sake of brevity, only the experimental implementation is presented. The paper is organized as follows: Section 2 presents the Hilbert marginal spectrum method for damage index estimation. Section 3 gives a brief overview of the bridge mock-up and describes its vibration characteristics in presence of active cables used for excitation. Section 4 mainly focuses on the experimental validation of the proposed method and discusses its shortcomings. Section 5 introduces a multiple active cable drive mode to improve the performance of the proposed method for cable damage detection. Finally, the ﬁndings and conclusions of this study are summarized at the end. 2.1. Hilbert–Huang Transform (HHT) Generally, a nonlinear and non-stationary complex signal is not suitable to be used to Hilbert transform. To use the HHT, the original signal should be decomposed into a number of intrinsic mode functions (IMFs) ci(t) (i = 1, 2, . . . n) and a residue rn(t). This process is known as empirical mode decomposition (EMD) [29]. x(t) = n ∑ i=1 ci(t) + rn(t), (1) (1) he IMF should be satisﬁed with the following two conditions [29]: The IMF should be satisﬁed with the following two conditions [29]: • In the whole data set, the number of extrema and the number of zero crossings must be equal or different at most by one. • In the whole data set, the number of extrema and the number of zero crossings must be equal or different at most by one. • At any point, the mean value of the envelope deﬁned by the local maxima and the envelope deﬁned by the local minima is zero. • At any point, the mean value of the envelope deﬁned by the local maxima and the envelope deﬁned by the local minima is zero. After using the Empirical Mode Decomposition (EMD) method, each intrinsic mode function component will satisfy the conditions of Hilbert transform. For each IMF component ci(t), the Hilbert transform is deﬁned as: 1 ∞ ( ) H[ci(t)] = 1 π P Z ∞ −∞ ci(τ) t −τ dτ, (2) (2) where P stands for the Cauchy principal value. This transform exists for all functions of class Lp [25]. 2. Theoretical Background The classical vibration-based damage detection techniques have in common that they rely on modal parameters. For the past two decades, many effective modal estimation techniques have been used in the ﬁeld of structural damage detection, such as Ibrahim time domain (ITD) method [31], eigensystem realization algorithm (ERA) [32], natural excitation technique (NExT) [33], stochastic subspace identiﬁcation (SSI) [34] and the newly developed blind source separation (BSS) [35–37]. The accuracy of modal parameter estimation is determined by the existing test conditions and technology. Hence, the modal parameter-based damage detection method may be complex in most practical applications in civil engineering. Alternatively, the HHT method can be directly used in damage detection and does not require knowing the modal parameters. For bridge-safety inspection, Huang [38] used a transient load to examine the nonlinear characteristics in a bridge vibration data to identify the damage. They used the HHT method to analyze the transient characteristic of the load and succeeded to locate the damaged spot. In this study, we consider the structural loading generated by the active tendons, intended to be used for active vibration damping [22]; this is more convenient as it enables the use of the same excitation proﬁle to study the healthy and the damaged structure. Hence, by simply comparing the Sensors 2018, 18, 3002 3 of 20 Hilbert marginal spectrum of the damaged structure and the undamaged structure, it is straightforward to identify an efﬁcient damage indicator. Hilbert marginal spectrum of the damaged structure and the undamaged structure, it is straightforward to identify an efﬁcient damage indicator. 2.1. Hilbert–Huang Transform (HHT) Using the Hilbert transform, the signal can be deﬁned as: qi(t) = ci(t) + i H[ci(t)]= Ai(t)e−iθ(t), (3) (3) With the instantaneous amplitude: With the instantaneous amplitude: Ai(t) = q c2 i (t) + H[ci(t)]2, (4) (4) The phase function: The phase function: θi(t) = arctan H[ci(t)] ci(t) , (5) (5) The instantaneous frequency is obtained by: The instantaneous frequency is obtained by The instantaneous frequency is obtained by: ωi(t) = dθi(t) dt , (6) (6) After performing the Hilbert transform on each IMF component, the original data can be expressed as the real part in the following form: After performing the Hilbert transform on each IMF component, the original data can be expressed as the real part in the following form: x(t) = Re ( n ∑ i=1 Ai(t)ei R ωi(t)dt ) , (7) (7) Equation (7) gives both the amplitude and the frequency of each component as a function of time. The same data expanded in a Fourier representation would be: Equation (7) gives both the amplitude and the frequency of each component as a function of time. The same data expanded in a Fourier representation would be: x(t) = Re " n ∑ i=1 Aieiωit # , (8) (8) where Ai and ωi are constants. The contrast between Equations (7) and (8) is clear: the IMF represents a generalized Fourier expansion. The variable amplitude and the instantaneous frequency improve the where Ai and ωi are constants. The contrast between Equations (7) and (8) is clear: the IMF represents a generalized Fourier expansion. The variable amplitude and the instantaneous frequency improve the 4 of 20 Sensors 2018, 18, 3002 efﬁciency of the expansion and enable the expansion to adapt to the nonlinear and non-stationary data. As the Hilbert transform on IMFs amplitude and frequency modulation are also clearly separated, the constant and ﬁxed frequency limits of Fourier expansion have been overcome with variable amplitude and frequency representation [29]. This frequency–time distribution of the amplitude is referred to as the “Hilbert amplitude spectrum” H(ω, t), or simply “Hilbert spectrum”. With the Hilbert spectrum deﬁned, the marginal spectrum, h(ω), is deﬁned as: h(ω) = Z T 0 H(ω, t)dt, (9) (9) The marginal spectrum provides a measure of the total amplitude (or energy) contribution of each frequency value. The spectrum represents the cumulative amplitude of the whole data span in the probability sense. 2.2. Damage Index The data collected from damage location may contain important information about the damage in the structural member. The special features of this signiﬁcant information are often reﬂected in the response amplitudes. In the instantaneous frequency range (ω1, ω2), the amplitude A, referred to as eigen amplitude, is deﬁned as: AD = Z ω2 ω1 h(ω)dω D , (10) AH = Z ω2 ω1 h(ω)dω H , (11) (10) (11) where h(ω) is the Hilbert marginal spectrum; ω is the instantaneous frequency and the instantaneous frequency range (ω1, ω2) is determined by the energy concentration range in the Hilbert marginal spectrum; and subscripts H and D stand for the healthy and damaged structure, respectively. It is worth mentioning that the input of the system should remain consistent in the test. In addition, the method assumes that the structure data (dynamics/signals) have been measured when it is healthy, where active cables are used for excitation. These data are then used as a reference. The eigen amplitude reﬂects the amplitude distribution of the signal in the corresponding instantaneous frequency region. When a local damage occurs, it may lead to the change of the local physical parameters such as stiffness (and eventually mass), which in turn causes the change of the Hilbert marginal spectrum. The eigen amplitude in the same instantaneous frequency range also changes due to damage. Thus, by simply comparing the eigen amplitudes before and after damage, one can compute a damage index DIv of the structure by combining Equations (10) and (11); it is given by: DIv = hR ω2 ω1 h(ω)dω i DV − hR ω2 ω1 h(ω)dω i HV hR ω2 ω1 h(ω)dω i HV , (12) (12) where the subscript v stands for the vertical direction of the eigen amplitude (as the structure motion is dominant in this direction). where the subscript v stands for the vertical direction of the eigen amplitude (as the structure motion is dominant in this direction). 2.1. Hilbert–Huang Transform (HHT) The Hilbert marginal spectrum can accurately represent the variation of the signal amplitude with the instantaneous frequency and has higher accuracy and resolution than the traditional FFT spectrum [29]. 2.3. Proposed Methodology Experimental Testing of the Suspe 3 1 L b t S i B id M d l The laboratory mock-up of the suspension bridge is shown in Figure 2. It consists of two articulated towers of 0.62 m distant of 2.2 m; the deck is free to rotate at both ends and is attached to the catenary by two rows of 10 hangers. The laboratory mock-up of the suspension bridge is shown in Figure 2. It consists of two articulated towers of 0.62 m distant of 2.2 m; the deck is free to rotate at both ends and is attached to the catenary by two rows of 10 hangers. 3.1. Laboratory Suspension Bridge Model The laboratory mock-up of the suspension bridge is shown in Figure 2. It consists of two articulated towers of 0.62 m distant of 2.2 m; the deck is free to rotate at both ends and is attached to the catenary by two rows of 10 hangers. Figure 2. Laboratory mock-up equipped with four active cables connecting the pylon to the deck. The catenary consists of a steel cable with a diameter of 1 mm and the hangers are made of steel Figure 2. Laboratory mock-up equipped with four active cables connecting the pylon to the deck. Figure 2. Laboratory mock-up equipped with four active cables connecting the pylon to the deck. Figure 2. Laboratory mock-up equipped with four active cables connecting the pylon to the deck. The catenary consists of a steel cable with a diameter of 1 mm and the hangers are made of steel Figure 2. Laboratory mock-up equipped with four active cables connecting the pylon to the deck. Figure 2. Laboratory mock-up equipped with four active cables connecting the pylon to the deck. 2.3. Proposed Methodology • Third, the Hilbert marginal spectrum of each measurement point is computed using the related IMFs (damaged and undamaged). • Third, the Hilbert marginal spectrum of each measurement point is computed using the related IMFs (damaged and undamaged). • Finally, the damage indices ܦܫ௩are calculated using Equations (10)–(12) for each damage considered bridge, the first five vibration modes). • Third, the Hilbert marginal spectrum of each measurement point is computed using the related IMFs (damaged and undamaged). • Finally, the damage indices DIv are calculated using Equations (10)–(12) for each damage scena • Finally, the damage indices ܦܫ௩ are calculated using Equations (10)–(12) for each damage scenario. IMFs (damaged and undamaged). • Finally, the damage indices ܦܫ௩ are calculated using Equations (10)–(12) for each damage • Finally, the damage indices DIv are calculated using Equations (10)–(12) for each damage scenario. The ﬂow chart of the proposal is shown in Figure 1. • Finally, the damage indices ܦܫ௩ are calculated using Equations (10) (12) for each damage scenario. The flow chart of the proposal is shown in Figure 1. I s ( a age a u a age ) • Finally, the damage indices ܦܫ௩ are calculated using Equations (10)–(12) for each damage scenario. y, g v g q ( ) ( ) g The ﬂow chart of the proposal is shown in Figure 1. scenario. The flow chart of the proposal is shown in Figure 1. • Finally, the damage indices ܦܫ௩ are calculated using Equations (10) (12) for each damage scenario. The ﬂow chart of the proposal is shown in Figure 1. The flow chart of the proposal is shown in Figure 1. scenario. Figure 1. Damage detection procedure. 3. Experimental Testing of the Suspension Bridge Mock-Up Figure 1. Damage detection procedure. 3. Experimental Testing of the Suspension Bridge Mock-Up The flow chart of the proposal is shown in Figure 1. Figure 1. Damage detection procedure. Figure 1. Damage detection procedure. Figure 1. Damage detection procedure. Fi 1 D d t ti d Figure 1. Damage detection procedure. Figure 1. Damage detection procedure. Fi 1 D d t ti d 3. Experimental Testing of the Suspension Bridge Mock-Up 3. Experimental Testing of the Suspension Bridge Mock-Up Figure 1. Damage detection procedu l f h d k 3.1. Laboratory Suspension Bridge Mode 3.1. Laboratory Suspension Bridge Model 3. 2.3. Proposed Methodology Once the detection algorithm parameters are well deﬁned (IMFs and the instantaneous frequency range), we calculate the damage index DIv as follow: 5 of 20 Sensors 2018, 18, 3002 • First, the bridge (damaged and undamaged) is excited with the active cables and the response is measured at several measurement points. For the considered bridge, it is measured on the deck, near to the hangers’ attachment. • First, the bridge (damaged and undamaged) is excited with the active cables and the response is measured at several measurement points. For the considered bridge, it is measured on the deck, near to the hangers’ attachment. • Second, the data from the same measurement points (damaged and undamaged) are decomposed into n-empirical modes (IMFs). By analyzing the central frequencies of the IMFs, the related IMFs are selected which contain the information of the dominant modes (for the considered bridge, the ﬁrst ﬁve vibration modes). Sensors 2018, 18, x FOR PEER REVIEW 5 of 20 related IMFs are selected which contain the information of the dominant modes (for the considered bridge, the first five vibration modes). Sensors 2018, 18, x FOR PEER REVIEW 5 of 20 related IMFs are selected which contain the information of the dominant modes (for the • Second, the data from the same measurement points (damaged and undamaged) are decomposed into n-empirical modes (IMFs). By analyzing the central frequencies of the IMFs, the related IMFs are selected which contain the information of the dominant modes (for the considered bridge, the ﬁrst ﬁve vibration modes). Sensors 2018, 18, x FOR PEER REVIEW 5 of 20 related IMFs are selected which contain the information of the dominant modes (for the considered bridge, the first five vibration modes). Sensors 2018, 18, x FOR PEER REVIEW 5 of 20 related IMFs are selected which contain the information of the dominant modes (for the • Third, the Hilbert marginal spectrum of each measurement point is computed using the related IMFs (damaged and undamaged). • Third, the Hilbert marginal spectrum of each measurement point is computed using the related IMFs (damaged and undamaged). • Finally, the damage indices ܦܫ௩are calculated using Equations (10)–(12) for each damage considered bridge, the first five vibration modes). • Third, the Hilbert marginal spectrum of each measurement point is computed using the related IMFs (damaged and undamaged). 3.2. Active Cables 3.2. Active Cables 3.2. Active Cables A band-limited white noise is used for excitation. Figure 4 contains important features of the bridge structure, namely, its symmetry and the tension in its cables. Indeed, such agreement between the transfer functions has been obtained after a complex tuning process of the bridge cables (the tension in the 2 catenaries, the 20 hangers, and the 4 active cables had to be tuned at the same time to reach a good symmetry). Figure 3. Detail of the active tendon. The transfer functions between the piezoelectric actuators (δi) and their corresponding force sensors are shown in Figure 4. A band-limited white noise is used for excitation. Figure 4 contains important features of the bridge structure, namely, its symmetry and the tension in its cables. Indeed, such agreement between the transfer functions has been obtained after a complex tuning process of the bridge cables (the tension in the 2 catenaries, the 20 hangers, and the 4 active cables had to be tuned at the same time to reach a good symmetry). Figure 3. Detail of the active tendon. The transfer functions between the piezoelectric actuators (ߜ௜) and their corresponding force sensors are shown in Figure 4. A band-limited white noise is used for excitation. Figure 4 contains important features of the bridge structure, namely, its symmetry and the tension in its cables. Indeed, such agreement between the transfer functions has been obtained after a complex tuning process of the bridge cables (the tension in the 2 catenaries, the 20 hangers, and the 4 active cables had to be tuned at the same time to reach a good symmetry). Figure 3. Detail of the active tendon. Figure 3. Detail of the active tendon. Figure 3. Detail of the active tendon. Figure 3. Detail of the active tendon. Figure 3. Detail of the active tendon. Figure 3. Detail of the active tendon. The transfer functions between the piezoelectric actuators (ߜ௜) and their corresponding force sensors are shown in Figure 4. A band-limited white noise is used for excitation. Figure 4 contains important features of the bridge structure, namely, its symmetry and the tension in its cables. Indeed, such agreement between the transfer functions has been obtained after a complex tuning process of the bridge cables (the tension in the 2 catenaries, the 20 hangers, and the 4 active cables had to be tuned at the same time to reach a good symmetry). 2.3. Proposed Methodology f being deduced from the lateral vibration of the cables, measured with a non-contact custom made laser sensor [39]. L is the length of the cable, ρ is its mass density and A is the cross-section. In this way, it was possible to distribute the tension in the hangers uniformly. ݂ being deduced from the lateral vibration of the cables, measured with a non-contact custom made laser sensor [39]. ܮ is the length of the cable, r is its mass density and ܣ is the cross-section. In this way, it was possible to distribute the tension in the hangers uniformly. ݂ being deduced from the lateral vibration of the cables, measured with a non-contact custom made laser sensor [39]. ܮ is the length of the cable, r is its mass density and ܣ is the cross-section. In this way, it was possible to distribute the tension in the hangers uniformly. 3.2. Active Cables 3.2. Active Cables 3.2. Active Cables Four active cables, with the same properties as the hangers, are added symmetrically to the mock-up in Figure 2. The chosen conﬁguration is the one connecting the deck to the top of the pylons. The control authority of the active cables over the targeted modes depends directly on their attachment point on the deck. Thus, to maximize the control authority over several low frequency modes, and thus excite most of them, the active cables are attached on the deck near the second or the third hangers, as shown in Figure 2. The tension in the active cables is controlled with four APA 50 s piezoelectric actuators, from Cedrat technologies, collocated with force sensors (for active control purposes only), as shown in Figure 3. Four active cables, with the same properties as the hangers, are added symmetrically to the mock-up in Figure 2. The chosen configuration is the one connecting the deck to the top of the pylons. The control authority of the active cables over the targeted modes depends directly on their attachment point on the deck. Thus, to maximize the control authority over several low frequency modes, and thus excite most of them, the active cables are attached on the deck near the second or the third hangers, as shown in Figure 2. The tension in the active cables is controlled with four APA 50 s piezoelectric actuators, from Cedrat technologies, collocated with force sensors (for active control purposes only), as shown in Figure 3. Four active cables, with the same properties as the hangers, are added symmetrically to the mock-up in Figure 2. The chosen configuration is the one connecting the deck to the top of the pylons. The control authority of the active cables over the targeted modes depends directly on their attachment point on the deck. Thus, to maximize the control authority over several low frequency modes, and thus excite most of them, the active cables are attached on the deck near the second or the third hangers, as shown in Figure 2. The tension in the active cables is controlled with four APA 50 s piezoelectric actuators, from Cedrat technologies, collocated with force sensors (for active control purposes only), as shown in Figure 3. Figure 3. Detail of the active tendon. The transfer functions between the piezoelectric actuators (ߜ௜) and their corresponding force sensors are shown in Figure 4. 2.3. Proposed Methodology y g cables of 0.5 mm; the tension ܶ଴ in the catenary and the hangers can be adjusted with screws; it is measured indirectly from the natural frequency ݂ of the lateral modes of the cabes, according to the string formula: ff = 1 ඨT0 (13) The catenary consists of a steel cable with a diameter of 1 mm and the hangers are made of steel cables of 0.5 mm; the tension ܶ଴ in the catenary and the hangers can be adjusted with screws; it is measured indirectly from the natural frequency ݂ of the lateral modes of the cabes, according to the string formula: 1 T The catenary consists of a steel cable with a diameter of 1 mm and the hangers are made of steel cables of 0.5 mm; the tension T0 in the catenary and the hangers can be adjusted with screws; it is measured indirectly from the natural frequency f of the lateral modes of the cabes, according to the string formula: s ff = 2L ඨρA , ff = 1 2L ඨT0 ρA , f = 1 2L s T0 ρA, ff = 2L ඨρA , ff = 1 2L ඨT0 ρA , f = 1 2L s T0 ρA, ff = 2L ඨρA , (13) ff = 1 2L ඨT0 ρA , (13) f = 1 2L s T0 ρA, (13) (13) 6 of 20 Sensors 2018, 18, 3002 f being deduced from the lateral vibration of the cables, measured with a non-contact custom made laser sensor [39]. L is the length of the cable, ρ is its mass density and A is the cross-section. In this way, it was possible to distribute the tension in the hangers uniformly. ݂ being deduced from the lateral vibration of the cables, measured with a non-contact custom made laser sensor [39]. ܮ is the length of the cable, r is its mass density and ܣ is the cross-section. In this way, it was possible to distribute the tension in the hangers uniformly. ݂ being deduced from the lateral vibration of the cables, measured with a non-contact custom made laser sensor [39]. ܮ is the length of the cable, r is its mass density and ܣ is the cross-section. In this way, it was possible to distribute the tension in the hangers uniformly. 3.2. Active Cables 3.2. Active Cables 3.2. Active Cables The transfer functions between the piezoelectric actuators (δi) and their corresponding force sensors are shown in Figure 4. A band-limited white noise is used for excitation. Figure 4 contains important features of the bridge structure, namely, its symmetry and the tension in its cables. Indeed, such agreement between the transfer functions has been obtained after a complex tuning process of the bridge cables (the tension in the 2 catenaries, the 20 hangers, and the 4 active cables had to be tuned at the same time to reach a good symmetry). The transfer functions between the piezoelectric actuators (ߜ௜) and their corresponding force sensors are shown in Figure 4. A band-limited white noise is used for excitation. Figure 4 contains important features of the bridge structure, namely, its symmetry and the tension in its cables. Indeed, such agreement between the transfer functions has been obtained after a complex tuning process of the bridge cables (the tension in the 2 catenaries, the 20 hangers, and the 4 active cables had to be tuned at the same time to reach a good symmetry). Figure 4. The transfer function of the four active cables ܶ௜/ߜ௜. Figure 4. The transfer function of the four active cables ܶ௜/ߜ௜. Figure 4. The transfer function of the four active cables Ti/δi. Figure 4. The transfer function of the four active cables ܶ௜/ߜ௜. Figure 4. The transfer function of the four active cables ܶ௜/ߜ௜. Figure 4. The transfer function of the four active cables Ti/δi. Sensors 2018, 18, 3002 Sensors 2018, 18, x FOR Sensors 2018, 18, x FOR 7 of 20 7 of 20 7 of 20 A small magnet is attached to the deck and a voice coil is used to apply a disturbance force to the structure (only for modal identiﬁcation, not for health monitoring) (Figure 2). The response of the deck is measured with four accelerometers, placed alternately along the deck. Due to the limitation of the hardware, the measurement is limited to four locations at the same time, by using four accelerometers. To cope with this limitation and obtain the response at the 20 locations, the sensors are moved along the bridge deck, where the same band-limited white noise signal is used for excitation. the structure (only for modal identification, not for health monitoring) (Figure 2). The response of the deck is measured with four accelerometers, placed alternately along the deck. 3.2. Active Cables 3.2. Active Cables 3.2. Active Cables Due to the limitation of the hardware, the measurement is limited to four locations at the same time, by using four accelerometers. To cope with this limitation and obtain the response at the 20 locations, the sensors are moved along the bridge deck, where the same band-limited white noise signal is used for excitation. the structure (only for modal identification, not for health monitoring) (Figure 2). The response of the deck is measured with four accelerometers, placed alternately along the deck. Due to the limitation of the hardware, the measurement is limited to four locations at the same time, by using four accelerometers. To cope with this limitation and obtain the response at the 20 locations, the sensors are moved along the bridge deck, where the same band-limited white noise signal is used for excitation. Figure 5 shows the experimental natural frequencies and mode shapes extracted from the Figure 5 shows the experimental natural frequencies and mode shapes extracted from the measured frequency response of the deck at 20 different points. The measured structural damping ratios range between 0.1% and 0.8%. Figure 5 shows the experimental natural frequencies and mode shapes extracted from the measured frequency response of the deck at 20 different points. The measured structural damping ratios range between 0.1% and 0.8%. Figure 5 shows the experimental natural frequencies and mode shapes extracted from the measured frequency response of the deck at 20 different points. The measured structural damping ratios range between 0.1% and 0.8%. Figure 5. Experimental natural frequencies and mode shapes. The natural modal damping ranges from 0 1% to 0 8% Figure 5. Experimental natural frequencies and mode shapes. The natural modal damping ranges from 0.1% to 0.8%. Figure 5. Experimental natural frequencies and mode shapes. The natural modal damping ranges from 0.1% to 0.8%. Figure 5. Experimental natural frequencies and mode shapes. The natural modal damping ranges from 0 1% to 0 8% Figure 5. Experimental natural frequencies and mode shapes. The natural modal damping ranges from 0.1% to 0.8%. Figure 5. Experimental natural frequencies and mode shapes. The natural modal damping ranges from 0.1% to 0.8%. 4 Experimental Implementation 4. Experimental Implementation 4. Experimental Implementation 4. Experimental Implementation First, consider a single active cable to excite the structure (Cable 1A). The active cable is attached at close to the third hanger to ensure an effective excitation. The excitation consists of a band-limited white noise voltage applied to the piezoelectric actuator; a set of accelerometers monitor the deck acceleration in the vertical direction which are laid as depicted in Figure 6 First, consider a single active cable to excite the structure (Cable 1A). The active cable is attached at close to the third hanger to ensure an effective excitation. The excitation consists of a band-limited white noise voltage applied to the piezoelectric actuator; a set of accelerometers monitor the deck acceleration in the vertical direction, which are laid as depicted in Figure 6. First, consider a single active cable to excite the structure (Cable 1A). The active cable is attached at close to the third hanger to ensure an effective excitation. The excitation consists of a band-limited white noise voltage applied to the piezoelectric actuator; a set of accelerometers monitor the deck acceleration in the vertical direction, which are laid as depicted in Figure 6. (a) (b) Figure 6. Layout of the accelerometers over the bridge deck (four accelerometers and multiple measurements): (a) the accelerometer; and (b) measuring position on the deck (one point near each (a) (b) Figure 6. Layout of the accelerometers over the bridge deck (four accelerometers and multiple measurements): (a) the accelerometer; and (b) measuring position on the deck (one point near each Figure 6. Layout of the accelerometers over the bridge deck (four accelerometers and multiple measurements): (a) the accelerometer; and (b) measuring position on the deck (one point near each hanger). (b) (b) (a) (a) (b) (b) (a) (a) ( ) ( ) Figure 6. Layout of the accelerometers over the bridge deck (four accelerometers and multiple measurements): (a) the accelerometer; and (b) measuring position on the deck (one point near each Figure 6. Layout of the accelerometers over the bridge deck (four accelerometers and multiple measurements): (a) the accelerometer; and (b) measuring position on the deck (one point near each Figure 6. Layout of the accelerometers over the bridge deck (four accelerometers and multiple measurements): (a) the accelerometer; and (b) measuring position on the deck (one point near each hanger). hanger). hanger). 4.1. Damage Detection with a Single Active Cable Drive: Scenarios A 4.1. Damage Detection with a Single Active Cable Drive: Scenarios A Generally, damage leads to a decrease of the structural stiffness, thus damage in the bridge hangers is simulated by reducing their pre-stress. Based on Equation (13), the damage levels are simply tuned by properly tuning the resonance frequency of the cables. The implemented damage scenarios are described in Table 1. After each damage scenario, the tension in the cables is tuned back to its original value (healthy configuration), by identifying the resonance frequency of the lateral vibration using a custom-made sensor (Equation (13)). Generally, damage leads to a decrease of the structural stiffness, thus damage in the bridge hangers is simulated by reducing their pre-stress. Based on Equation (13), the damage levels are simply tuned by properly tuning the resonance frequency of the cables. The implemented damage scenarios are described in Table 1. After each damage scenario, the tension in the cables is tuned back to its original value (healthy conﬁguration), by identifying the resonance frequency of the lateral vibration using a custom-made sensor (Equation (13)). Table 1. Single cable drive: damage scenarios A. Table 1. Single cable drive: damage scenarios A. Table 1. Single cable drive: damage scenarios A. Table 1. Single cable drive: damage scenarios A. Table 1. Single cable drive: damage scenarios A. Damage Case Location Severity of Damage (Tension Reduction) Single damage scenario Case A1 Cable 5 50% Case A2 Cable 5 95% Case A3 Cable 3 50% Case A4 Cable 3 95% Case A5 Cable 2 95% Multiple damage scenario Case A6 Cable 15 and Cable 16 95% Case A7 Cable 4 and Cable 18 95% C A8 C bl 6 d C bl 9 95% Table 1. Single cable drive: damage scenarios A. Damage Case Location Severity of Damage (Tension Reduction) Single damage scenario Case A1 Cable 5 50% Case A2 Cable 5 95% Case A3 Cable 3 50% Case A4 Cable 3 95% Case A5 Cable 2 95% Multiple damage scenario Case A6 Cable 15 and Cable 16 95% Case A7 Cable 4 and Cable 18 95% Case A8 Cable 6 and Cable 9 95% Sensors 2018, 18, 3002 The minimu Sensors 2018, 18, 3002 The minimu 1000 ∼2000 fmin , where f min is the lowest natural frequency in Hz (i.e., at least, the measurement must cover 1000–2000 oscillations associated with the ﬁrst mode). Based on the preliminary FRF analysis, f min is close to 8 Hz and Tmin is selected as 250 s. To minimize the size of data, we selected a sampling rate of 1000 Hz (the highest resonance frequency is about 33 Hz). A low-pass analog ﬁlter at 100 Hz has been used to remove the high frequency noise and avoid any temporal aliasing. y q y [ , ], Tmin= 1000 ~ 2000 fmin , where fmin is the lowest natural frequency in Hz (i.e., at least, the measurement must cover 1000–2000 oscillations associated with the first mode). Based on the preliminary FRF analysis, fmin is close to 8 Hz and Tmin is selected as 250 s. To minimize the size of data, we selected a sampling rate of 1000 Hz (the highest resonance frequency is about 33 Hz). A low-pass analog filter at 100 Hz has been used to remove the high frequency noise and avoid any temporal aliasing. 1000 ∼2000 fmin , where f min is the lowest natural frequency in Hz (i.e., at least, the measurement must cover 1000–2000 oscillations associated with the ﬁrst mode). Based on the preliminary FRF analysis, f min is close to 8 Hz and Tmin is selected as 250 s. To minimize the size of data, we selected a sampling rate of 1000 Hz (the highest resonance frequency is about 33 Hz). A low-pass analog ﬁlter at 100 Hz has been used to remove the high frequency noise and avoid any temporal aliasing. y q y Tmin= 1000 ~ 2000 fmin , where fmin is the lowest natural frequency in Hz (i.e., at least, the measurement must cover 1000–2000 oscillations associated with the first mode). Based on the preliminary FRF analysis, fmin is close to 8 Hz and Tmin is selected as 250 s. To minimize the size of data, we selected a sampling rate of 1000 Hz (the highest resonance frequency is about 33 Hz). A low-pass analog filter at 100 Hz has been used to remove the high frequency noise and avoid any temporal aliasing. 4 Experimental Implementation 4. Experimental Implementation 4. Experimental Implementation The minimum length of time series Tmin used for this experiment covers almost the whole dynamic behavior of the structure within the frequency band of interest [40,41], such that Tmin = hanger). hanger). The minimum length of time series Tmin used for this experiment covers almost the whole dynamic behavior of the structure within the frequency band of interest [40,41], such that Tmin = 8 of 20 whole Sensors 2018, 18, 3002 The minimu Finally, the cables are numbered as shown in Figure 7: from 1 to 10 on one side, starting from the right, and from 11 to 20 on the opposite side. 1A, 2A, 1B, and 2B indicate the active cables. has been used to remove the high frequency noise and avoid any temporal aliasing. Finally, the cables are numbered as shown in Figure 7: from 1 to 10 on one side, starting from the right, and from 11 to 20 on the opposite side. 1A, 2A, 1B, and 2B indicate the active cables. Figure 7. Cable number of mock-up. The hangers are numbered from 1 to 10 on one side of the deck, and from 11 to 20 on the other side. The active cables are 1A, 1B, 2A, and 2B. Figure 7. Cable number of mock-up. The hangers are numbered from 1 to 10 on one side of the deck, and from 11 to 20 on the other side. The active cables are 1A, 1B, 2A, and 2B. Figure 7. Cable number of mock-up. The hangers are numbered from 1 to 10 on one side of the deck, and from 11 to 20 on the other side. The active cables are 1A, 1B, 2A, and 2B. Figure 7. Cable number of mock-up. The hangers are numbered from 1 to 10 on one side of the deck, and from 11 to 20 on the other side. The active cables are 1A, 1B, 2A, and 2B. 4.1. Damage Detection with a Single Active Cable Drive: Scenarios A 4.1. Damage Detection with a Single Active Cable Drive: Scenarios A 4.1. Damage Detection with a Single Active Cable Drive: Scenarios A 4.1. Damage Detection with a Single Active Cable Drive: Scenarios A Case A8 Cable 6 4.1.1. Intrinsic Mode Functions (IMFs) To illustrate the process of the algorithm, consider the signal measured by the accelerometer located near the damaged Cable 5 (Case A2) as an example. Based on the empirical mode decomposition method (EMD), the signal is decomposed in several steps until (for this example, into 14 IMFs) a monotonous/constant residue is obtained (IMF 15). The detail process of EMD can 9 of 20 mode le, into 9 of 20 mode ple, into Sensors 2018, 18, 3002 located near the decomposition me found in [29]. For the considered example, the IMFs in the time domain are shown in Figure 8a. Each IMF contains different dynamic information of the structure and the central frequency is an effective way to evaluate the main component of each IMF. By analyzing the central frequencies of the IMFs (Figure 8a), observe that the main component of IMFs 4–6 contain the information of the ﬁrst ﬁve vibration modes, which is also reﬂected in their Hilbert marginal spectrum, as shown in Figure 8b (although IMFs 3 and 7 count for a small contribution). These IMFs are chosen, as they exhibit a signiﬁcant contribution of the structure modes, and the response of the structure is simply a combination of these modes (as the excitation is a white noise). in [29]. For the considered example, the IMFs in the time domain are shown in Figure 8a. Each IMF contains different dynamic information of the structure and the central frequency is an effective way to evaluate the main component of each IMF. By analyzing the central frequencies of the IMFs (Figure 8a), observe that the main component of IMFs 4–6 contain the information of the first five vibration modes, which is also reflected in their Hilbert marginal spectrum, as shown in Figure 8b (although IMFs 3 and 7 count for a small contribution). These IMFs are chosen, as they exhibit a significant contribution of the structure modes, and the response of the structure is simply a combination of these modes (as the excitation is a white noise). (a) (b) Figure 8. Results of HHT. The signal from the accelerometer at position 5 (Case A2): (a) IMFs in the time domain; and (b) Hilbert marginal spectrum. Figure 8. Results of HHT. The signal from the accelerometer at position 5 (Case A2): (a) IMFs in the time domain; and (b) Hilbert marginal spectrum. 4.1.2. Case A8 Cable 6 4.1.1. Intrinsic Mode Functions (IMFs) Instantaneous Frequency Range (ω1, ω2) To select a reasonable frequency range, we analyze the Hilbert marginal spectrums (Figure 8b) (a) (b) (b) Figure 8. Results of HHT. The signal from the accelerometer at position 5 (Case A2): (a) IMFs in the time domain; and (b) Hilbert marginal spectrum. Figure 8. Results of HHT. The signal from the accelerometer at position 5 (Case A2): (a) IMFs in the time domain; and (b) Hilbert marginal spectrum. 4.1.2. Instantaneous Frequency Range (ω1, ω2) 4.1.2. Instantaneous Frequency Range (ω1, ω2) To select a reasonable frequency range, we analyze the Hilbert marginal spectrums (Figure 8b); one can ﬁnd that the energy is concentrated in the range of 5–15 Hz, which includes the ﬁrst ﬁve vibration modes. Although the natural frequencies have a little sensitivity to the damage [42,43], 10 of 20 ure 8b); irst five Sensors 2018, 18, 3002 To select a rea one can find that Figure 8 indicates that the Hilbert marginal spectrum exhibits some obvious changes in the frequency range 5–15 Hz. Therefore, the frequency range (5 Hz, 15 Hz) is used in all the examples below. The calculation process of the damage index DIv is found in Section 2.3. g q y g [ , ], Figure 8 indicates that the Hilbert marginal spectrum exhibits some obvious changes in the frequency range 5–15 Hz. Therefore, the frequency range (5 Hz, 15 Hz) is used in all the examples below. The calculation process of the damage index ܦܫ௩ is found in Section 2.3. 4.1.3. Effect of Signal Error 4.1.3. Effect of Signal Error The signal quality may lead to different damage detection results. To ﬁgure out the extent of the error (the process noise from the disturbances and the measurement noise from the sensors), we compare two samples of signals at a different time from the same position of the healthy bridge. The results are shown in Figure 9, where the maximum error is close to 3%. In a perfect conﬁguration, without any noise, the damage indices would be zero. However, due to the measurement noise and the different excitations, small levels of damage indices are extracted (less than 3%); this can be considered as the noise on the damage index. The signal quality may lead to different damage detection results. To figure out the extent of the error (the process noise from the disturbances and the measurement noise from the sensors), we compare two samples of signals at a different time from the same position of the healthy bridge. The results are shown in Figure 9, where the maximum error is close to 3%. In a perfect configuration, without any noise, the damage indices would be zero. However, due to the measurement noise and the different excitations, small levels of damage indices are extracted (less than 3%); this can be considered as the noise on the damage index. Figure 9. Effect of signal error. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 9. Effect of signal error. Figure 9. Effect of signal error. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 9. Effect of signal error. Figure 9. Effect of signal error. Figure 9. Effect of signal error. 4.1.4. Results and Discussion 4.1.4. Results and Discussion 4.1.4. Results and Discussion 4.1.4. Results and Discussion ( ) Si l D S i A C A5 • Single Damage Scenario A: Case A5 • Single Damage Scenario A: Case A5 • Single Damage Scenario A: Case A5 In Case A5, the tension of Cable 2 is reduced by 95%. Contrary to expectation, we do not find a significant peak in Figure 12 and the vertical eigen amplitude method with a single active cable drive fails to detect the 95% damage. This can be associated with the fact that Cable 2 is located near the edge of the bridge, and, by observing the mode shapes in Figure 5, we can see that the edge of the deck has smaller modal contribution compared to the mid-span (for most of the excited modes), which explains the insensitivity of the method to the damage of Cable 2. Thus, to detect the damage in Cable 2, one needs to excite the vibration modes where the cable has a higher modal contribution. Thi b f d b i l ti bl d ib d f th i S ti 4 2 In Case A5, the tension of Cable 2 is reduced by 95%. Contrary to expectation, we do not find a significant peak in Figure 12 and the vertical eigen amplitude method with a single active cable drive fails to detect the 95% damage. This can be associated with the fact that Cable 2 is located near the edge of the bridge, and, by observing the mode shapes in Figure 5, we can see that the edge of the deck has smaller modal contribution compared to the mid-span (for most of the excited modes), which explains the insensitivity of the method to the damage of Cable 2. Thus, to detect the damage in Cable 2, one needs to excite the vibration modes where the cable has a higher modal contribution. This can be performed by using several active cables, as described further in Section 4.2. In Case A5, the tension of Cable 2 is reduced by 95%. Contrary to expectation, we do not ﬁnd a signiﬁcant peak in Figure 12 and the vertical eigen amplitude method with a single active cable drive fails to detect the 95% damage. • Single Damage Scenario A: Case A1 and Case A2 • Single Damage Scenario A: Case A1 and Case A2 • Single Damage Scenario A: Case A1 and Case A2 • Single Damage Scenario A: Case A1 and Case A2 • Single Damage Scenario A: Case A1 and Case A2 • Single Damage Scenario A: Case A1 and Case A2 g g The first two damage cases assume Cable 5 is damaged. The results of the damage indices are shown in Figure 10, for both cases. In Figure 10a, the damage index ܦܫ௩ refers to the various positions of the accelerometers along the deck; it is maximum for the accelerometer located at the position of the damaged hanger. However, Number 18 and Number 20 have similar levels too, which may lead to confusion. In Figure 10b, where the damage level is about 95%, the ܦܫ௩ shows a maximum value at the damaged location. In this damage case, the vertical damage index is able to detect 95% of damage to the cable successfully at the center of the bridge span and confirm the actual The ﬁrst two damage cases assume Cable 5 is damaged. The results of the damage indices are shown in Figure 10, for both cases. In Figure 10a, the damage index DIv refers to the various positions of the accelerometers along the deck; it is maximum for the accelerometer located at the position of the damaged hanger. However, Number 18 and Number 20 have similar levels too, which may lead to confusion. In Figure 10b, where the damage level is about 95%, the DIv shows a maximum value at the damaged location. In this damage case, the vertical damage index is able to detect 95% of damage to the cable successfully at the center of the bridge span and conﬁrm the actual location of the damage. nsors 2018, 18, 3002 11 of 20 nsors 2018, 18, x FOR PEER REVIEW 11 of 2 (a) (b) Figure 10. Damage indices for a damage at Cable 5: (a) Case A1 (50% reduction of tension); and (b) Case A2 (95% reduction of tension). 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 10. • Single Damage Scenario A: Case A3 and Case A4 • Single Damage Scenario A: Case A3 and Case A4 • Single Damage Scenario A: Case A3 and Case A4 Figure 11 illustrates the ܦܫ for Cases A3 and Figure 11 illustrates the ܦܫ௩ for Cases A3 and A4, where the tension of Cable 3 has been reduced. It can be seen that ܦܫ௩ peaks at the exact damage location for Case A4, but less precisely Figure 11 illustrates the DIv for Cases A3 and A4, where the tension of Cable 3 has been reduced. It can be seen that DIv peaks at the exact damage location for Case A4, but less precisely for Case A3. Figure 11 illustrates the ܦܫ௩ for Cases A3 and A4, where the tension of Cable 3 has been reduced. It can be seen that ܦܫ௩ peaks at the exact damage location for Case A4, but less precisely for Case A3. r Case A3. (a) (b) Figure 11. Damage indices for damage Cable 3: (a) Case A3 (50% reduction of tension); and (b) Case A4 (95% reduction of tension) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) (a) (b) Figure 11. Damage indices for damage Cable 3: (a) Case A3 (50% reduction of tension); and (b) Case A4 (95% reduction of tension). 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 11. Damage indices for damage Cable 3: (a) Case A3 (50% reduction of tension); and (b) Case A4 (95% reduction of tension). Case A3. • Single Damage Scenario A: Case A3 and Case A4 • Single Damage Scenario A: Case A3 and Case A4 • Single Damage Scenario A: Case A3 and Case A4 Figure 11 illustrates the ܦܫ for Cases A3 and (a) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) (a) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) (b) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) (b) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) (b) Cable num (b) (a) Cable num (a) (a) (b) Figure 11. Damage indices for damage Cable 3: (a) Case A3 (50% reduction of tension); and (b) Case A4 (95% reduction of tension) Figure 11. Damage indices for damage Cable 3: (a) Case A3 (50% reduction of tension); and (b) Case A4 (95% reduction of tension). Figure 11. Damage indices for damage Cable 3: (a) Case A3 (50% reduction of tension); and (b) Case A4 (95% reduction of tension). • Single Damage Scenario A: Case A1 and Case A2 • Single Damage Scenario A: Case A1 and Case A2 Damage indices for a damage at Cable 5: (a) Case A1 (50% reduction of tension); and (b) Case A2 (95% reduction of tension). nsors 2018, 18, x FOR PEER REVIEW 11 of 2 (a) (b) Figure 10. Damage indices for a damage at Cable 5: (a) Case A1 (50% reduction of tension); and (b) Case A2 (95% reduction of tension). A A A 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) 11 of 20 11 of 20 11 of 20 11 of 20 11 of 20 11 of 20 Sensors 2018, 18, 3002 Sensors 2018 18 x FOR Sensors 2018, 18, x FOR (b) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) (b) (a) Case A1 (50% reduction of tension); and (b) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) (a) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) (a) Figure 10. Damage indices for a damage at Cab 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) (b) ( ) uction (a) ( ) indice Figure 10. Damage indices for a damage at Cable 5: (a) Case A1 (50% reduction of tension); and (b) Case A2 (95% reduction of tension). Figure 10. Damage indices for a damage at Cable 5: (a) Case A1 (50% reduction of tension); and (b) Case A2 (95% reduction of tension). Figure 10. Damage indices for a damage at Cable 5: (a) Case A1 (50% reduction of tension); and (b) Case A2 (95% reduction of tension). ( ) Si l D S i A C A5 • Single Damage Scenario A: Case A5 • Single Damage Scenario A: Case A5 This can be associated with the fact that Cable 2 is located near the edge of the bridge, and, by observing the mode shapes in Figure 5, we can see that the edge of the deck has smaller modal contribution compared to the mid-span (for most of the excited modes), which explains the insensitivity of the method to the damage of Cable 2. Thus, to detect the damage in Cable 2, one needs to excite the vibration modes where the cable has a higher modal contribution. This can be performed by using several active cables, as described further in Section 4.2. 12 of 20 Sensors 2018, 18, 3002 Figure 12. Damage indices for 95% damage Cable 2. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 12. Damage indices for 95% damage Cable 2. R REVIEW Figure 12. Damage indices for 95% damage Cable 2. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) R REVIEW Figure 12. Damage indices for 95% damage Cable 2. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 12. Damage indices for 95% damage Cable 2. Figure 12. Damage indices for 95% damage Cable 2. Figure 12. Damage indices for 95% damage Cable 2. Figure 12. Damage indices for 95% damage Cable 2 Cable number • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 • Multiple Damage Scenario A: Case A6 In this damage case, a tension reduction of 95% is made at two cables located at mid-span (Cables 15 and 16). The results are shown in Figure 13 and it can be seen that ܦܫ௩ locates precisely the damage. In this damage case, a tension reduction of 95% is made at two cables located at mid-span (Cables 15 and 16). The results are shown in Figure 13 and it can be seen that DIv locates precisely the damage. p g In this damage case, a tension reduction of 95% is made at two cables located at mid-span (Cables 15 and 16). The results are shown in Figure 13 and it can be seen that ܦܫ௩ locates precisely the damage. Mu tip e a age Sce a io A: Case A6 In this damage case, a tension reduction of 95% is made at two cables located at mid-span (Cables 15 and 16). The results are shown in Figure 13 and it can be seen that ܦܫ௩ locates precisely the damage Figure 13. Damage indices for 95% Damage Case A6. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 13. Damage indices for 95% Damage Case A6. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 13. Damage indices for 95% Damage Case A6. Figure 13. Damage indices for 95% Damage Case A6. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 13. Damage indices for 95% Damage Case A6. Figure 13. Damage indices for 95% Damage Case A6. Figure 13. Damage indices for 95% Damage Case A6. Figure 13. Damage indices for 95% Damage Case A6. 4.2. Damage Detection with Multiple Active Cables Drive: Scenarios B 4.2. Damage Detection with Multiple Active Cables Drive: Scenarios B 4.2. Damage Detection with Multiple Active Cables Drive: Scenarios B Due to the low modal contribution of some modes when the structure is excited with a single active cable, the ܦܫ௩ is unable to detect the damage in the cables located near the edge of the bridge. To deal with such a problem, we propose to use two active cables for exciting the deck instead of one single cable (it is also possible to excite with four active cables). This enables the possibility of exciting the structure with a spatial forcing shape similar to the mode shapes of interest, and thus excite efficiently the modes with high modal amplitude near the clamp. Two active cables (1A and 1B) are used to drive the mock-up. The whole drive process is divided into two parts: synchronous drive, where the two cables are driven with the same signal (e.g., more suitable for exciting Mode 1), and the antiphase drive, where the two cables are driven with opposite sign signals, as shown in Figure 16. We use the same band-limited white noise as before. Due to the low modal contribution of some modes when the structure is excited with a single active cable, the DIv is unable to detect the damage in the cables located near the edge of the bridge. To deal with such a problem, we propose to use two active cables for exciting the deck instead of one single cable (it is also possible to excite with four active cables). This enables the possibility of exciting the structure with a spatial forcing shape similar to the mode shapes of interest, and thus excite efﬁciently the modes with high modal amplitude near the clamp. Two active cables (1A and 1B) are used to drive the mock-up. The whole drive process is divided into two parts: synchronous drive, where the two cables are driven with the same signal (e.g., more suitable for exciting Mode 1), and the antiphase drive, where the two cables are driven with opposite sign signals, as shown in Figure 16. We use the same band-limited white noise as before. Due to the low modal contribution of some modes when the structure is excited with a single active cable, the ܦܫ௩ is unable to detect the damage in the cables located near the edge of the bridge. • Multiple Damage Scenario A: Case A8 • Multiple Damage Scenario A: Case A8 Sensors 2018, 18, x FOR PEER REVIEW The last damage case studied under the multiple damage scenarios are with 95% tension reductions at the middle and quarter span (Cables 6 and 9); the results are shown in Figure 15. It is evident that DIv is able to locate the damage in the middle span but gives an incorrect prediction in the quarter span of the cable near the edge of the bridge. The last damage case studied under the multiple damage scenarios are with 95% tension reductions at the middle and quarter span (Cables 6 and 9); the results are shown in Figure 15. It is evident that ܦܫ௩ is able to locate the damage in the middle span but gives an incorrect prediction in the quarter span of the cable near the edge of the bridge. • Multiple Damage Scenario A: Case A8 The last damage case studied under the multiple damage scenarios are with 95% tension reductions at the middle and quarter span (Cables 6 and 9); the results are shown in Figure 15. It is evident that ܦܫ௩ is able to locate the damage in the middle span but gives an incorrect prediction in the quarter span of the cable near the edge of the bridge Figure 15. Damage indices for 95% Damage Case A8. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 15. Damage indices for 95% Damage Case A8. g g Figure 15. Damage indices for 95% Damage Case A8. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 15. Damage indices for 95% Damage Case A8. Figure 15. Damage indices for 95% Damage Case A8. Figure 15. Damage indices for 95% Damage Case A8. The foregoing results indicate that ܦܫ௩ is appropriate to locate multiple damages provided the damage location is located near an element whose contribution to the excited modes is significant. Moreover, it has been shown that damages located near the deck supports are difficult to detect. This situation is further analyzed below. The foregoing results indicate that DIv is appropriate to locate multiple damages provided the damage location is located near an element whose contribution to the excited modes is signiﬁcant. • Multiple Damage Scenario A: Case A8 • Multiple Damage Scenario A: Case A8 Sensors 2018, 18, x FOR PEER REVIEW Moreover, it has been shown that damages located near the deck supports are difﬁcult to detect. This situation is further analyzed below. The foregoing results indicate that ܦܫ௩ is appropriate to locate multiple damages provided the damage location is located near an element whose contribution to the excited modes is significant. Moreover, it has been shown that damages located near the deck supports are difficult to detect. This situation is further analyzed below. • Multiple Damage Scenario A: Case A7 I D C A7 h 95 • Multiple Damage Scenario A: Case A7 • Multiple Damage Scenario A: Case A7 • Multiple Damage Scenario A: Case A7 • Multiple Damage Scenario A: Case A7 • Multiple Damage Scenario A: Case A7 • Multiple Damage Scenario A: Case A7 • Multiple Damage Scenario A: Case A7 In Damage Case A7, there are two 95% tension reductions at two cables near the middle and third span (Cables 4 and 18). The results are shown in Figure 14 and it is evident that ܦܫ௩ shows two peaks corresponding to the two damage locations. In Damage Case A7, there are two 95% tension reductions at two cables near the middle and third span (Cables 4 and 18). The results are shown in Figure 14 and it is evident that ܦܫ௩ shows two peaks corresponding to the two damage locations. In Damage Case A7, there are two 95% tension reductions at two cables near the middle and third span (Cables 4 and 18). The results are shown in Figure 14 and it is evident that DIv shows two peaks corresponding to the two damage locations. In Damage Case A7, there are two 95% tension reductions at two cables near the middle and third span (Cables 4 and 18). The results are shown in Figure 14 and it is evident that ܦܫ௩ shows two peaks corresponding to the two damage locations. Figure 14. Damage indices for 95% Damage Case A7. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 14. Damage indices for 95% Damage Case A7. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 14. Damage indices for 95% Damage Case A7. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 1 2 3 4 5 6 7 8 9 10 DI (%) Figure 14. Damage indices for 95% Damage Case A7. igu e . a age i dices o 95% a age Case A Figure 14. Damage indices for 95% Damage Case A7. Figure 14. Damage indices for 95% Damage Case A7. Figure 14. Damage indices for 95% Damage Case A7. Sensors 2018, 18, 3002 13 of 20 13 f 20 • Multiple Damage Scenario A: Case A8 • Multiple Damage Scenario A: Case A8 Sensors 2018, 18, x FOR PEER REVIEW 4.2. Damage Detection with Multiple Active Cables Drive: Scenarios B 4.2. Damage Detection with Multiple Active Cables Drive: Scenarios B 4.2. Damage Detection with Multiple Active Cables Drive: Scenarios B To deal with such a problem, we propose to use two active cables for exciting the deck instead of one single cable (it is also possible to excite with four active cables). This enables the possibility of exciting the structure with a spatial forcing shape similar to the mode shapes of interest, and thus excite efficiently the modes with high modal amplitude near the clamp. Two active cables (1A and 1B) are used to drive the mock-up. The whole drive process is divided into two parts: synchronous drive, where the two cables are driven with the same signal (e.g., more suitable for exciting Mode 1), and the antiphase drive, where the two cables are driven with opposite sign signals, as shown in Figure 16. We use the same band-limited white noise as before. Fi 16 M l i l i bl d i Figure 16. Multiple active cables drive process. Figure 16. Multiple active cables drive process. Figure 16. Multiple active cables drive process. Figure 16. Multiple active cables drive process. 14 of 20 14 of 20 Sensors 2018, 18, 3002 Sensors 2018, 18, x FOR As shown in Figure 16, Modes 1 and 3 tend to be well excited by a synchronous drive of Cables 1A and 1B, while the antiphase drive tends to excite Mode 2. By using the two drive modes alternatively, a new enhanced Damage Index can be deﬁned as the sum of the two damage indices DI1 and DI2 associated to each drive mode. Table 2 shows the details of the various damage scenarios considered with multiple active cables drive. g y y 1A and 1B, while the antiphase drive tends to excite Mode 2. By using the two drive modes alternatively, a new enhanced Damage Index can be defined as the sum of the two damage indices ܦܫଵ and ܦܫଶ associated to each drive mode. Table 2 shows the details of the various damage scenarios considered with multiple active cables drive. T bl 2 D i B Table 2. Damage scenarios B. • Single Damage Scenario B: Case B1, B2, and B3 • Single Damage Scenario B: Case B1, B2, and B3 • Single Damage Scenario B: Case B1, B2, and B3 • Single Damage Scenario B: Case B1, B2, and B3 In these three damage cases, 95%, 80%, and 50% tension reductions are made at the cable located at the third span (Cable 3) and the results are shown in Figures 17–19. In these three damage cases, 95%, 80%, and 50% tension reductions are made at the cable located at the third span (Cable 3) and the results are shown in Figures 17–19. In these three damage cases, 95%, 80%, and 50% tension reductions are made at the cable located at the third span (Cable 3) and the results are shown in Figures 17–19. In these three damage cases, 95%, 80%, and 50% tension reductions are made at the cable located at the third span (Cable 3) and the results are shown in Figures 17–19. t the third span (Cable 3) and the results are shown in Figures 17–19. g t the third span (Cable 3) and the results are shown in Figures 17–19. (a) (b) (c) Figure 17. Enhanced damage indices for 95% Damage Case B1: (a) results of the synchronous drive; (b) results of the antiphase drive; and (c) results of synchronous drive + antiphase drive. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 2 4 6 8 10 12 14 DI (%) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 2 4 6 8 10 12 14 DI (%) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 17. Enhanced damage indices for 95% Damage Case B1: (a) results of the synchronous drive; (b) results of the antiphase drive; and (c) results of synchronous drive + antiphase drive. (a) (b) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 2 4 6 8 10 12 14 DI (%) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 2 4 6 8 10 12 14 DI (%) (a) (a) (b (c) 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) (b) (c) Figure 17. 4.2. Damage Detection with Multiple Active Cables Drive: Scenarios B 4.2. Damage Detection with Multiple Active Cables Drive: Scenarios B 4.2. Damage Detection with Multiple Active Cables Drive: Scenarios B Damage Case Location Severity of Damage (Tension Reduction) Single damage scenario Case B1 Cable 3 95% Case B2 Cable 3 80% Case B3 Cable 3 50% Case B4 Cable 2 90% Case B5 Cable 2 80% Multiple damage scenario Case B6 Cable 4 and Cable 18 80% Case B7 Cable 6 and Cable 9 80% g Damage Case Location Severity of Damage (Tension Reduction) Single damage scenario Case B1 Cable 3 95% Case B2 Cable 3 80% Case B3 Cable 3 50% Case B4 Cable 2 90% Case B5 Cable 2 80% Multiple damage scenario Case B6 Cable 4 and Cable 18 80% Case B7 Cable 6 and Cable 9 80% Table 2. Damage scenarios B. Location Severity o • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 Figure 20 shows a significant outcome of Case B4, from which it is evident that the enhanced damage index ܦܫ௩ gives a correct prediction of the damage location. This result could not be achieved with a single active cable drive (Damage Case A5). However, once the damage intensity reduces to 80%, the enhanced ܦܫ௩ is unable to locate the damage, as shown in Figure 21. Figure 20 shows a significant outcome of Case B4, from which it is evident that the enhanced damage index ܦܫ௩ gives a correct prediction of the damage location. This result could not be achieved with a single active cable drive (Damage Case A5). However, once the damage intensity reduces to 80%, the enhanced ܦܫ௩ is unable to locate the damage, as shown in Figure 21. Figure 20 shows a signiﬁcant outcome of Case B4, from which it is evident that the enhanced damage index DIv gives a correct prediction of the damage location. This result could not be achieved with a single active cable drive (Damage Case A5). However, once the damage intensity reduces to 80%, the enhanced DIv is unable to locate the damage, as shown in Figure 21. g g Figure 20 shows a significant outcome of Case B4, from which it is evident that the enhanced damage index ܦܫ௩ gives a correct prediction of the damage location. This result could not be achieved with a single active cable drive (Damage Case A5). However, once the damage intensity reduces to 80%, the enhanced ܦܫ௩ is unable to locate the damage, as shown in Figure 21. Figure 20. Enhanced damage indices for 90% Damage Case B4. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 20. Enhanced damage indices for 90% Damage Case B4. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 20. Enhanced damage indices for 90% Damage Case B4. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 20. Sensors 2018, 18, 3002 15 of 20 Sensors 2018, 18, 3002 Figure 17 shows the results of each step in the damage detection process for Case B1. Compared with the results of Case A4, the detection capability has been signiﬁcantly improved. In Figure 18, the enhanced DIv shows an evident peak at the damage location. Under this hybrid drive, the enhanced damage index becomes more sensitive to lower damage levels located near the clamp. However, the enhanced damage index still fails to detect accurately a damage with low severity of 50%, as shown in Figure 19. Figure 17 shows the results of each step in the damage detection process for Case B1. Compared with the results of Case A4, the detection capability has been significantly improved. In Figure 18, the enhanced ܦܫ௩ shows an evident peak at the damage location. Under this hybrid drive, the enhanced damage index becomes more sensitive to lower damage levels located near the clamp. However, the enhanced damage index still fails to detect accurately a damage with low severity of 50%, as shown in Figure 19. Figure 17 shows the results of each step in the damage detection process for Case B1. Compared with the results of Case A4, the detection capability has been significantly improved. In Figure 18, the enhanced ܦܫ௩ shows an evident peak at the damage location. Under this hybrid drive, the enhanced damage index becomes more sensitive to lower damage levels located near the clamp. However, the enhanced damage index still fails to detect accurately a damage with low severity of 50%, as shown in Figure 19. Sensors 2018, 18, x FOR PEER REVIEW 15 of 20 Figure 17 shows the results of each step in the damage detection process for Case B1. Compared with the results of Case A4, the detection capability has been significantly improved. In Figure 18, the enhanced ܦܫ௩ shows an evident peak at the damage location. Under this hybrid drive, the enhanced damage index becomes more sensitive to lower damage levels located near the clamp. However, the enhanced damage index still fails to detect accurately a damage with low severity of 50% as shown in Figure 19 Figure 18. Enhanced damage indices for 80% Damage Case B2. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 18. Enhanced damage indices for 80% Damage Case B2. Figure 18. Sensors 2018, 18, 3002 Enhanced damage indices for 80% Damage Case B2. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) g Figure 18. Enhanced damage indices for 80% Damage Case B2. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 18. Enhanced damage indices for 80% Damage Case B2. Figure 18. Enhanced damage indices for 80% Damage Case B2. Figure 18. Enhanced damage indices for 80% Damage Case B2. Figure 18. Enhanced damage indices for 80% Damage Case B2. Figure 19. Enhanced damage indices for 50% Damage Case B3. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 19. Enhanced damage indices for 50% Damage Case B3. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 19. Enhanced damage indices for 50% Damage Case B3. Figure 19. Enhanced damage indices for 50% Damage Case B3. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 19. Enhanced damage indices for 50% Damage Case B3. Figure 19. Enhanced damage indices for 50% Damage Case B3. Figure 19. Enhanced damage indices for 50% Damage Case B3. Figure 19. Enhanced damage indices for 50% Damage Case B3. • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B1, B2, and B3 • Single Damage Scenario B: Case B1, B2, and B3 Enhanced damage indices for 95% Damage Case B1: (a) results of the synchronous drive; (b) results of the antiphase drive; and (c) results of synchronous drive + antiphase drive. Figure 17. Enhanced damage indices for 95% Damage Case B1: (a) results of the synchronous drive; (b) results of the antiphase drive; and (c) results of synchronous drive + antiphase drive. • Multiple Damage Scenario B: Case B7 • Multiple Damage Scenario B: Case B7 • Multiple Damage Scenario B: Case B7 • Multiple Damage Scenario B: Case B7 This damage case also considers multiple damage scenarios with 80% tension reduction of the cables located in the middle and near the quarter span (Cables 6 and 9); the results are shown in Figure 23. The ܦܫ௩ is able to locate the damage in the middle span of the Cable 6 but gives an incorrect prediction at the quarter span of Cable 9, located near the edge of the bridge (exhibiting a small modal amplitude). This damage case also considers multiple damage scenarios with 80% tension reduction of the cables located in the middle and near the quarter span (Cables 6 and 9); the results are shown in Figure 23. The ܦܫ௩ is able to locate the damage in the middle span of the Cable 6 but gives an incorrect prediction at the quarter span of Cable 9, located near the edge of the bridge (exhibiting a small modal amplitude). This damage case also considers multiple damage scenarios with 80% tension reduction of the cables located in the middle and near the quarter span (Cables 6 and 9); the results are shown in Figure 23. The DIv is able to locate the damage in the middle span of the Cable 6 but gives an incorrect prediction at the quarter span of Cable 9, located near the edge of the bridge (exhibiting a small modal amplitude). This damage case also considers multiple damage scenarios with 80% tension reduction of the cables located in the middle and near the quarter span (Cables 6 and 9); the results are shown in Figure 23. The ܦܫ௩ is able to locate the damage in the middle span of the Cable 6 but gives an incorrect prediction at the quarter span of Cable 9, located near the edge of the bridge (exhibiting a small modal amplitude). Figure 23. Enhanced damage indices for 80% Damage Case B7. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 23. Enhanced damage indices for 80% Damage Case B7. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 23. Enhanced damage indices for 80% Damage Case B7. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 23. • Multiple Damage Scenario B: Case B6 • Multiple Damage Scenario B: Case B6 • Multiple Damage Scenario B: Case B6 • Multiple Damage Scenario B: Case B6 • Multiple Damage Scenario B: Case B6 • Multiple Damage Scenario B: Case B6 • Multiple Damage Scenario B: Case B6 • Multiple Damage Scenario B: Case B6 Figure 22 shows the enhanced ܦܫ௩, where two cables have seen their tension reduced by 80%. The enhanced ܦܫ௩ locates precisely the damaged cables. Figure 22 shows the enhanced DIv, where two cables have seen their tension reduced by 80%. The enhanced DIv locates precisely the damaged cables. Figure 22 shows the enhanced ܦܫ௩, where two cables have seen their tension reduced by 80%. The enhanced ܦܫ௩ locates precisely the damaged cables. p g Figure 22 shows the enhanced ܦܫ௩, where two cables have seen their tension reduced by 80%. The enhanced ܦܫ௩ locates precisely the damaged cables. Figure 22. Enhanced damage indices for 80% Damage Case B6. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 22. Enhanced damage indices for 80% Damage Case B6. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 22. Enhanced damage indices for 80% Damage Case B6. Figure 22. Enhanced damage indices for 80% Damage Case B6. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 DI (%) Figure 22. Enhanced damage indices for 80% Damage Case B6. Figure 22. Enhanced damage indices for 80% Damage Case B6. Figure 22. Enhanced damage indices for 80% Damage Case B6. Figure 22. Enhanced damage indices for 80% Damage Case B6. • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 • Single Damage Scenario B: Case B4 and B5 Enhanced damage indices for 90% Damage Case B4. Figure 20. Enhanced damage indices for 90% Damage Case B4. Figure 20. Enhanced damage indices for 90% Damage Case B4. Figure 20. Enhanced damage indices for 90% Damage Case B4. Figure 20. Enhanced damage indices for 90% Damage Case B4. 16 of 20 16 of 20 Sensors 2018, 18, 3002 Figure 21. Enhanced damage indices for 80% Damage Case B5. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 Figure 21. Enhanced damage indices for 80% Damage Case B5. Figure 21. Enhanced damage indices for 80% Damage Case B5. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 R PEER REVIEW Figure 21. Enhanced damage indices for 80% Damage Case B5. 0 2 4 6 8 10 12 14 16 18 20 Cable number 0 5 10 15 20 25 Figure 21. Enhanced damage indices for 80% Damage Case B5. Figure 21. Enhanced damage indices for 80% Damage Case B5. Figure 21. Enhanced damage indices for 80% Damage Case B5. Figure 21. Enhanced damage indices for 80% Damage Case B5. • Multiple Damage Scenario B: Case B7 • Multiple Damage Scenario B: Case B7 • Multiple Damage Scenario B: Case B7 • Multiple Damage Scenario B: Case B7 Enhanced damage indices for 80% Damage Case B7. Figure 23. Enhanced damage indices for 80% Damage Case B7. Figure 23. Enhanced damage indices for 80% Damage Case B7. Figure 23. Enhanced damage indices for 80% Damage Case B7. Figure 23. Enhanced damage indices for 80% Damage Case B7. 17 of 20 17 of 20 Sensors 2018, 18, 3002 4.3. Comparison with Traditional Methods 4.3. Comparison with Traditional Methods In this section, the traditional modal parameters-based damage detection methods: Coordinate Modal Assurance Criterion (COMAC) [44], Enhanced Coordinate Modal Assurance Criterion (ECOMAC) [45], Mode Shape Curvature (MSC) [46], Modal Strain Energy (MSE) [47] and Modal Flexibility (MF) [48] are introduced to detect the damage in the bridge. Only few scenarios are considered. The experimental results are summarized in Table 3. These results are extracted from a separate study under consideration [49]. As one observes in Table 3, none of the traditional methods can detect the low-level damages, corresponding to a loss of 80% of the hanger’s tension (Cases B2, B6 and B7). When the damage intensity is high, up to 95% tension loss, two traditional methods (MSE and MF) are able to detect the damage successfully and conﬁrm the actual location of the damage (Case B1). However, by simply observing these results (Table 3), one can ﬁnd that, once the damage intensity reduces to less than 90% and the damage position deviates to the edge of deck, only the MF method locates precisely the damaged cable (Case B4). For the 80% damage level near the mid-span (Cases B2 and B6), the performance of the enhanced DIv is superior to the traditional methods. Table 3. Comparison of damage detection methods. Case # COMAC ECOMAC MSC MSE MF Enhanced DIv B1 X X OO O O O B2 X X OO OO OO O B4 X X X X O O B6 X OO X OO OO O B7 X OO OO OO OO OO O, damage detected and located; OO, damage detected but not located; X, damage not detected. Table 3. Comparison of damage detection methods. Table 3. Comparison of damage detection methods. 5. Conclusions In this paper, we investigate and validate experimentally the possibility of using active cables to excite the structure to detect and locate damages in the hangers of a suspension bridge. The proposed method, based on the Hilbert marginal spectrum, has been successfully implemented and validated through an extensive set of damage scenarios. The damage index has been found very effective for single and multiple damages provided the damage location is sufﬁciently excited. The sensitivity of the damage index has been enhanced by using several cables in multiple driving modes. the damage index has been enhanced by using several cables in multiple driving mode Based on the presented results and interpretations, the main ﬁndings are summarized • The active cable drive is an effective way to excite the suspension bridge. Combined with Hilbert–Huang transform (HHT), a damage index (DIv) is constructed for damage detection of suspension bridge hangers. The proposed damage index (DIv) showed promising damage detection capability. • The active cable drive is an effective way to excite the suspension bridge. Combined with Hilbert–Huang transform (HHT), a damage index (DIv) is constructed for damage detection of suspension bridge hangers. The proposed damage index (DIv) showed promising damage detection capability. • The hybrid drive with multiple active cables can improve signiﬁcantly the damage detection capability and is able to detect single and multiples damages with small modal amplitudes. For the 80% damage level near the mid-span, the performance of the enhanced DIv is superior to the traditional modal parameters-based damage detection methods (COMAC, ECOMAC, MSC, MSE and MF). However, for some conﬁgurations, the enhanced damage index fails to detect damages, mainly for low-level damage. • This paper validates the feasibility of the proposed method under laboratory conditions; however, some limitations are inevitable. Indeed, reducing the tension in the hangers is not representative of damage as it modiﬁes only the lateral stiffness of the cable and does not affect its longitudinal stiffness, which signiﬁcantly contributes to the deck vertical stiffness. Thus, a more representative damage scenario must be used to conclude on the quality of the algorithm (e.g., cutting slightly the cable). 18 of 20 Sensors 2018, 18, 3002 • Finally, in this paper, we consider the inﬂuence of noise and ignored the inﬂuence of environmental and operational variability on the dynamic behavior. It is well known that suspension bridges are insensitive to tiny damages. 5. Conclusions Thus, a small damage intensity would be detectable by this method (equivalent to 80% reduction of the hanger tension), which is a satisfactory result. Nevertheless, to conﬁrm these ﬁndings, the extension to ﬁeld experiments under realistic conditions should be further investigated. More importantly, some quantitative results can be obtained. A potential test case would be the Seriate footbridge, located in Bergamo, Italy, and considered as a test case in a previous study [22]. Author Contributions: All authors discussed and agreed upon the idea and made scientiﬁc contributions. The original idea of the article was provided by F.M. and A.P. designed the methodology, realized its MATLAB program, and wrote the manuscript. B.M. and D.A. provided with the support of the entire study and revised the manuscript. J.Y. and A.P. gave suggestions in the theoretical analyses and tests. Funding: This research was funded by the National Science Foundations of China (Grant Nos. 91748205 and 51575017). Funding: This research was funded by the National Science Foundations of China (Grant Nos. 91748 nd 51575017). Acknowledgments: The authors thank M. Horodinca and I. Romanescu from Ghorge Asachi University, Iasi, Romania for the realization of the experimental set-up; and M. Voltan and A. 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https://openalex.org/W1994929676	https://europepmc.org/articles/pmc4433656?pdf=render	English	null	An Activity of Thioacyl Derivatives of 4-Aminoquinolinium Salts towards Biofilm Producing and Planktonic Forms of Coagulase-Negative Staphylococci	BioMed research international	2,015	cc-by	8,171	Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 725939, 10 pages http://dx.doi.org/10.1155/2015/725939 Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 725939, 10 pages http://dx.doi.org/10.1155/2015/725939 Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 725939, 10 pages http://dx.doi.org/10.1155/2015/725939 Correspondence should be addressed to Robert D. Wojtyczka; rwojtyczka@sum.edu Received 18 July 2014; Revised 15 October 2014; Accepted 22 November 2014 Received 18 July 2014; Revised 15 October 2014; Accepted 22 November 2014 Academic Editor: Madhab K. Chattopadhyay Academic Editor: Madhab K. Chattopadhyay Academic Editor: Madhab K. Chattopadhyay Copyright © 2015 Robert D. Wojtyczka et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Microorganisms present in different environments have developed specific mechanisms of settling on various abiotic and biotic surfaces by forming a biofilm. It seems to be well justified to search for new compounds enabling biofilm reduction, which is highly resistant to antibiotics. This study was thus an initial assessment of the antibacterial activity of two new quinoline derivatives of a structure of 3-thioacyl 1-methyl 4-arylaminoquinolinium salts against coagulase-negative staphylococci (CoNS) isolated from a hospital environment, in a form of both biofilms and in planktonic form. Thirty-three stains of CoNS isolated from the hospital environment (air, surfaces) and seven reference strains from the ATCC collection were selected for the study. The mean MIC value for 1-methyl-3-benzoylthio-4-(4-chlorophenylamino)quinolinum chloride (4-chlorophenylamino derivative) was 42.60 ± 19.91 𝜇g/mL, and in the case of strains subjected to 1-methyl-3-benzoylthio-4-(4-fluorophenylamino)quinolinum chloride (4-fluorophenylamino derivative) activity, the mean MIC value was 43.20 ± 14.30 𝜇g/mL. The mean concentration of 4-chlorophenylamino derivative that inhibited biofilm formation was 86.18 ± 30.64 𝜇g/mL. The mean concentration of 4- fluorophenylamino derivatives that inhibited biofilm formation was higher and amounted to 237.09 ± 160.57 𝜇g/mL. Based on the results, both derivatives of the examined compounds exhibit high antimicrobial activity towards strains growing both in planktonic and biofilm form. Robert D. Wojtyczka,1 Andrzej Ziwba,2 Arkadiusz Dziedzic,3 MaBgorzata Kwpa,1 and Danuta Idzik1 1Department and Institute of Microbiology and Virology, School of Pharmacy and Division of Laboratory Medicine in Sos Medical University of Silesia, Jagiello´nska 4, Sosnowiec, 41-200 Katowice, Poland 2Department of Organic Chemistry, School of Pharmacy and Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Jagiello´nska 4, Sosnowiec, 41-200 Katowice, Poland 3Department of Conservative Dentistry with Endodontics, Medical University of Silesia, Plac Akademicki 17, Bytom, 41-902 Katowice, Poland Correspondence should be addressed to Robert D. Wojtyczka; rwojtyczka@sum.edu.pl 1. Introduction Staphylococcus epidermidis is believed to be an important microorganism responsible for infections following surgical vascular grafts, infections after implantation of central venous catheters, heart valves, ventricular assist devices, coronary stents, neurological ventricular shunts, surgical wounds, arthroprotheses, or equipment used for fracture stabilization [2–6]. Coagulase-negative staphylococci (CoNS) constitute a con- siderable part of the commensal flora of human skin and mucous membranes. These microorganisms are often isolated from nosocomial infections of the bloodstream, cardiovas- cular system, as well as infections of the eye, ear, nose, and throat [1]. Over the recent decades, the main representative of CoNS, Staphylococcus epidermidis, has become a significant opportunistic pathogen due to an increasing number of specialist medical procedures, including implanted objects. Microorganisms present in various environments have developed mechanisms of settling on various abiotic and biotic surfaces. The adhesive properties of bacteria may influence the degree of their invasiveness and the likelihood BioMed Research International 2 leading to canal formation and biofilm organization are not as well understood as those regulating intercellular adhesion. The results obtained from studies of Pseudomonas aeruginosa suggest a role in cell-cell signaling as part of a quorum- sensing system [24]. In staphylococci, expression of PIA exopolysaccharide in biofilms may to some degree contribute to biofilm formation. Various mechanisms of resistance to antibiotics are well known, such as antibiotic removal by antibiotic pumps, modification of their structure by enzymes or mutations of target sites [25, 26].i of infection. Bacteria connecting to the surface form micro- colonies anchored in the extracellular matrix [7]. Bacte- ria present in biofilms have developed a strategy of long- lasting microecosystem, representing a nonaggressive viru- lence. Therefore, biofilm-related infections may last for many months, extending even for the whole life; however, they rarely directly cause death [8].i A biofilm is a cyclically maturing, three-dimensional structure composed of about 85% of extracellular matrix and of about 15% of microorganism cell aggregates. The matrix is composed of polysaccharides, proteins, enzymes, DNA, bac- terial glycolipids, water, and other environmental elements [9]. Because biofilm provides a reservoir for microbial cells, its dispersion enhances the risk of chronic and persistent infections. Likewise the matrix confers a protection against drugs and has environmental promoters that induce biofilm formation and contributes to drug resistance development [10, 11]. Biofilm structure promotes the antibiotic resistance through facilitated horizontal gene transfer due to the high microbial population density. 1. Introduction Several mechanisms have been reported to contribute an increased antimicrobial resistance in biofilm structures [11, 12], including low diffusion, trans- membrane passage of antibiotics across the polysaccharide matrix, physiological changes of bacteria due to slow growth rate and starvation responses (oxygen, nutrient deprivation, or environmental stress), phenotypic change of the cells forming the biofilm, the expression of efflux pumps that decrease intracellular antimicrobial concentration, and the emergence of persister cells which are multidrug-tolerant cells that have not acquired genetic resistance [13]. These fac- tors contribute to biofilm cells being 1000-fold more resistant to antimicrobial agents than planktonic cells [11, 14, 15]. A well-known genetic element among the staphylococci with regard to biofilm formation is the ica operon, which encodes a polysaccharide intercellular adhesin (PIA), which is also often called poly-N-acetyl glucosamine (PNAG) according to its chemical structure [16–18]. The operon contains the icaADBC genes, in addition to the icaR gene, which exerts a regulatory function and is transcribed in the opposite direction. Earlier studies suggested a direct link between the presence of ica and biofilm formation. Once this operon is activated, four proteins are transcribed, IcaA, IcaD, IcaB, and IcaC, which are necessary for the biosynthesis of PIA [17, 19, 20]. IcaA encodes N-acetylglucosaminyltransferase, which synthesises the PIA polymer. Sole expression of icaA induces only low enzymatic activity, but coexpression with icaD significantly increases the activity due to the phenotypic expression of the capsular polysaccharide. IcaC is responsible for formation of long chains, and icaB deacetylates the poly- N-acetylglucosamine molecule [17, 20]. Expression of locus ica genes is regulated by various environmental factors and regulatory proteins. PIA production and its deacetylation have been considered as key factors of virulence in S. epidermidis [20–22]. Mature biofilms have a specific three-dimensional struc- ture, which is described as “towers” or “mushrooms” [23] Various mechanisms of resistance of biofilm-forming strains to antibiotics are suggested. The first hypothesis is the slow or incomplete antibiotic penetration inside the biofilm structure. The second hypothesis conditions antibi- otic activity on the chemical differentiation of the biofilm microenvironment. According to the third hypothesis, a subpopulation of highly protected microorganisms similar to spores is formed in biofilm structures. This hypothesis is confirmed in a study on newly formed biofilm structures, which are still too thin to present a mechanical barrier against antibiotic penetration [27, 28]. 1. Introduction This hypothesis of spore formation by some cells living in a form of biofilm may be an explanation of lowered susceptibility of biofilm bacteria to various antibiotics, disinfection means, or a wide range of different chemical compounds [8, 25]. f Modern chemotherapeutic compounds such as quinoline derivatives exhibit a wide antibacterial activity towards gram- positive bacteria such as Staphylococcus aureus, as well as towards gram-negative bacteria [29, 30] and also antifungal and antimalarial activity [29, 31]. Modification of the main structural fragment of a drug may lead to an improvement in its antimicrobial efficiency and strength as well as mode and direction of interaction.hi The current treatment and control of biofilm is compli- cated, because antimicrobials have been developed against planktonically grown bacteria and microorganisms in meta- bolically active stage. Therefore, we have made an attempt to assess the activity of newly synthetized antibacterial agents 1- methyl-3-benzoylthio-4-(4-chlorophenylamino)quinolinum chloride (4-chlorophenylamino derivative) and 1-methyl-3- benzoylthio-4-(4-fluorophenylamino) quinolinum chloride (4-fluorophenylamino derivative) towards the strains of coagulase-negative staphylococci (CoNS), growing in a form of biofilm (BF) and in planktonic form (PF), isolated from a hospital environment. The investigated compounds were obtained as a result of an acylation of suitable deriv- atives of 1-methyl-4-aminoquinolinium-3-thiolates using benzoyl chloride [29, 30]. These compounds exhibit strong nucleophilic properties and easily undergo alkylation and acylation reactions on thiolate sulfur atom. The new compounds described, 4-chlorophenylamino derivative and 4-fluorophenylamino derivative, similar to 4-quinolones, are the quinoline derivatives; however their structure, compared to chinolones, is significantly different. 4-Aminoquinoline derivatives have been used as antimalarial drugs, while fluorine derivatives of 4-quinolones are an important class of antibiotics. The evaluated quinoline compounds contain the chlorine or fluoride in phenylamine substituent and not in quinoline ring as it is in case of quinolones. The examined Mature biofilms have a specific three-dimensional struc- ture, which is described as “towers” or “mushrooms” [23]. There are canals filled with liquid between the towers, and it is believed they play a significant function in nutrient supply to the cells in the deeper biofilm layers. Mechanisms 3 BioMed Research International 3 N N H S C O F CH3 Cl− + (a) N N H S C O Cl CH3 Cl− + (b) Figure 1: Structure of quinoline derivatives: (a) 1-methyl-3-benzoylthio-4-(4-fluorophenylamino) quinolinum chloride (4-fluorophenylam- ino derivative), (b) 1-methyl-3-benzoylthio-4-(4-chlorophenylamino)quinolinum chloride (4-chlorophenylamino derivative). 1. Introduction N N H S C O F CH3 Cl− + (a) N N H S C O Cl CH3 Cl− + (b) Cl− Cl− Cl− (a) (b) Figure 1: Structure of quinoline derivatives: (a) 1-methyl-3-benzoylthio-4-(4-fluorophenylamino) quinolinum chloride (4-fluorophenylam- ino derivative), (b) 1-methyl-3-benzoylthio-4-(4-chlorophenylamino)quinolinum chloride (4-chlorophenylamino derivative). salts are the sulfate derivatives of 4-aminoquinoline, and considering their structure, they seem to be more similar to chloroquine (used for preventing malaria from Plasmodium vivax, P. ovale, and P. malariae) or ammonium salt. 2.2. Investigation of Biofilm Production by the Congo Red Agar (CRA) Method. Phenotypic characterization of biofilm production was performed by culturing the CoNS isolates on CRA plates as described by Freeman et al. [33]. A special medium was prepared of brain-heart infusion broth (BHI- BTL Poland) supplemented with 5% sucrose and Congo red. Plates were inoculated and incubated aerobically for 24 to 48 hours at 37∘C. According to the authors, biofilm producers form black, crusty colonies on CRA, whereas nonproducers form red colonies. A darkening of the colonies with the absence of a dry crystalline colonial morphology indicated an intermediate result. Quinoline is a heterocyclic aromatic organic compound with the chemical formula C9H7N. This compound forms part of the structure of quinine, the malaria remedy found in cinchona bark and known since the time of the Incas. Quinolines and their derivatives occur in numerous natural products, many of which possess interesting physiological and biological properties. Design and preparation of poten- tial quinoline-based antibacterial agents consist partially in a molecular hybridization approach that involves the coupling of two or more groups with relevant biological properties [32]. Figure 1 presents the structure of two examined com- pounds.h 2.3. Biofilm Formation Studied by Microtiter Plate Assay (MP). We performed the microtiter plate assay described by Christensen et al. [34] with modifications. A suspension equivalent to McFarland 0.5 turbidity standard was prepared in Muller-Hinton broth (MHB-BTL, Poland) for each strain. The accuracy of bacterial counts in the suspension was confirmed by serial dilution in log steps. Aliquots of 100 𝜇L from each bacterial suspension were inoculated onto 96-well tissue microtiter plates. These were incubated at 37∘C for 24 h in a normal atmosphere. The medium was then removed, and the wells were washed three times with phosphate-buffered saline (PBS, pH 7.2) to remove free-floating “planktonic” bacteria. 1. Introduction An amount of 150 𝜇L of 0.1% crystal violet (Sigma, USA) was added to each well and left for 30 min at room temperature. The dye was removed, and this was followed by five washes with sterile deionized water. The preparations were destained with 200 𝜇L of 95% isopropanol in 1 M HCl for 5 min. Finally, 100 𝜇L of coloured isopropanol from each sample was transferred to another microtiter plate. The optical density (OD) of the suspension was measured at a wavelength of 490 nm with a Multitec SX microplate reader.h The search for new antibacterial compounds continues and other quinolines with similar structures may become available as efficient antibiotics against resistant microorgan- isms. The key reason of all these structural changes of quino- line derivatives was to improve physicochemical parameters of quinoline molecules, which may lead to an enhanced fit into the binding site. As the hospital strains become increasingly resistant to the standard antibiotics, there is an urgent need to understand the molecular mechanisms for new drug action and resistance so that novel antibacterial drugs can be designed. A number of modified quinolines compounds show some promise in this regard. 2. Material and Methods 2.1. Bacterial Strain. Thirty-three isolated of coagulase- negative staphylococci obtained from a hospital environment (surfaces, air) and eight reference strains from the ATCC collection (S. epidermidis ATCC 12228, S. epidermidis ATCC 35984, S. saprophyticus ATCC 15305, S. hominis ATCC 27844, S. haemolyticus ATCC 29970, S. capitis subsp. capitis ATCC 35661, S. warneri ATCC 49454, and S. lugdunensis ATCC 49567) were selected for the study. The species of the strains were identified according to standard methodology with the use of biochemical APIStaph tests (bioMerieux, France) and stored for further analysis on TBE medium with the addition of 20% glycerol (Sigma) at −20∘C reference strains from the ATCC collection were obtained from LCG Standards (Lomianki, Poland). g The ODs obtained were compared with those of the nega- tive control (well without bacterial inoculum). We considered the OD > 0.17 to be positive. All the strains were tested four times, and the average value for each sample was calculated. The mean A490 ± SD values are presented.hi h The isolates were classified into two categories: nonadher- ent, optical density to or lower than 0.17 and adherent, optical density higher than 0.17. When the cut-off corresponded to nonadherent, the isolates were classified as negative and as positive when the cut-off corresponded to adherent. 4 BioMed Research International was used as negative control. The bioassay was performed in triplicate.if 2.4. Detection of icaA and icaD Genes Specific for Biofilm Production in S. epidermidis Strains. All isolates were stored at −80∘C pending analysis and were subcultured on blood agar plates and checked for purity prior to DNA preparation. An Isolate Genomic DNA Mini Kit (BLIRT S.A., Poland) was used to isolate DNA from strains with the following modifications. Pure DNA was stored at −20∘C. 2.4. Detection of icaA and icaD Genes Specific for Biofilm Production in S. epidermidis Strains. All isolates were stored at −80∘C pending analysis and were subcultured on blood agar plates and checked for purity prior to DNA preparation. An Isolate Genomic DNA Mini Kit (BLIRT S.A., Poland) was used to isolate DNA from strains with the following modifications. Pure DNA was stored at −20∘C. For assessing the biofilm growth inhibition effects of the 4-chlorophenylamino derivative and 4-fluorophenylamino derivative, they were added to the growth medium at the time of in oculation and the cells were allowed to form biofilm. 2. Material and Methods i An aliquot of twofold serial dilutions (200 𝜇L) was prepared in the 96-well microtiter plate containing tryptic soy broth (TSB, BTL, Ł´od´z, Poland) supplemented with 2% D-glucose (TSBGlc), with final concentrations of 4- chlorophenylamino derivative ranging from 1000 𝜇g/mL to 1 𝜇g/mL for 4-fluorophenylamino derivative. Bacterial sus- pensions (100 𝜇L; 5× 105 CFU/mL, final concentration) were then transferred into the plate. TSBGlc containing 0.2% DMSO was employed as a negative control. TSBGlc without the extract was used as the nontreated well and the medium with each concentration of the extracts was used as the blank control [40].tf i PCR was used to detect the presence of icaA and icaD. The primer sequences and predicted product lengths for icaA and icaD were described by Ziebuhr et al. [35] and de Silva et al. [36]. The PCR reactions were performed in separate reactions using a 10x PCR RED master mix kit. The PCR cycling conditions used were 30 cycles of 30 s of denaturation at 95∘C and 3 min of elongation at 72∘C for all reactions, with an annealing for 1 min at 60∘C (icaA) or 59∘C (icaD). PCR was performed by using an MJ Mini Personal thermal cycler (BIORAD, Germany).h The PCR products were electrophoresed through 1.5% agarose gels and checked for size against molecular weight markers using 1 Kb HypeLadderIV (BLIRT S.A., Poland). After incubation in 37∘C for 24 hours the inhibitory effect was evaluated using MTT reduction assay. The supernatant was discarded and replaced with 200 𝜇L of PBS supplemented with 50 𝜇g MTT (Sigma-Aldrich, USA). The formazan crys- tals were dissolved in dimethyl sulfoxide (DMSO, Sigma- Aldrich, USA), and the absorbance was determined at 570 nm (OD = 570 nm) with microplate reader (Thermo Electron Corp., Finland). 2.5. Examination of Antistaphylococcal Activity of 1-Methyl-4- aminoquinolinium-3-thiolates. Determination of minimum inhibitory concentrations (MIC) of microorganisms was performed using the method of microdilutions on 96-well plates according to obligatory methodology [37].h 2.5. Examination of Antistaphylococcal Activity of 1-Methyl-4- aminoquinolinium-3-thiolates. Determination of minimum inhibitory concentrations (MIC) of microorganisms was performed using the method of microdilutions on 96-well plates according to obligatory methodology [37].h The MIC value was determined by an incubation of the examined strains in 96-well plates for 20 hours at 37∘C. Bacterial inocula were prepared in 0.9% sodium chloride from fresh cultures up to the moment of 0.5 McFarland’s turbidity. 2. Material and Methods Next, the inocula were diluted (1 : 100) in sterile Mueller-Hinton broth medium just before addition to the plates. The microorganisms were exposed to a serial dilution of 4-chlorophenylamino derivative and 4-fluorophenylamino derivative in the range from 1000 to 1 𝜇g/mL. In addition, the values of minimum and maximum MIC as well as MIC50 and MIC90 were determined. The examinations were conducted both for the strains forming biofilm and those not forming it. 2.7. Statistical Analysis. All examinations of MIC and biofilms were performed four times, and the results were expressed as mean values ± standard deviation (SD). Results from correlation assays were submitted to the 𝑈Mann- Whitney nonparametric comparing biofilm formation status and ica status, MP test, and CRA plate test. Results were considered significant when 𝑃< 0.05. For comparison of the MIC and MBIC results, the respective 50th percentile values (MIC50 and MBIC50) and 90th percentile values (MIC90 and MBIC90) were calculated, and the ranges were compared. Statistical analyses were performed using Statistica 10.0 PL (StatSoft). 2.6. Inhibition of Staphylococcal Biofilm Formation by 1-Methyl-4-aminoquinolinium-3-thiolates. The 1-methyl-4- aminoquinolinium-3-thiolates were tested for their potential to prevent biofilm formation of a biofilm producing S. epidermidis strain. The evaluation of minimum biofilm inhibiting concentrations (MBIC) was done colorimetrically from the metabolic activity of surviving CoNS strains cells treated with new quinoline compounds [38, 39]. The presence of viable cells in biofilm was tested using 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). This “static,” indirect viability assay measures enzymatic activity in actively respiring cells and is based on the formation of insoluble purple formazan due to the reduction of MTT by respiratory reductase of living staphylococcal cells. Therefore, it is a measure of cell viability and/or relative numbers of viable cells. The minimum biofilm inhibitory concentration (MBIC) is defined as the lowest concentration of an antimicrobial agent required to inhibit the formation of biofilms. Nonbiofilm-forming clinical isolate S. epidermidis 4. Discussion An assessment of the bactericidal activity of six selected antibiotics on the planktonic and biofilm forms of three S. epidermidis strains and three S. aureus strains was conducted by Nishimura et al. [41]. In the case of clarithromycin, cefo- taxime, erythromycin, and benzylpenicillin, the planktonic forms of the S. epidermidis strains demonstrated susceptibil- ity to antibiotic concentrations below 0.5 𝜇g/mL, while in the case of vancomycin and cefmetazole this value was estimated at 1 𝜇g/mL. Conducting a similar analysis of biofilm-forming bacteria, Nishimura et al. demonstrated their susceptibility in concentrations not lower than 512 𝜇g/mL to 1024 𝜇g/mL [41].h Analyses of the MIC values for 40 strains of coagulase- negative staphylococci (Tables 2 and 3) showed a mean MIC value for 4-chlorophenylamino derivative of 42.60 ± 19.91 𝜇g/mL, with a minimum value of 16 𝜇g/mL and max- imum of 64 𝜇g/mL. In the case of the strains subjected to 4-fluorophenylamino derivative, the mean MIC value was 43.20 ± 14.30 g/mL, the minimum value was 16 𝜇g/mL, and the maximum was 64 𝜇g/mL. The values of MIC50 and MIC90 for all examined strains were 46 𝜇g/mL and 64 𝜇g/mL, respectively, and were the same for 4-chlorophenylamino derivative and 4-fluorophenylamino derivatives. The activity of various antibiotics including dapto- mycin, vancomycin, gentamicin, and rifampicin applied as a monotherapy and combined polytherapy against two clinical MRSA strains forming biofilm l was investigated by LaPlante and Woodmansee [45]. They noted that daptomycin and rifampicin had only slight differences in the ratio between MIC and MBEC. In the case of vancomycin, the MIC values for two examined strains forming biofilms were 2 𝜇g/mL and 4 𝜇g/mL, respectively, while the MBEC values for the same strains were considerably higher and amounted to 128 mg/L and 64 mg/L, respectively. These strains were more suscep- tible to gentamicin with even higher differences between the MIC and MBEC values, and they were estimated as 0.5 𝜇g/mL and 256 mg/L, respectively, for both strains. Assessing the ability of biofilm inhibition by 4-chloro- phenylamino derivative and 4-fluorophenylamino derivative, 11 strains exhibiting such ability phenotypically were selected for analysis using the MP method (Table 4). The analysis showed that biofilms grown in the presence of compounds had significantly lower activity as compared to an inhibitor control. 4. Discussion The results of our study demonstrated the presence of the icaA and/or icaD genes and subsequent biofilm production in most S. epidermidis isolates (sensitivity 0.909; specificity 0.724). Taking into account the fact that biofilm structures are the main factor of virulence involved in infections with coagulase-negative staphylococci [1], it seems to be essential to determine their antibiotic activity considering infections caused by these microorganisms occur both in planktonic forms and those forming a biofilm. Scanning electron microscopy demonstrated that biofilm is visible as soon as after 24 hours of bacterial adhesion to the surface [41]. According to some observations, 99% of bacteria live in a form of bacterial biofilm, and only 1% in a free form not attached to the surface, for example, in body liquids [7]. Among 40 strains of coagulase-negative staphylococci analyzed using the MP method, 11 (27.5%) strains were classified as forming a biofilm (A490 > 0.17). The other 22 examined strains and 7 reference strains did not form biofilms (Table 1). Among the 11 strains forming biofilms, 10 (90.9%) belonged to S. epidermidis species, while 1 strain (9.1%) was identified as S. kloosii. After examining the presence of icaA and icaD genes in S. epidermidis isolated strains, the icaAD gene was noted in 11 strains (57.9%) (Table 1). Analyzing the results of an assessment of an ability of biofilm formation using CRA, MP methods, and determination of the presence of icaA and icaD genes, it may be concluded that 9 (90%) out of 10 S. epidermidis strains exhibiting phenotypical ability of biofilm formation using the MP method demonstrated the presence of at least one of these genes (icaA). In this one analyzed case, this may suggest the possibility of biofilm formation independent of the icaADBC operon. In one case, the presence of the icaA gene did not induce the phenotypical presence of biofilm in any of the methods used. Statistical analysis by the Kruskal-Wallis test showed a correlation among the phenotypical presence of biofilm and ica status (𝑃= 0.0017). Numerous studies were carried out with the attempt to compare the susceptibility of bacteria growing in a form of biofilm on selected antibiotics, as well as their equivalents in planktonic form; however the results were not validated and/or were unequivocal [42, 43]. Bacteria growing in a form of biofilm are from 100- up to 1000-fold more resistant than their planktonic forms [42–44]. BioMed Research International BioMed Research International reached a value of 192 𝜇g/mL, and the MBIC90 value was 448 𝜇g/mL. reached a value of 192 𝜇g/mL, and the MBIC90 value was 448 𝜇g/mL. By assessing the ability of biofilm formation using the CRA method, characteristic black crusty colonies were obtained only in the case of two (5%) strains, and interme- diate colonies, grey ones in three (7.5%) cases. The examined reference strains did not exhibit an ability for biofilm forma- tion using the CRA method (Table 1). 3. Results Analyses of the antimicrobial activity of 4-chlorophenyl- amino derivatives and 4-fluorophenylamino derivatives were conducted on 40 strains of coagulase-negative staphylococci, of which 33 strains were isolated from a hospital environment (air, surfaces) and seven reference strains were derived from the American Type Culture Collection. Among 33 of the identified CoNS environmental strains, 19 were identified as S. epidermidis, 5 strains as S. haemolyticus, 3 strains as S. warneri, 2 strains as S. cohnii subsp. cohnii, and 1 strain as S. saprophyticus, S. kloosii, S. cohni subsp. urealyticum, and as S. capitis subsp. capitis. Among the ATCC strains, the following were used in the study: S. epidermidis ATCC 12228, S. saprophyticus ATCC 15305, S. hominis subsp. hominis ATCC 27844, S. haemolyticus ATCC 29970, S. capitis subsp. capitis ATCC 35661, S. warneri ATCC 49454, and S. lugdunensis ATCC 49567. 5 4. Discussion Biofilm status MP test∗1 (𝐴490 ± SD) CRA plate test∗∗2 Presence of icaA/icaD gene∗∗∗2 Biofilm-negative 0.07 ± 0.01 r ND Biofilm-positive 0.44 ± 0.08 b ND Biofilm-negative 0.11 ± 0.01 r ND Biofilm-negative 0.08 ± 0.02 r ND Biofilm-negative 0.10 ± 0.04 g ND Biofilm-positive 0.48 ± 0.06 r +/− Biofilm-positive 0.44 ± 0.10 r +/− Biofilm-positive 0.61 ± 0.03 r +/+ Biofilm-negative 0.07 ± 0.02 r −/+ Biofilm-negative 0.08 ± 0.01 r −/− Biofilm-negative 0.06 ± 0.01 r ND Biofilm-positive 0.19 ± 0.01 r +/− Biofilm-negative 0.13 ± 0.01 r +/+ Biofilm-negative 0.07 ± 0.02 g ND Biofilm-negative 0.08 ± 0.02 r −/− Biofilm-positive 0.27 ± 0.08 r −/− Biofilm-positive 0.47 ± 0.05 r +/− Biofilm-positive 0.53 ± 0.10 r +/− Biofilm-negative 0.09 ± 0.02 r −/− Biofilm-negative 0.07 ± 0.01 g −/− Biofilm-positive 0.45 ± 0.04 b +/+ Biofilm-negative 0.05 ± 0.00 r ND Biofilm-negative 0.10 ± 0.05 r −/− Biofilm-negative 0.11 ± 0.02 r ND Biofilm-positive 0.46 ± 0.01 r +/+ Biofilm-positive 0.55 ± 0.05 r +/+ Biofilm-negative 0.06 ± 0.01 r ND Biofilm-negative 0.06 ± 0.00 r −/+ Biofilm-negative 0.14 ± 0.03 r ND Biofilm-negative 0.10 ± 0.03 r ND Biofilm-negative 0.07 ± 0.02 r −/− Biofilm-negative 0.08 ± 0.03 r ND Biofilm-negative 0.10 ± 0.04 r ND Biofilm-negative 0.11 ± 0.02 r −/− Biofilm-positive 0.56 ± 0.11 b +/+ Biofilm-negative 0.09 ± 0.03 r ND Biofilm-negative 0.06 ± 0.01 r ND Biofilm-negative 0.08 ± 0.03 r ND Biofilm-negative 0.08 ± 0.01 r ND Biofilm-negative 0.08 ± 0.02 r ND Biofilm-negative 0.06 ± 0.01 r ND onies, b: black colonies, and g: grey colonies; d ND: not detection. .001; ∗∗2CRA plate test 𝑟= 0.13 (𝑃= 0.785); ∗∗∗2ica status 𝑟= 0.43 (𝑃= 0.015). Results w nparametric test; 2Rang Spearman test. yp g yp Strain number Bacterial species Biofilm status MP test∗1 (𝐴490 ± SD) CRA plate test∗∗2 Presence o icaA/icaD gene∗∗∗2 1 S. saprophyticus Biofilm-negative 0.07 ± 0.01 r ND 2 S. klosii Biofilm-positive 0.44 ± 0.08 b ND 4 S. cohnii subsp. urealyticum Biofilm-negative 0.11 ± 0.01 r ND 5 S. haemolyticus Biofilm-negative 0.08 ± 0.02 r ND 14 S. cohnii subsp. cohnii Biofilm-negative 0.10 ± 0.04 g ND 24 S. epidermidis Biofilm-positive 0.48 ± 0.06 r +/− 25 S. epidermidis Biofilm-positive 0.44 ± 0.10 r +/− 26 S. epidermidis Biofilm-positive 0.61 ± 0.03 r +/+ 27 S. icaA/D: (+) presence of the gene, ( ): absence of the gene, and ND: not detection. Correlation between biofilm status and ∗1MP test 𝑟= 𝑃< 0.001; ∗∗2CRA plate test 𝑟= 0.13 (𝑃= 0.785); ∗∗∗2ica status 𝑟= 0.43 (𝑃= 0.015). Results were considered significant when 𝑃< 0.05; 1𝑈Mann-Whitney nonparametric test; 2Rang Spearman test. 4. Discussion The mean concentration of 4-chlorophenylamino deriva- tive inhibiting biofilm formation for the examined strains of coagulase-negative staphylococci was 86.18 ± 30.64 𝜇g/mL, with a minimum of 40 𝜇g/mL and maximum of 128 𝜇g/mL. In the case of 4-fluorophenylamino derivatives the mean concentration inhibiting biofilm formation was higher and amounted to 237.09±160.57 𝜇g/mL, while the minimum and maximum values were 56 𝜇g/mL and 448 𝜇g/mL, respectively (𝑃< 0.001). Values of MBIC50 and MBIC90 for 4-chlo- rophenylamino derivative were 96 𝜇g/mL and 128 𝜇g/mL. In the case of 4-fluorophenylamino derivatives the MBIC50 Analysis of the susceptibility of eight S. aureus and CoNS strains forming biofilms with respect to azithromycin in a form of eye solution was conducted by Wu et al. [46]. The MIC values for eight examined strains ranged from 0.75 𝜇g/mL to >256 𝜇g/mL, and both strains were susceptible and resistant to azithromycin. Analyses of the susceptibility of biofilm forms of these strains to azithromycin in a form of eye 6 BioMed Research International 6 Table 1: Phenotypic and genotypic characterization data from 41 CoNS strains. d genotypic characterization data from 41 CoNS strains. icaA/D: (+) presence of the gene, (−): absence of the gene, and ND: not detection.i 1 2 CRA: type of growth on Congo Red Agar medium, r: red colonies, b: black colonies, and g: grey colonies; 4. Discussion epidermidis Biofilm-negative 0.07 ± 0.02 r −/+ 28 S. epidermidis Biofilm-negative 0.08 ± 0.01 r −/− 32 S. warneri Biofilm-negative 0.06 ± 0.01 r ND 33 S. epidermidis Biofilm-positive 0.19 ± 0.01 r +/− 53 S. epidermidis Biofilm-negative 0.13 ± 0.01 r +/+ 62 S. cohnii subsp. cohnii Biofilm-negative 0.07 ± 0.02 g ND 64 S. epidermidis Biofilm-negative 0.08 ± 0.02 r −/− 65 S. epidermidis Biofilm-positive 0.27 ± 0.08 r −/− 76 S. epidermidis Biofilm-positive 0.47 ± 0.05 r +/− 78 S. epidermidis Biofilm-positive 0.53 ± 0.10 r +/− 84 S. epidermidis Biofilm-negative 0.09 ± 0.02 r −/− 90 S. epidermidis Biofilm-negative 0.07 ± 0.01 g −/− 91 S. epidermidis Biofilm-positive 0.45 ± 0.04 b +/+ 93 S. haemolyticus Biofilm-negative 0.05 ± 0.00 r ND 95 S. epidermidis Biofilm-negative 0.10 ± 0.05 r −/− 96 S. haemolyticus Biofilm-negative 0.11 ± 0.02 r ND 97 S. epidermidis Biofilm-positive 0.46 ± 0.01 r +/+ 99 S. epidermidis Biofilm-positive 0.55 ± 0.05 r +/+ 101 S. haemolyticus Biofilm-negative 0.06 ± 0.01 r ND 102 S. epidermidis Biofilm-negative 0.06 ± 0.00 r −/+ 103 S. capitis subsp. capitis Biofilm-negative 0.14 ± 0.03 r ND 105 S. haemolyticus Biofilm-negative 0.10 ± 0.03 r ND 107 S. epidermidis Biofilm-negative 0.07 ± 0.02 r −/− 125 S. warneri Biofilm-negative 0.08 ± 0.03 r ND 139 S. warneri Biofilm-negative 0.10 ± 0.04 r ND 12228 S. epidermidis ATCC 12228 Biofilm-negative 0.11 ± 0.02 r −/− 35984 S. epidermidis ATCC 35984 Biofilm-positive 0.56 ± 0.11 b +/+ 15305 S. saprophyticus ATCC 15305 Biofilm-negative 0.09 ± 0.03 r ND 27844 S. hominis ATCC 27844 Biofilm-negative 0.06 ± 0.01 r ND 29970 S. haemolyticus ATCC 29970 Biofilm-negative 0.08 ± 0.03 r ND 35661 S. capitis subsp. capitis ATCC 35661 Biofilm-negative 0.08 ± 0.01 r ND 49454 S. warneri ATCC 49454 Biofilm-negative 0.08 ± 0.02 r ND Strain number Bacterial species 1 S. saprophyticus 2 S. klosii 4 S. cohnii subsp. urealyticum 5 S. haemolyticus 14 S. cohnii subsp. cohnii 24 S. epidermidis 25 S. epidermidis 26 S. epidermidis 27 S. epidermidis 28 S. epidermidis 32 S. warneri 33 S. epidermidis 53 S. epidermidis 62 S. cohnii subsp. cohnii 64 S. epidermidis 65 S. epidermidis 76 S. epidermidis 78 S. epidermidis 84 S. epidermidis 90 S. epidermidis 91 S. epidermidis 93 S. haemolyticus 95 S. epidermidis 96 S. haemolyticus 97 S. epidermidis 99 S. epidermidis 101 S. haemolyticus 102 S. g g Correlation between biofilm status and ∗1MP test 𝑟= 𝑃< 0.001; ∗∗2CRA plate test 𝑟= 0.13 (𝑃= 0.785); ∗∗∗2ica status 𝑟 considered significant when 𝑃< 0.05; 1𝑈Mann-Whitney nonparametric test; 2Rang Spearman test. 4. Discussion epidermidis 103 S. capitis subsp. capitis 105 S. haemolyticus 107 S. epidermidis 125 S. warneri 139 S. warneri 12228 S. epidermidis ATCC 12228 35984 S. epidermidis ATCC 35984 15305 S. saprophyticus ATCC 15305 27844 S. hominis ATCC 27844 29970 S. haemolyticus ATCC 29970 35661 S. capitis subsp. capitis ATCC 3566 49454 S. warneri ATCC 49454 49576 S. lugdunensis ATCC 49576 CRA: type of growth on Congo Red Agar medium, r: red icaA/D: (+) presence of the gene, (−): absence of the gen Correlation between biofilm status and ∗1MP test 𝑟= 𝑃 1 7 BioMed Research International BioMed Research International Table 2: Mean MIC values for CoNS strains subjected to an activity of 4-chlorophenylamino derivative and 4-fluorophenylamino derivative (𝜇g/mL). Bacterial species (number of strains) 4-Chlorophenylamino derivative MIC ± SD 4-Fluorophenylamino derivative MIC ± SD S. epidermidis (19) 43 ± 7.13 41 ± 6.41 S. haemolyticus (5) 42 ± 8.75 39 ± 20.65 S. warneri (3) 31 ± 8.83 43 ± 0.00 S. cohnii subsp. cohnii (2) 28 ± 4.62 36 ± 13.90 S. klosii (1) 48 ± 18.48 16 ± 0.00 S. cohnii subsp. urealyticum (1) 64 ± 0.00 56 ± 16.00 S. saprophyticus (1) 24 ± 9.24 32 ± 0.00 S. capitis subsp. capitis (1) 48 ± 18.48 48 ± 18.48 S. epidermidis ATCC 12228 28 ± 8.00 64 ± 0.00 S. epidermidis ATCC 35984 64 ± 0.00 48 ± 18.48 S. saprophyticus ATCC 15305 24 ± 9.24 56 ± 16.00 S. hominis ATCC 27844 64 ± 0.00 48 ± 18.48 S. haemolyticus ATCC 29970 64 ± 0.00 56 ± 16.00 S. capitis subsp. capitis ATCC 35661 48 ± 18.48 48 ± 18.48 S. warneri ATCC 49454 64 ± 0.00 48 ± 18.48 S. lugdunensis ATCC 49567 48 ± 18.48 64 ± 0.00 Table 3: Statistical analysis of obtained MIC values (𝜇g/mL) 4-chlorophenylamino derivative and 4-fluorophenylamino derivative with respect to CoNS strains depending on growth form. Biofilm-negative strains MIC ± SD (𝜇g/mL) 𝑛= 29 Biofilm-positive strains MIC ± SD (𝜇g/mL) 𝑛= 12 𝑃 4-Chlorophenylamino derivative 40.14 ± 15.73 49.09 ± 15.19 0.016 4-Fluorophenylamino derivative 45.52 ± 13.79 37.09 ± 14.43 0.018 Results were considered significant when 𝑃< 0.05; 𝑈Mann-Whitney nonparametric test. ed MIC values (𝜇g/mL) 4-chlorophenylamino derivative and 4-fluorophenylamino derivative with growth form. Table 4: Values of minimum biofilm inhibitory concentration (MBIC) of the examined strains. References [1] C. Vuong and M. Otto, “Staphylococcus epidermidis infections,” Microbes and Infection, vol. 4, no. 4, pp. 481–489, 2002. [2] J. W. Costerton, L. Montanaro, and C. R. Arciola, “Biofilm in implant infections: its production and regulation,” International Journal of Artificial Organs, vol. 28, no. 11, pp. 1062–1068, 2005. [3] E. Heikens, A. Fleer, A. Paauw, A. Florijn, and A. C. Fluit, “Comparison of genotypic and phenotypic methods for species- level identification of clinical isolates of coagulase-negative staphylococci,” Journal of Clinical Microbiology, vol. 43, no. 5, pp. 2286–2290, 2005. The molecular basis of the action of these new com- pounds is not completely understood, but they are thought to interfere with bacterial DNA synthesis. Structure-activity relationship studies revealed that the antimicrobial activity in this heterocyclic class of quinoline molecules depends on the nature of the peripheral substituents and their spatial relationship within the quinoline skeleton. This type of quinolines may inhibit DNA synthesis by promoting cleavage of bacterial DNA in the DNA-enzyme complexes of type II topoisomerase DNA gyrase and the topoisomerase IV, resulting in rapid bacterial death [32].h [4] J. Huebner and D. A. Goldmann, “Coagulase-negative staphy- lococci: role as pathogens,” Annual Review of Medicine, vol. 50, pp. 223–236, 1999. [5] F. 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MBIC values of thioacyl derivatives of 4aminoquinolinium salts against hospital isolates are being documented for the first time in this study which outlines a very sturdy basis for future investigations in pursuit to discover new anti-infectious agents. Further study is advised to elucidate the complex mode of action of newly synthetized compounds against biofilms of CoNS, particularly S. epider- midis and other clinically relevant microbes. [7] A. Wo´zniak-Kosek, J. Kosek, and J. Mierzejewski, “Biofilm as multicellular organism created by bacteria. Microbiologist and doctor’s perspective,” Military Pharmacy and Medicine, vol. 4. Discussion Bacterial species (number of strains) 4-Chlorophenylamino derivative MBIC ± SD (𝜇g/mL) 4-Fluorophenylamino derivative MBIC ± SD (𝜇g/mL) S. klosii (1) 44 ± 24.00 56 ± 16.00 S. epidermidis (9) 100 ± 27.91 283 ± 96.72 S. epidermidis ATCC 35984 128 ± 0.00 384 ± 147.80 Table 4: Values of minimum biofilm inhibitory concentration (MBIC) of the examined strains. cells in a biofilm is predominantly related to the mechanism of antibiotic activity.i solution (concentration of 0.25–1.0%) showed an inhibition of biofilm formation irrespective of the strains’ susceptibility or resistance to azithromycin. A significant variation in bactericidal activity between planktonic and biofilm forms of bacteria was also reported by El-Banna et al. [47]. Analyzing eight strains of staphy- lococci (three strains of S. aureus and five CoNS strains), they showed that, considering ciprofloxacin, the bactericidal value represented by the MIC was significantly different. For biofilm-forming strains, the MIC values were from 2- to 512- fold higher than those obtained for planktonic forms. These authors also obtained similar results by analyzing the activity of gentamicin towards the same bacterial strains. In the case of amoxicillin with clavulanic acid, the value of MBC for An analysis of various antibiotic (cefazolin, vancomycin, dicloxacillin, tetracycline, and rifampicin) activity mecha- nisms towards six strains of coagulase-negative staphylococci was conducted in turn by Cerca et al. [44]. When the target site of the antibiotic activity was the cell membrane, a reduction in biofilm formation was observed irrespective of the size of the antibiotic particle. Moreover, when the antibiotic target site was RNA or protein synthesis, a similar activity was noted for planktonic and biofilm forms. They concluded that phenotypical antibiotic resistance of bacterial BioMed Research International 8 8 biofilm forms was 4 to 64 times higher than the MBC results obtained for bacteria growing in planktonic form. discussed in the paper are completely disclosed (e.g., employ- ment, consultancies, honoraria, expert testimony, grants, and royalties). Comparing the minimum concentration inhibiting microorganism growth and the minimum concentration inhibiting biofilm formation for the examined CoNS strains, we demonstrated that in the case of 4-chlorophenylamino derivative the MIC values are only about 2-fold higher, and for 4-fluorophenylamino derivative they were approximately 6- to 10-fold higher (𝑃= 0.009). Therefore, both derivatives of the examined compounds demonstrate high activity with respect to strains growing in a biofilm, and a clear predominance is demonstrated for 4-chlorophenylamino derivative. References 3, no. 3, pp. 21–26, 2010. [8] P. S. Stewart and J. W. 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https://openalex.org/W2905701061	https://jneurodevdisorders.biomedcentral.com/track/pdf/10.1186/s11689-018-9259-4	English	null	The reach-to-grasp movement in infants later diagnosed with autism spectrum disorder: a high-risk sibling cohort study	Journal of neurodevelopmental disorders	2,018	cc-by	10,608	RESEARCH Open Access © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Although autism spectrum disorder (ASD) is characterized by impairments in social communication and the presence of repetitive behavior and/or restricted interests, there is evidence that motor impairments may be a contributing factor to the ASD phenotype. The purpose of this study was to examine the motor act of reaching-to-grasp in children at high risk (HR; with an older sibling diagnosed with ASD) and low-risk (LR; no family history of ASD) for ASD. Methods: Children were compared for differences in reaching-to-grasp based on sibling status and diagnostic outcome. Children were enrolled between 6 and 12 months of age and the reach-to-grasp movement was scored at 6, 9, (where available) 12, 15, 18, 24, and 36 months of age using the qualitative Skilled Reaching Rating Scale to determine the presence of any group-, age-, or sex-related differences in the mechanics of the reach-to-grasp movement using a Mixed Models analysis. At 36 months, all children underwent a gold-standard diagnostic assessment, which resulted in three outcome groups: HR children diagnosed with ASD (HR-ASD; n = 10), HR children not diagnosed with ASD (HR-N; n = 10), and low-risk children not diagnosed with ASD (LR; n = 10). Results: The group of children who were later diagnosed with ASD (HR-ASD group) showed higher (worse) total scores on the reach-to-grasp movement, as well as higher scores on the components of Orient, Lift, and Pronate compared to children in the LR and HR-N groups. Conclusions: Our results support the growing literature indicating that children who are later diagnosed with ASD show impaired early motor performance. These results highlight the importance of early surveillance of children who are at elevated risk for ASD, and early initiatives should focus on early signs of the phenotype, including both movement and sensory differences (prodromal signs) prior to the emergence of diagnostic characteristics. Keywords: Autism spectrum disorder, Autism, Reaching, Motor, Motor skills, Infant siblings, Baby siblings The reach-to-grasp movement in infants later diagnosed with autism spectrum disorder: a high-risk sibling cohort study Lori-Ann R. Sacrey1,2* , Lonnie Zwaigenbaum1,2, Susan Bryson3,6, Jessica Brian4,5 and Isabel M. Smith3,6 Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 https://doi.org/10.1186/s11689-018-9259-4 Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 https://doi.org/10.1186/s11689-018-9259-4 * Correspondence: sacrey@ualberta.ca 1Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada 2Autism Research Centre, Glenrose Rehabilitation Hospital, (E209) 10230 - 111 Avenue, Edmonton, Alberta T5G 0B7, Canada Full list of author information is available at the end of the article Background motor learning [1, 30, 31, 34, 77], and disturbances in the kinematics of reach-to-grasp (i.e., velocity; [47]), as well as the ability to perform a peg-board placement (reaching and manipulation) task [57]. Much of this work was conducted with children or adults who already had a diagnosis of ASD, and the quality of different compo- nents of the movement was not compared between groups. Growing evidence indicates that motor impairments appear in the first year of life [4, 43, 80, 87], preceding differences more directly related to ASD diagnostic criteria (reviewed by [88]). Autism spectrum disorder (ASD) is characterized by impairments in social communication and the presence of repetitive behavior and/or restricted interests [2]. There is evidence that motor impairments may be a contributing factor to the ASD phenotype. Many indi- viduals with ASD have gross and fine motor deficits [26, 29, 49, 50, 61, 86], including difficulty performing motor gestures [35, 51, 72], impairments in motor control and * Correspondence: sacrey@ualberta.ca 1Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada 2Autism Research Centre, Glenrose Rehabilitation Hospital, (E209) 10230 - 111 Avenue, Edmonton, Alberta T5G 0B7, Canada Full list of author information is available at the end of the article Prospective assessment of younger siblings of children with a diagnosis of ASD (“baby siblings”) who are at Participants Thirty infants from a longitudinal study of infant siblings of children with ASD (see [10, 89]) participated in the current study. The main study tracked HR infant siblings (each with an older biological sibling diagnosed with ASD) and LR controls (no family history of ASD) to document differences in early development potentially associated with ASD. All infants were enrolled between 6 and 12 months of age and underwent comprehensive assessments of communication, social, cognitive, and motor abilities at ages 6, 9 (where available), 12, 15, 18, 24, and 36 months. Infants in the present study were a subsample of participants from the Autism Research Centre at the Glenrose Rehabilitation Hospital (GRH), Edmonton, Alberta. Infants were selected at random to comprise three groups of equal size: (1) 10 non-sibling controls (LR-control; 7 boys); (2) 10 HR siblings without an ASD diagnosis (i.e., with an older sibling with ASD but did not receive an ASD diagnosis themselves at 36 months; HR-N; 3 boys); and (3) 10 HR siblings with an ASD diagnosis (i.e., with an older sibling with ASD and also received an ASD diagnosis at 36 months; HR-ASD; 6 boys). The HR siblings were recruited from families following assessment of the older sibling with ASD at the GRH. The diagnosis of the older sibling (or “proband”) was based on evaluation by a multi-disciplinary team and expert clinical review using DSM-IV-TR criteria, supported by a comprehensive developmental history and the Autism Diagnostic Observation Schedule (ADOS; [44]). The LR controls were recruited from the local community on the basis of having no first- or second-degree relatives with an ASD diagnosis. All participants were born at 36 to 42 weeks gestation, had a birth weight greater than 2500 g, and no known genetic or neurological disorders. Table 1 presents detailed participant characteristics. The local insti- tutional review board approved the research protocol and parents provided written informed consent. g The reach-to-grasp movement, in which a hand advances towards an object to grasp with the digits, is a natural act and is displayed in a variety of forms by developing infants and adults. Newborn infants will automatically grasp objects that have been placed in the hand [11, 81], 4-month-old infants begin to reach for distal objects [12, 24, 59], and 12-month-old infants display arm movements and hand grasps that approximate those of adults [22, 67, 79, 85]. Participants The topography of the reach-to- grasp movement has been standardized using healthy young and older adults [66], adults with Huntington’s disease [36], Parkinson’s disease [66, 68], and recovering from stroke [23]. Thus, reaching-to-grasp is a robust movement that can be used to study the development of skilled hand movement and its sensory control, beginning in infancy and continuing across the lifespan. Detailed description of the development of the reach-to-grasp movement beyond 12 months in typically developing infants is still warranted, in addition to potential devel- opmental differences in the movement in children at HR for ASD. The purpose of this study was to examine whether the reach-to-grasp movement is impaired in children at high risk (HR; with an older sibling diagnosed with ASD) for ASD, relative to controls. Infants were recruited from HR and low-risk (LR; no family history of ASD) families to compare potential differences in reaching-to-grasp based on sibling status and diagnostic outcome. The reach-to-grasp movement was recorded from two ASD assessments, the Autism Observation Scale for Infants (AOSI; ages 6, 9 (where available), 12, and 15 months; Assessments administered Several assessments were administered to track cognitive and ASD-specific characteristics over time; Table 1 pre- sents a summary of these. Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 Page 2 of 11 Page 2 of 11 Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 [10]) and the Autism Diagnostic Observation Schedule (ADOS; ages 18, 24, and 36 months; [44]). The movement was analyzed using the Skilled Reaching Rating Scale [67] to determine the presence of any group- or age-related differences in the mechanics of the reach-to-grasp movement. heightened risk for also being diagnosed with the dis- order (approximately 18%; [58]) provides an opportunity to explore ASD-related behavioral differences early in life. With respect to motor impairment, research using the baby sibling design has compared composite scores between high-risk children who were diagnosed versus not diagnosed with ASD [8, 19, 37, 41] and the attain- ment of gross or fine motor skills on developmental measures, such as the Mullen Scales of Early Learning [33, 55]. Results of these studies indicate that children who were later diagnosed with ASD had lower (i.e., poorer) overall composite scores on measures of motor skills/development compared to community controls and/ or other high-risk children who were not diagnosed with ASD. However, examining composite scores or the absence of specific motor behaviors only provides part of the picture—it is also important to understand whether the mechanics of motor behavior differ based on risk status. The reach-to-grasp movement provides an opportunity for a more detailed examination of motor development, in terms of movement quality and developmental timings for different components of the reach, and is also of func- tional significance. Diagnostic procedure At 3 years of age, each participant underwent an inde- pendent diagnostic evaluation, conducted by an expert clinician blind to assessments from previous study visits. ASD diagnoses were assigned using DSM-IV-TR criteria, based on the best judgment of the clinician (developmental pediatrician with over 10 years of diagnostic experience), considering information from all concurrent assessments. Because the majority of our sample was diagnosed prior to 2013 (when DSM-5 was published), we continued to use DSM-IV-TR criteria for diagnoses to remain consistent. Autism Observation Scale for Infants (AOSI; [10]) The AOSI is a semi-structured, observational measure designed to detect and monitor early signs of ASD in infants aged 6 to 18 months. The AOSI uses “presses” to elicit various target behaviors, including visual tracking and attention disengagement, coordination of eye gaze and action, imitation, affective responses, early social- Autism Diagnostic Observation Schedule (ADOS; [44]) Autism Diagnostic Observation Schedule (ADOS; [44]) The ADOS includes standardized activities and presses to elicit communication, social interaction, imaginative use of play materials, and repetitive behavior. Inter-rater reliability for the ADOS is excellent [44]. The scoring algorithm is organized into two domains, Social Affect (including Communication and Social items), and Restricted Repetitive Behaviors [28]. The ADOS consists of four mod- ules, each appropriate for individuals of differing language levels (module 1 = minimal or no language, module 2 = regular use of non-echoed three-word phrases, module 3 = child with fluent language, and module 4 = adolescent or adult with fluent language), the first three of which were used to assess participants in this study. To compare across modules (and thus, across language levels), we used the ADOS severity metric [27]. The ADOS was administered at 18, 24, and 36 months of age. LR low-risk, HR-N high-risk and not diagnosed with ASD, HR-ASD high-risk and diagnosed with ASD, N total sample, mos months, AOSI Autism Observation Scale for Infants, ADOS Autism Diagnostic Observation Schedule, ADI Autism Diagnostic Interview—Revised, MSEL ELC Mullen Scales of Early Learning Early Learning Composite ELC, VABS ABC Vineland Adaptive Behavior Scales Adaptive Behavior Composite ABC Autism Diagnostic Interview—Revised (ADI-R; [45]) g The ADI-R is an investigator-directed interview that elicits information regarding social development, verbal and non- verbal communication skills, and the presence of repetitive, stereotyped interests, and behavior required to make an ICD-10 or DSM-IV-TR diagnosis of ASD. The questions are designed to distinguish qualitative impairments from developmental delays. The ADI-R discriminates well between ASD and other forms of developmental disability, and inter-rater reliability is excellent [45]. The ADI-R was administered to parents when the children were at 36 months of age. Fine Motor) assess nonverbal, cognitive, and language ability, while the fifth scale measures gross motor develop- ment (from 0 to 29 months only). An Early Learning Composite is calculated based on scores from the first four scales for children aged 0–69 months. Inter-rater and test-retest reliability are excellent [54]. The MSEL was administered at 36 months of age. Fine Motor) assess nonverbal, cognitive, and language ability, while the fifth scale measures gross motor develop- ment (from 0 to 29 months only). An Early Learning Composite is calculated based on scores from the first four scales for children aged 0–69 months. Inter-rater and test-retest reliability are excellent [54]. The MSEL was administered at 36 months of age. Vineland Adaptive Behavior Scales I/II (Vineland; [74]) The Vineland is a semi-structured parent interview designed to assess daily living, communication, social, and motor functioning in everyday life, outlined by typical developmental milestones anchored to specific ages. The Vineland has excellent reliability and concurrent validity and is reported to be sensitive to impairments experienced by children with ASD [13, 83]. The Vineland was adminis- tered at 36 months of age. Mullen Scales of Early Learning (MSEL; [54]) Mullen Scales of Early Learning (MSEL; [54]) The MSEL consists of five scales, four of which (Visual Reception, Receptive Language, Expressive Language, and Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 Page 3 of 11 Table 1 Participant characteristics LR controls HR-N HR-ASD Number 10 10 10 Age first visit (mos) 6.94 ± 1.82 7.06 ± 1.29 8.56 ± 2.14 Age diagnostic visit (mos) 36.84 ± 1.13 37.23 ± 1.8 37.66 ± 1.98 Sex 7 boys; 3 girls 3 boys; 7 girls* 6 boys; 4 girlso AOSI total score 6 months 11.11 ± 4.13 10.38 ± 3.58 14.75 ± 2.50 9 months 6.89 ± 1.83 8.89 ± 3.55 7.22 ± 2.77 12 months 5.00 ± 2.05 7.60 ± 3.41 9.70 ± 5.21* 15 months 4.50 ± 3.72 8.40 ± 4.86 10.0 ± 5.16* ADOS severity score 18 months 7.70 ± 5.29 9.20 ± 5.40 14.80 ± 5.90o 24 months 5.50 ± 3.03 6.70 ± 4.90 14.90 ± 6.10o 36 months 4.20 ± 4.94 5.00 ± 3.50 17.30 ± 4.10o ADI–R total score 6.60 ± 2.76 9.10 ± 2.28 19.80 ± 10.80o MSEL ELC 123.60 ± 15.71 106.60 ± 11.31 96.44 ± 21.18* VABS ABC 102.20 ± 7.21 89.88 ± 12.03 75.83 ± 11.94* Values reported are means + standard deviations. Significant difference from LR control; oHR-N, ps < .0167. Note: reaches were scored at 6 and 9 months (where available) and at 12, 15, 18, 24, and 36 months LR low-risk, HR-N high-risk and not diagnosed with ASD, HR-ASD high-risk and diagnosed with ASD, N total sample, mos months, AOSI Autism Observation Scale for Infants, ADOS Autism Diagnostic Observation Schedule, ADI Autism Diagnostic Interview—Revised, MSEL ELC Mullen Scales of Early Learning Early Learning Composite ELC, VABS ABC Vineland Adaptive Behavior Scales Adaptive Behavior Composite ABC Table 1 Participant characteristics communicative behaviors, and behavioral reactivity. A “press” is either a verbal or non-verbal request with the goal of eliciting behavior in the child. For example, a press for a “social smile” would involve the assessor looking at the child and smiling, with the goal of elicit- ing a reciprocal smile from the child. The AOSI was administered at 6, 9, 12, and 15 months of age. Autism Diagnostic Observation Schedule (ADOS; [44]) Autism Diagnostic Observation Schedule (ADOS; [44]) Reach-to-grasp measures were coded from the ADOS at 18, 24, and 36 months of age using the Birthday Party routine. The Birthday Party routine occurs at the end of the ADOS (followed only by “snack”) and is approxi- mately 10 min. During this routine, the child sits at a table across from the examiner with hands and arms free to grasp and manipulate objects. Briefly, during the Birthday Party, the examiner “makes a cake” from play-dough and encourages the child to grasp and place candles in the cake. After singing “Happy Birthday,” the child is encouraged to feed cake and offer drinks to the birthday baby (i.e., the examiner says, “baby’s hungry” with the expectation that the child will “feed” the baby). The ADOS Birthday Party routine was chosen for scor- ing because (1) the child is encouraged to pick up small items (candles, fork, knife) for manipulation; (2) this is one of the longer manipulation sections of the ADOS, providing opportunities to gather grasps; and (3) the Birthday Party routine is included in the two ADOS modules used to assess 18- to 36-month-olds in this study. The movement is divided into five components: 1. Orient: participant moves the head and eyes in order to fixate the target visually prior to reach onset and visually disengage from the target at grasp. 2. Lift: hand is lifted and supinated towards the midline of the body as the digits close and semi-flex 3. Advance: hand is carried towards the target and stops above the target 4. Pronation: hand pronates over target item and digits shape to target size 1. Orient: participant moves the head and eyes in order to fixate the target visually prior to reach onset and visually disengage from the target at grasp. 2. Lift: hand is lifted and supinated towards the midline of the body as the digits close and semi-flex 4. Assessments scored for reach-to-grasp Reach-to-grasp was coded from the video recordings of the AOSI and the ADOS by an individual who was not Page 4 of 11 Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 Page 4 of 11 Sacrey et al. Journal of Neurodevelopmental Disorders involved in the assessments and was blind to sibling status and diagnostic outcomes. substrate, and make an overt eye movement to disengage from the target. Some infants did not perform five successful reaches in a given session (e.g., did not lift or manipulate an object). Table 2 displays the total number of reaches scored at each time-point, for each infant. Autism Diagnostic Observation Schedule (ADOS; [44]) Pronation: hand pronates over target item and digits shape to target size Table 2 Total number of reaches scored Table 2 Total number of reaches scored Age Assessment scored LR control HR-N HR-ASD Total 6 months AOSI 40 40 20 100 9 months AOSI 40 40 45 125 12 months AOSI 45 50 50 145 15 months AOSI 45 45 45 135 18 months ADOS 42 50 50 142 24 months ADOS 47 46 43 136 36 months ADOS 37 49 38 124 Total 296 320 291 907 LR low-risk, HR-N high-risk and not diagnosed with ASDASD, HR-ASD high-risk and diagnosed with ASD, AOSI Autism Observation Scale for Infants, ADOS Autism Diagnostic Observation Schedule. Note: reaches were scored at 6 and 9 months (where available) and at 12, 15, 18, 24, and 36 months Autism Observation Scale for Infants (AOSI; [10]) Reach-to-grasp measures were coded from the AOSI at 6, 9 (where available), 12, and 15 months of age using the two Free Play sections. These together are approxi- mately 10 min, with the first section occurring at the beginning and the second at the end of the AOSI. The two sections are separated by two tasks (peek-a-boo and imitation), lasting approximately 5 min. During the free play sessions, the child sits at a table across from the examiner, either seated in her/his parent’s lap or alone in a posture supportive chair, with hands and arms free to grasp and manipulate objects. Briefly, the examiner places a variety of graspable items on the table in front of the infant and encourages the child to reach for and grasp the items. The Free Play sections of the AOSI were chosen because (1) the child is encouraged to pick up small items (blocks, rings) for manipulation; (2) inter- ruptions by the examiner are minimal; and (3) the two sections constitute the majority of the AOSI, allowing ample opportunity to sample grasps sufficiently (see below). Infants reached for the same set of toys at each age. Reach-to-grasp coding g p g The Skilled Reaching Rating Scale is based on a descrip- tion of reaching derived from a conceptual framework using Eshkol-Wachman Movement Notation (EWMN; [18, 76, 77]). The topography of the reach-to-grasp movement has been standardized using healthy young and older adults [66], adults with Huntington’s disease [36], Parkinson’s disease [66, 68], and stroke [23]. Sacrey et al. [67] adapted the scoring system to assess its utility in infant development by comparing the absence/pres- ence of subcomponents of the movement compared to the typical adult construct. The results showed that the reach-to-grasp movement undergoes dramatic changes from 6 to 12 months of age, from an uncoordinated, immature movement at 6 months to an act that closely resemble the adult reach-to-grasp by 12 months. This study extends this research by examining the development of the reach-to-grasp movement beyond 12 months in typically developing infants, but also aims to explore potential developmental differences in the movement in children at HR for ASD. Reaches sampled The first five successful reach-to-grasp movements were sampled for each infant at each time point. To be in- cluded as a sampled reach, the infant had to make an overt eye movement towards the target, reach his/her hand towards the target, grasp it, lift the target from the LR low-risk, HR-N high-risk and not diagnosed with ASDASD, HR-ASD high-risk and diagnosed with ASD, AOSI Autism Observation Scale for Infants, ADOS Autism Diagnostic Observation Schedule. Note: reaches were scored at 6 and 9 months (where available) and at 12, 15, 18, 24, and 36 months Page 5 of 11 Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 Statistical analysis The first five successful reaches per infant per time point were included in the analysis. Given instances in which an infant made less than five successful reach and grasp move- ments, particularly for the HR-ASD group at 6 months, scores of each component of the reach for each infant were transformed into averages across trials. The Statistical Package for the Social Sciences (SPSS) v. 21 was used to run a Linear Mixed Model analysis with group (LR control, HR-N, HR-ASD), age (6, 9, 12, 15, 18, 24, and 36 months), and sex (boy, girl; due to the disproportionate number of boys in the HR-ASD and LR control groups) as the inde- pendent variables and reach-to-grasp components as the within-subjects measures. Benjamini and Hochberg [7] corrections were applied to all post hoc comparisons. This method controls the expected proportion of rejected null hypotheses that are false. The p values are ordered smallest to largest. The alpha level for each test is then set at kα m with k corresponding to the p value’s rank (e.g., lowest p value = 1) and m corresponding to the number of comparisons, which in this case was 21. The comparisons stop once one of the t tests is rejected and a resultant q value is generated. The q value is then used as the new “alpha” for post hoc comparisons. Effect sizes were calculated using Cohen’s d, with 0.2–0.49 = small effect, 0.5–0.79 = medium effect, and 0.8+ = large effect [14]. Table 3 Skilled Reaching Rating Scale Phase Component Sub- component Description Orient Orient A Eyes locate target prior to movement of head/reach B Eyes disengage target at tactile contact Transport Lift A Initial hand lift due to flexion of the elbow B Digits semi-flex C Hand supinates approximately 30 degrees D Tips of digits are brought towards the midline of the body Advance A Hand takes shortest path to target B Hand stops directly above the target C Trunk leans to the side opposite the reach Pronation A Digits open and extend over the target B Knuckle on reaching hand form horizontal line C Elbow opens to full arm length as participant reaches Grasp Grasp A Thumb and index finger/proper grasp for target size/shape B Wrist extends to lift target from platform Procedure Video recordings of AOSI administration at 6, 9 (where available), 12, and 15 months of age and of the ADOS at 18, 24, and 36 months of age were collected and given to the coder, who was blind to both sibling status and diag- nostic outcome until all reaches were scored. The children were scored for reach-to-grasp measures at each time point in chronological order; that is, records were scored for one child at a time until all were scored, and each infant’s videos were scored at 6 months, then 9, through to 36 months. The scores from each time point were stored in a binder and were not referenced during the following scoring in an attempt to minimize the influence of the previous scoring on subsequent time points. After all records were scored at each of the seven time points, participants were placed into groups based on sibling status and 36-month diagnostic outcomes. The five components are further divided into 14 sub- components (for a complete description, see Table 3). The developmental trajectory of reaching was scored based on infant performance relative to healthy adult performance. For rating, a score of “0” was given if the movement was present and resembled the adult con- struct, a score of “0.5” was given if the movement was present, but differed from the adult construct, and a score of “1” was given if the movement was absent. Thus, lower scores reflect better movement quality rela- tive to that of adults [84]. The first study to standardize reaching-to-grasp for humans reported no significant difference between scores from four different raters [84]. The blind coder established reliability on a standard set of infant reach-to-grasp reliability videos (a total of 168 reaches). Inter-rater reliability was assessed using Pearson’s r, resulting in r = 0.916, p < 0.002, suggesting high reliability. For a detailed description of the video recording procedure, EWMN, and movement onset and offset definitions, see Additional file 1. Total score As displayed in Fig. 1, significant effects were seen for group (F(2,144) = 7.66, p = .001), age (F(6, 144 = 72.72, p < .001), and sex (F(1, 144) = 5.41, p = .02), but no sig- nificant interactions emerged (ps > .05). GPower was used to calculate estimated power for the overall group effect, resulting in a value of 0.74. Post hoc analyses showed that children in the HR-ASD group had higher (worse) reach-to-grasp scores compared to children in the HR-N and LR groups (qs < .033; d = .48, d = .74, respectively), who did not differ (d = .26). Post hoc analyses on the age effect revealed improvements in the overall reach-to-grasp score for all time point comparisons at 6, 9, and 12 months, suggesting overall improvement in reaching in the first year of life, which plateaus for each successive time point (qs < .038). The sex effect showed that boys (mean = 4.35) had significantly lower (better) overall reach-to-grasp scores than girls (mean = 4.87; p < .02; d = 1.96). Participant characteristics Groups were compared on cognitive/developmental (MSEL), adaptive behavior (VABS), and ASD symptom (AOSI, ADOS, and ADI-R) scores. As shown in Table 1, children who were diagnosed with ASD differed from the LR controls and HR-N groups on these measures. Ages at first visit and diagnostic visit were compared. As Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 Page 6 of 11 Fig. 1 Main effect for reach-to-grasp score. LR, low-risk controls; HR- N, high-risk siblings without autism spectrum disorder; HR-ASD, high-risk siblings with autism spectrum disorder. p < .05; p <. 01 shown in Table 1, groups did not differ in age at initial assessment (F(2,27) = 2.43, p > 0.05) or diagnostic assess- ment (F(2,27) = 0.59, p > 0.05). More girls were in the HR-N group compared to the LR control group and the HR-ASD groups (χ2 = 31.08, p < .001; χ2 = 25.47.08, p < .001, respect- ively), who did not differ (χ2 = .50, p = .49). Reach-to-grasp movement The group means, standard deviations, and 95% confi- dence intervals for the components, sub-components, and total reach-to-grasp scores are provided in Table 4. Fig. 1 Main effect for reach-to-grasp score. LR, low-risk controls; HR- N, high-risk siblings without autism spectrum disorder; HR-ASD, high-risk siblings with autism spectrum disorder. p < .05; *p <. 01 Discussion The present study examined mechanical differences between the reach-to-grasp movements of infants and toddlers who were at high and low risk for ASD. Children performed the movement at 6, 9 (where available), 12, 15, 18, 24, and 36 months of age and the movement was scored using the qualitative Skilled Reaching Rating Scale [84]. Children who were later diagnosed with ASD showed higher (worse) total scores on the reach-to-grasp move- ment, as well as higher scores on the components of Orient, Lift, and Pronate compared to children in the LR and HR-N groups. These results suggest that such move- ment mechanics are relevant to monitoring motor devel- opment in children at risk for or diagnosed with ASD. Lift. Significant effects were obtained for group (F(2, 144) = 6.51, p = .002) and age (F(6, 144) = 46.60, p < .001), but not sex (F(1, 144) = .3.66, p = .06) or interaction effects (ps > .05). Post hoc analyses on the group effect showed that children in the HR-ASD group had a higher Lift score than the LR-control group (q < .017). Children in the HR-N group did not differ from the other two groups. Post hoc analyses on the age effect showed that Lift improved for all time-point comparisons at 6, 9, and 12 months and plateaued for each successive time point (qs < .036). Our study of the reach-to-grasp movement is, to our knowledge, the first to examine the quality of various components of the movement prior to the diagnosis of ASD. Much of the research examining reach-to-grasp movements in ASD has either been completed with chil- dren or adults who already have a diagnosis or employed kinematics (velocity/acceleration) to study potential group differences [16, 25, 47, 75, 82]. Nevertheless, our results are consistent with previous reports, indicating these movements are less well-coordinated in children with ASD compared to age-matched typically developing con- trols [20, 31]. The emerging reach-to-grasp movements of children in our study who were later diagnosed with ASD appeared less well-coordinated than the more adult-like movements of LR controls and also differed from those of HR children who were not diagnosed with ASD by age 3. Advance. A significant effect was seen for age (F(6, 144) = 67.27, p < .001), but not group (F(2, 144) = 2.41, p = .09), sex (F(1, 144) = .1.25, p = .27), or interaction effects (ps > .05). Discussion Post hoc analyses on the age effect showed that Advance improved for all time-point com- parisons at 6, 9, and 12 months and plateaued for each successive time point (qs < .036). Pronate. Significant effects were obtained for group (F(2, 144) = 6.17, p = .003), age (F(6, 144) = 33.14, p < .001), and sex (F(1, 144) = 10.77, p = .001), with no significant interactions (ps > .05). Post hoc analyses on the group effect showed that children in the HR-ASD group had a higher (worse) Pronate score compared to children in the LR and HR-N groups (qs < .033; d = .66; d = .41, respectively), who did not differ (d = .21). Post hoc analyses on the age effect showed that Pronate improved for all time point compari- sons at 6, 9, and 12 months and plateaued for each succes- sive time point thereafter (qs < .036). The results of the sex effect showed that boys (mean = .83) had lower (better) Pronate scores than girls (mean = 1.05; p = .001; d = 2.75). Previous research examining motor impairments, specifically reaching and grasping, which identified group differences often had only LR comparison groups [20, 25, 47, 75]. Additional comparison groups were children with various developmental disabilities (DD; [16, 31]) or devel- opmental coordination disorder (DCD; [82]). Results from these studies are mixed, some finding differences between ASD and DD or DCD, and others not. This is problematic, as other control groups (e.g., developmental delay, HR- non-ASD siblings, in addition to LR controls) are neces- sary to separate putative ASD-specific impairments from more general delays [71]. Grasp. A significant effect was seen for age (F(6, 144) = 18.59, p < .001), but not group (F(2, 144) = .61, p = .55), sex (F(1, 144) = 1.39, p = .24), or interaction effects (ps > .05). Post hoc analyses on the age effect showed that Grasp improvements began between 9 and 12 months and plateaued by age 15 months (qs < .029). Children with ASD show impairments in the disengage subcomponent of orienting. This finding is consistent with the literature, noting the presence of “sticky attention” in children who are diagnosed, or HR infant siblings who are later diagnosed, with ASD (reviewed in [64]). Component analyses Group comparisons were made on components to deter- mine if different aspects of the reach-to-grasp movement differed between children with ASD and the other two groups. Overall, children with ASD showed differences in Table 4 Group means, standard deviations, and 95% CI for the reach-to-grasp measures Measure LR HR-N HR-ASD Orient .65 ± .26 (.56–.75) .65 ± .26 (.56–.75) .81 ± .26 (.72–.91) Engage .20 ± .14 (.15–.25) .24 ± .15 (.19–.29) .20 ± .13 (.16–.25) Disengage .41 ± .17 (.35–.47) .51 ± .18 (.44–.57) .51 ± .16 (.45–.57) LIFT 1.03 ± .50 (1.12–1.48) 1.39 ± .54 (1.19–1.58) 1.73 ± .48 (1.56–1.90) Elbow flex .10 ± .11 (.06–.13) .13 ± .12 (.09–.17) .18 ± .10 (.14–.21) Digits flex .40 ± .18 (.34–.47) .44 ± .19 (.37–.51) .53 ± .17 (.47–.59) Supinate .36 ± .17 (.30–.42) .34 ± .17 (.27–.41) .46 ± .17 (.40–.52) Tips to midline .44 ± .18 (.38–.51) .48 ± .19 (.41–.55) .57 ± .17 (.51–.63) Advance .45 ± .21 (.38–.53) .49 ± .22 (.41–.57) .56 ± .20 (.49–.64) Hand path .25 ± .13 (.20–.29) .28 ± .14 (.23–.33) .28 ± .13 (.24–.33) Hand to target .20 ± .12 (.15–.24) .21 ± .12 (.16–.25) .26 ± .11 (.22–.30) Trunk lean .01 ± .05 (.00–.03) .00 ± .05 (.00–.02) .02 ± .05 (.01–.04) Pronation .82 ± .32 (.70–.93) .91 ± .35 (.78–1.03) 1.10 ± .31 (.99–1.21) Digits open .44 ± .20 (.37–.52) .51 ± .22 (.43–.59) .59 ± .19 (.52–.66) Knuckle .37 ± .16 (.32–.43) .40 ± .17 (.33–.46) .51 ± .15 (.45–.56) Elbow extend .001 ± .02 (.0–.01) .01 ± .01 (.00–.01) .00 ± .01 (.00–.01) Grasp .93 ± .23 (.85–1.01) .89 ± .25 (.81–.98) .96 ± .22 (.88–1.04) Appropriate grasp .23 ± .14 (.18–.28) .24 ± .15 (.18–.29) .25 ± .13 (.20–.30) Wrist extend .70 ± .19 (.63–.76) .66 ± .20 (.59–.73) .71 ± .18 (.65–.76) Reach-to-grasp 4.15 ± 1.04 (3.78–4.53) 4.52 ± 1.12 (4.11–4.92) 5.17 ± 0.99 (4.81–5.53) Table 4 Group means, standard deviations, and 95% CI for the reach-to-grasp measures Measure LR HR-N Page 7 of 11 Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 supinate, and tips to midline of Lift, and digits open, knuckle, and elbow of Pronation. the Orient, Lift, and Pronate components. These results are described below. Orient. Component analyses Significant effects were seen for group (F(2,144) = 4.08, p = .019) and age (F(6, 144) = 22.69, p < .001), but not sex (F(1, 144) = 1.97, p = .16), nor significant interactions (ps > .05). Post hoc analyses on the group effect showed that children in the LR group had a lower (better) Orient score compared to children in the HR-N and HR-ASD groups (qs < .033; d = .51; d = .47), who did not differ (d = .05). Post hoc analyses on the age effect showed that im- provements in Orient began between 9 and 12 months and continued to age 3 (qs < .043). Discussion Delays in disengagement may affect joint attention, spontaneous gaze to faces, orienting to name, and making eye contact, which all involve orienting visual attention to biologically relevant information in the environment [38, 39, 58, 89]. Sub-component analysis Future research could link behavioral and neu- roimaging data to determine whether impairments in the reach-to-grasp movement are associated with specific brain areas/abnormalities. Furthermore, sticky attention may combine with motor impairments to further impact an individual’s ability to participate in collaborative activities (i.e., delays in responding to visual information in addition to motor deficits). Our finding here, combined with previous re- search suggesting impaired disengagement of attention is apparent at 12 months of age [9, 65], can distinguish between HR siblings who later receive an ASD diagno- sis [9, 17, 65], and can distinguish between ASD and Down syndrome [39], suggests that “sticky attention” can be a marker in the early identification of children at risk for ASD. Motor ability is linked to cognitive outcomes in indi- viduals with ASD [3, 60, 73]. This relationship may be mediated by the effects of motor impairments on oppor- tunities for learning through everyday experience. As an illustration, consider the motor milestone of independ- ent sitting. If a child is delayed in achieving independent sitting and therefore spends a large portion of his/her time on the tummy, this reduces opportunities to use the hands to engage in reaching and grasping for objects, showing objects to caregivers, and even requesting objects. As a consequence, the onset of these “social” behaviors may in turn be delayed. This is supported by a 2011 study from Libertus and Needham, who found that typically de- veloping babies who engage in active reaching movements also show spontaneous orienting to faces. This is in con- trast to babies who watch others play with objects, having not yet achieved active reaching skills, and show less spon- taneous orienting to faces. Like sitting, reaching-to-grasp is typically acquired in the first year [67] and disruptions may have further implications for more sophisticated object exploration (e.g., [63]). Reaching provides infants with the opportunity to play with toys spontaneously, and the ability to grasp toys and refine the grasp to adjust to the shape or Neuroimaging research has identified several cortical and subcortical abnormalities in areas known to be involved in motor behavior [5, 6, 15, 48, 52, 56, 62]. For example, cere- bellar abnormalities have been identified in individuals with ASD (reviewed in [21]). Sub-component analysis The results of the sub-component analyses are summarized in Table 5. Briefly, eight sub-components showed group differences: disengage of Orient, elbow flex, digits flex, Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 Page 8 of 11 Table 5 Sub-component analysis results Component Sub-component Group Age Sex Interaction effects Post hoc groupO F p F p F p Orient A. Engage .63 .53 5.33 .001 2.27 .13 No NA B. Disengage 3.38 .04* 18.65 .001* 1.12 .29 No a < b, c Lift A. Elbow flex 4.61 .01* 44.56 .001* .62 .43 No a < c B. Digits flex 4.33 .02* 23.79 .001* 2.57 .11 No a < c C. Supinate 3.77 .03* 34.01 .001* 6.53 .01^ No b < c D. Tips to midline 3.91 .02 24.12 .001* 2.88 .09 No a < c Advance A. Hand path .86 .43 37.47 .001* 1.76 .19 No NA B. Hand to target 2.53 .08 56.89 .001* .001 .98 Yes1 NA C. Trunk lean 1.58 .21 1.04 .40 1.41 .24 No NA Pronation A. Digits open 4.02 .02* 15.87 .001* 8.71 .004^ No a < c B. Knuckle 6.57 .002 43.81 .001* 9.24 .003^ No c > a, b C. Elbow extend 3.39 .04 4.17 .001* 5.65 .019^ Yes1234 b < c Grasp A. Appropriate grasp .13 .88 13.03 .001 .11 .75 Yes1 NA B. Wrist extenda .60 .55 10.51 .001* 2.95 .09 No NA a LR control; b HR-N, c HR-ASD, NA not applicable; *Significantly different at p < .05, OBenjamini and Hochberg corrected p value 1Age × sex interaction 2Group × sex interaction 3Age × sex interaction 4Group × age × sex interaction ^Boys < girls Furthermore, sticky attention may combine with motor impairments to further impact an individual’s ability to participate in collaborative activities (i.e., delays in responding to visual information in addition to motor deficits). Our finding here, combined with previous re- search suggesting impaired disengagement of attention is apparent at 12 months of age [9, 65], can distinguish between HR siblings who later receive an ASD diagno- sis [9, 17, 65], and can distinguish between ASD and Down syndrome [39], suggests that “sticky attention” can be a marker in the early identification of children at risk for ASD. motor planning and execution in individuals with ASD [70, 75]. Sub-component analysis The cerebellum, a key structure required to form internal models of motor actions, shares reciprocal connections with motor cortices to carry out on- line corrections during movement execution [53, 78], and these abnormalities likely contribute to the impaired online movement correction seen in ASD. Abnormal connectivity between adjacent primary sensory and motor cortices has also been recorded in ASD [70, 75] and may also contribute to the impairments seen during the online control of movement [46]. Of note here, reduced inter-connectivity between distal areas of the motor system, such as visual and motor regions subserving action, may lead to impaired Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 Page 9 of 11 Page 9 of 11 Page 9 of 11 Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 Sacrey et al. Journal of Neurodevelopmental Disorders (2018) 10:41 features of the toy allows the child to maintain control while engaging in exploratory behavior. As such, difficulties with reaching and grasping may constrain these interac- tions and limit the infant’s ability to explore objects effect- ively [33]. Furthermore, infants impaired in their ability to grasp and manipulate objects often also show delays in speech onset and development (reviewed in [32]), suggest- ing that the ability to reach for and grasp objects may have cascading effects on other emerging skills. frame-by-frame analyses. Our smaller sample is similar to that of other papers analyzing reaching movements in ASD (e.g., [47, 57]); yet, those papers only tested their participants at one time point whereas our participants performed reaching movements over multiple time points between 6 (where available) and 36 months of age. Importantly, a large number of reaches were scored (approximately 300 per group) and the power analyses indicated that our study could detect small-to-medium effects as statistically significant because of multiple sampling per participant at each assessment and over time. How do motor impairments relate to social-commu- nicative impairments? A typically developing child can use a full movement repertoire to engage in social in- teractions. Yet, many children with ASD have impaired motor behavior, detectable as early as 3 months of age [8]. As proposed by Leary and Hill [40], motor ability might have a significant impact on the core characteristics of ASD. Specifically, when a person is unable to respond to another’s action in a timely fashion, he/she will miss the positive reinforcement associated with interpersonal inter- actions. Funding This project was supported by Canadian Institutes of Health Research (CIHR), Alberta Innovates – Health Solutions (AI-HS), the Stollery Children’s Hospital Foundation, and Kids Brain Health Network (formerly NeuroDevNet). The funding agencies did not provide feedback on the study design, analysis, or write up of the report. Abbreviations ADI-R: Autism Diagnostic Interview—Revised; ADOS: Autism Diagnostic Observation Schedule; AOSI: Autism Observation Scale for Infants; ASD: Autism spectrum disorder; DCD: Developmental coordination disorder; DD: Developmental delay; EWMN: Eshkol-Wachman movement notation; HR: High-risk; HR-ASD: High-risk sibling with autism spectrum disorder; HR-N: High-risk sibling without autism spectrum disorder; LR: Low-risk; MSEL: Mullen Scales of Early Learning; SPSS: Statistical Package for the Social Sciences Sub-component analysis As well, the child’s suboptimal response may negatively affect their relationship with that person (espe- cially with a peer), reducing the likelihood of subsequent positive interactions. Such consequences may be overt during an interaction centered on motor activities (e.g., team sports such as baseball, which centers on cooperative motor tasks such as catching and throwing a ball), but may also occur during a broader range of social interac- tions that involve responding to another’s actions. Thus, experiences throughout development may be drastically altered if, at an early age, a child is unable to remain in- volved in social interaction and, as a result, may withdraw from social activities [40]. This “motor cognition” perspec- tive does not imply that social-communicative impairments directly result from motor impairments, but rather that impaired movements may interfere with opportunities for positive social experiences and thus social learning. Conversely, reduced social interaction opportunities may also contribute to poor action un- derstanding. Thus, the relationship between social and motor competencies/impairments may be reciprocal in ASD, a hypothesis that remains to be explored in future longitudinal research. Additional file Additional file 1: Table S1. Comparison of Main Effects for including all reaches completed between 6 and 36 months versus reaches completed only between 12 and 36 months. (DOCX 22 kb) Acknowledgements This study was supported by Canadian Institute of Health Research (CIHR), Alberta Innovates – Health Solutions (AI-HS), the Stollery Children’s Hospital Foundation, and Kids Brain Health Network (formerly NeuroDevNet). The authors thank all the families who participated in the Canadian Infant Siblings Study and members of the Canadian Infant Siblings Study Team. Although our results add to the recent literature of motor impairments in children with ASD, it is not without limita- tions. First, the number of children in the HR-ASD group is low at the earliest age examined (6 months), due to later enrollment in the study, reducing the number of codable reaches. Nevertheless, when we directly compared results inclusive of all data versus data from 12 to 36 months, the pattern of significance was identical, suggesting the overall group results are quite robust (see Additional file 1: Table S1). Second, we acknowledge that the data pre- sented in this paper are based on a relatively small sample (10 participants per group) coded using labor-intensive Conclusion Our results support the growing literature indicating that children who are later diagnosed with ASD show impaired early motor performance. Results such as these highlight the importance of early surveillance of children who are at elevated risk for the disorder. 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Author details 1 1Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada. 2Autism Research Centre, Glenrose Rehabilitation Hospital, (E209) 10230 - 111 Avenue, Edmonton, Alberta T5G 0B7, Canada. 3Dalhousie University/IWK Health Centre, Halifax, Nova Scotia, Canada. 4Bloorview Research Institute, Toronto, Ontario, Canada. 5University of Toronto, Toronto, Ontario, Canada. 1Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada. 23. Foroud A, Whishaw IQ. Reaching-to-eat in humans post-stroke: fluctuating components within a constant pattern. Behav Neurosci. 2010;124(6):851–67. 2Autism Research Centre, Glenrose Rehabilitation Hospital, (E209) 10230 - 111 Avenue, Edmonton, Alberta T5G 0B7, Canada. 3Dalhousie University/IWK Health Centre, Halifax, Nova Scotia, Canada. 4Bloorview Research Institute, 2Autism Research Centre, Glenrose Rehabilitation Hospital, (E209) 10230 - 111 Avenue, Edmonton, Alberta T5G 0B7, Canada. 3Dalhousie University/IWK Health Centre, Halifax, Nova Scotia, Canada. 4Bloorview Research Institute, 24. Foroud A, Whishaw IQ. The consummatory origins of visually guided reaching in human infants: a dynamic integration of whole-body and upper-limb movements. Behav Brain Res. 2012;231:343–55. Toronto, Ontario, Canada. 5University of Toronto, Toronto, Ontario, Canada. 6Autism Research Centre, IWK Health Centre, Halifax, Nova Scotia, Canada. Toronto, Ontario, Canada. 5University of Toronto, Toronto, Ontario, Canada. 6Autism Research Centre, IWK Health Centre, Halifax, Nova Scotia, Canada. 25. 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https://openalex.org/W4289552558	https://infoscience.epfl.ch/record/271321/files/s10703-019-00338-9.pdf	English	null	Synthesizing Adaptive Test Strategies from Temporal Logic Specifications	arXiv (Cornell University)	2,018	cc-by	20,800	Formal Methods in System Design (2019) 55:103–135 https://doi.org/10.1007/s10703-019-00338-9 Formal Methods in System Design (2019) 55:103–135 https://doi.org/10.1007/s10703-019-00338-9 Synthesizing adaptive test strategies from temporal logic speciﬁcations Roderick Bloem1 · Goerschwin Fey2,3 · Fabian Greif3 · Robert Könighofer1 · Ingo Pill1 · Heinz Riener3,4 · Franz Röck1 Roderick Bloem1 · Goerschwin Fey2,3 · Fabian Greif3 · Robert Könighofer1 · Ingo Pill1 · Heinz Riener3,4 · Franz Röck1 Roderick Bloem1 · Goerschwin Fey2,3 · Fabian Greif3 · Robert Könighofer1 · Ingo Pill1 · Heinz Riener3,4 · Franz Röck1 Published online: 14 October 2019 © The Author(s) 2019 Published online: 14 October 2019 © The Author(s) 2019 Abstract Constructing good test cases is difﬁcult and time-consuming, especially if the system under test is still under development and its exact behavior is not yet ﬁxed. We propose a new approach to compute test strategies for reactive systems from a given temporal logic speciﬁ- cation using formal methods. The computed strategies are guaranteed to reveal certain simple faults in every realization of the speciﬁcation and for every behavior of the uncontrollable part of the system’s environment. The proposed approach supports different assumptions on occurrences of faults (ranging from a single transient fault to a persistent fault) and by default aims at unveiling the weakest one. We argue that such tests are also sensitive for more complex bugs. Since the speciﬁcation may not deﬁne the system behavior completely, we use reactive synthesis algorithms with partial information. The computed strategies are adaptive test strategies that react to behavior at runtime. We work out the underlying theory of adaptive test strategy synthesis and present experiments for a safety-critical component of a real-world satellite system. We demonstrate that our approach can be applied to industrial B Franz Röck franz.roeck@iaik.tugraz.at Roderick Bloem roderick.bloem@tugraz.at Goerschwin Fey goerschwin.fey@tuhh.de Fabian Greif fabian.greif@dlr.de Robert Könighofer robert.koenighofer@iaik.tugraz.at Ingo Pill ipill@ist.tugraz.at Heinz Riener heinz.riener@epﬂ.ch 1 Graz University of Technology, Graz, Austria 2 Hamburg University of Technology, Hamburg, Germany 3 German Aerospace Center, Bremen, Germany 4 EPFL, Lausanne, Switzerland 123 B Franz Röck franz.roeck@iaik.tugraz.at Roderick Bloem roderick.bloem@tugraz.at Goerschwin Fey goerschwin.fey@tuhh.de Fabian Greif fabian.greif@dlr.de Robert Könighofer robert.koenighofer@iaik.tugraz.at Ingo Pill ipill@ist.tugraz.at Heinz Riener heinz.riener@epﬂ.ch 1 Graz University of Technology, Graz, Austria 2 Hamburg University of Technology, Hamburg, Germany 3 German Aerospace Center, Bremen, Germany 4 EPFL, Lausanne, Switzerland B Franz Röck franz.roeck@iaik.tugraz.at Roderick Bloem roderick.bloem@tugraz.at Goerschwin Fey goerschwin.fey@tuhh.de Fabian Greif fabian.greif@dlr.de Robert Könighofer robert.koenighofer@iaik.tugraz.at Ingo Pill ipill@ist.tugraz.at Heinz Riener heinz.riener@epﬂ.ch 1 Graz University of Technology, Graz, Au 2 Hamburg University of Technology, Ham 3 German Aerospace Center, Bremen, Ger 4 EPFL, Lausanne, Switzerland 1 Introduction Model checking [12,48] is an algorithmic approach to prove that a model of a system adheres to its speciﬁcation. However, model checking cannot always be applied effectively to obtain conﬁdence in the correctness of a system. Possible reasons include scalability issues, third- party IP components for which no code or detailed model is available, or a high effort for building system models that are sufﬁciently precise. Moreover, model checking cannot verify the ﬁnal and “live” product but only an (abstracted) model. Testing is a natural alternative to complement formal methods like model checking, and automatic test case generation helps keeping the effort acceptable. Black-box testing tech- niques, where tests are derived from a speciﬁcation rather than the implementation, are particularly attractive: ﬁrst, tests can be computed before the implementation phase starts, and thus guide the development. Second, the same tests can be reused across different realizations of a given speciﬁcation. Third, a speciﬁcation is usually much simpler than its implemen- tation, which gives a scalability advantage. At the same time, the speciﬁcation focuses on critical functional aspects that require thorough testing. Fault-based techniques [29] are par- ticularly appealing, where the computed tests are guaranteed to reveal all faults in a certain fault class—after all, the foremost goal in testing is to detect bugs. Methods to derive tests from declarative requirements (see, e.g., [25]) are sparse. One issue in this setting is controllability: the requirements leave plenty of implementation freedom, so they cannot be used to fully predict the system behavior for all given inputs. Consequently, test cases have to be adaptive, i.e., able to react to observed behavior at runtime, rather than being ﬁxed input sequences. This is particularly true for reactive systems that continuously interact with their environment. Existing methods often work around this complication by requiring a deterministic system model as additional input [24]. Even a probabilistic model ﬁxes the behavior in a way not necessarily required by the speciﬁcation. In previous work, we presented a fault-based approach to compute adaptive test strategies for reactive systems [10]. This approach generates tests that enforce certain coverage goals for every implementation of a provided speciﬁcation. The generated tests can be used across realizations of the speciﬁcation that differ not only in implementation details but also in their observable behavior. 123 104 Formal Methods in System Design (2019) 55:103–135 speciﬁcations and that the synthesized test strategies are capable of detecting bugs that are hard to detect with random testing. Keywords Automatic test case generation · System testing · Speciﬁcation testing · Adaptive tests · Synthesis · Reactive systems · Mutation testing 123 1 Introduction This is, e.g., useful for standards and protocols that are implemented by multiple vendors or for systems under development, where the exact behavior is not yet ﬁxed. Figure 1 outlines the assumed testing setup and shows how the approach for synthesizing adaptive test strategies (illustrated in black) can be integrated in an existing testing ﬂow. The user provides a speciﬁcation ϕ, which describes the requirements of the system under test (SUT) and additionally a fault model δ, which deﬁnes the coverage goal in terms of a class of faults for which the tests shall cause a speciﬁcation violation. Both the speciﬁcation and the coverage goal are expressed in Linear Temporal Logic (LTL) [46]. By default, our approach supports the detection of transient and permanent faults and distinguishes four fault occurrence frequencies: faults that occur at least (1) once, (2) repeatedly, (3) from some point 123 123 Formal Methods in System Design (2019) 55:103–135 105 Speciﬁcaon Fault Model Synthesis Oracle Synthesis Adapve Test Strategy System Under Test (SUT) Oracle input output Test case generaon Test case execuon Input Verdict Pass/ Fail/ Inconcl. Fig. 1 Testing setup: this paper focuses on test strategy synthesis Fig. 1 Testing setup: this paper focuses on test strategy synthesis on, or (4) permanently. Besides the four default fault occurrence frequencies, a user can also provide a custom frequency using LTL. Our approach then automatically synthesizes a test strategy to reveal a fault for the lowest frequency possible. Such a test strategy guarantees to cause a speciﬁcation violation if the fault occurs with the deﬁned fault occurrence (and all higher fault occurrence frequencies) and the test is executed long enough. Although test oracles can be synthesized from the speciﬁcation ϕ, in this paper, we do not explicitly consider test oracle synthesis, but assume that the oracles are available or manually generated for the test strategies. Under the hood, reactive synthesis [47] with partial information [33] is used, which pro- vides strong guarantees about all uncertainties: if synthesis is successful and if the computed tests are executed long enough, they reveal all faults from the fault model for every realization of the speciﬁcation and every behavior of the uncontrollable part of the system’s environ- ment. Uncontrollable environment aspects can be seen as part of the system for the purpose of testing. 1 While the semantics of LTL are deﬁned over inﬁnite execution traces, we can only run the tests for a ﬁnite amount of time. This can result in inconclusive verdicts [6]. We exclude this issue from the scope of this paper, relying on the user to judge when tests have been executed long enough, and on existing research on interpreting LTL over ﬁnite traces [14,15,27,39]. 2 PARTYStrategy, https://www.iaik.tugraz.at/content/research/scos/tools/. 2 Motivating example Let us develop a trafﬁc light controller for the scenario depicted in Fig. 2. For this highway and farmroad crossing, the controller’s Boolean input signal c describes whether a car is idling at the farmroad. Boolean outputs h and f control the highway and farmroad trafﬁc lights respectively, where a value of true means a green light. Output p controls a camera that takes a picture if a car on the farmroad makes a fast start, i.e., races off immediately when the farmroad light turns green. The controller then should implement the following critical properties: 1. The trafﬁc lights must never be green simultaneously. 1. The trafﬁc lights must never be green simultaneously. 2. If a car is waiting at the farmroad, f eventually turns true. 2. If a car is waiting at the farmroad, f eventually turns true. 2. If a car is waiting at the farmroad, f eventually turns true. g , f y 3. If no car is waiting at the farmroad, h eventually becomes true. 4. A picture is taken if a car on the farmroad makes a fast start. We model the four properties in Linear Temporal Logic (LTL) [46] as We model the four properties in Linear Temporal Logic (LTL) [46] as ϕ1 = G(¬f ∨¬h) (1) ϕ2 = G(c →F f) (2) ϕ3 = G(¬c →F h) (3) ϕ4 = G (¬f ∧X(c ∧f ∧X ¬c)) ↔X X p (4) (4) where the operator G denotes always, F denotes eventually, and X denotes in the nextstep. The resulting speciﬁcation is then: where the operator G denotes always, F denotes eventually, and X denotes in the nextstep. Th lti iﬁ ti i th where the operator G denotes always, F denotes eventually, and X denotes in the nextstep Th l i iﬁ i i h ere the operator G denotes always, F denotes eventually, and X denotes in the nextstep. The resulting speciﬁcation is then: The resulting speciﬁcation is then: ϕ = ϕ1 ∧ϕ2 ∧ϕ3 ∧ϕ4 To compute a test strategy (only from the speciﬁcation) that enforces a speciﬁcation violation by the system under the existence of a certain fault (or class of faults), we have some requirements for our approach. Enforcing test objectives To mitigate scalability issues, we compute test cases directly from the speciﬁcation ϕ. Fig. 2 Trafﬁc light example The remainder of this paper is organized as follows: Sect. 2 illustrates our approach and presents a motivating example. Section 3 discusses related work. Section 4 gives preliminaries and notation. Our test case generation approach is then worked out in detail in Sect. 5. Section 6 presents the case study and discusses results. Section 7 concludes. The remainder of this paper is organized as follows: Sect. 2 illustrates our approach and presents a motivating example. Section 3 discusses related work. Section 4 gives preliminaries and notation. Our test case generation approach is then worked out in detail in Sect. 5. Section 6 presents the case study and discusses results. Section 7 concludes. 1 Introduction Finally, existing techniques from runtime veriﬁcation [6] can be used to build an oracle that checks the system behavior against the speciﬁcation while tests are executed.1 This paper is an extension of [10]. In summary, this paper presents the following contri- butions: This paper is an extension of [10]. In summary, this paper presents the following contri- butions: – An approach to compute adaptive test strategies for reactive systems from temporal speciﬁcations that provide implementation freedom. The tests are guaranteed to reveal certain bugs for every realization of the speciﬁcation. – The underlying theory is considered in detail, i.e., we show that the approach is sound and complete for many interesting cases and provide additional solutions for other cases that may arise in practice. – The underlying theory is considered in detail, i.e., we show that the approach is sound and complete for many interesting cases and provide additional solutions for other cases that may arise in practice. – A proof of concept tool, called PARTYStrategy,2 that is capable of generating multiple different test strategies, implemented on top of the synthesis tool PARTY [31]. – A proof of concept tool, called PARTYStrategy,2 that is capable of generating multiple different test strategies, implemented on top of the synthesis tool PARTY [31]. – A post-processing procedure to generalize a test strategy by eliminating input constraints not necessary to guarantee a coverage goal. – A case study with a safety-critical software component of a real-world satellite system developed in the German Aerospace Center (DLR). We specify the system in LTL, synthesize test strategies, and evaluate the generated adaptive test strategies using code coverage and mutation coverage metrics. Our synthesized test strategies increase both the mutation coverage as well as the code coverage of random test cases by activating behaviors that require complex input sequences that are unlikely to be produced by random testing. 123 Formal Methods in System Design (2019) 55:103–135 106 SUT f p c h Highway Farm Road Fig. 2 Trafﬁc light example The remainder of this paper is organized as follows: Sect. 2 illustrates our approach and presents a motivating example. Section 3 discusses related work. Section 4 gives preliminaries and notation. Our test case generation approach is then worked out in detail in Sect. 5. Section 6 presents the case study and discusses results. Section 7 concludes. 2 Motivating example Note that ϕ focuses on the desired properties only, and allows for plenty of implementation freedom. Our goal is to compute tests that enforce certain coverage objectives independent of this implementation freedom. Some uncertainties about the SUT behavior may actually be rooted in uncontrollable environment aspects (such as weather conditions) rather than implementation freedom inside the system. But for our testing approach, this 123 Formal Methods in System Design (2019) 55:103–135 107 ¬c c ¬c f ¬f ¬f f true ¬c c f ¬f ¬f f Fig. 3 Two adaptive test strategies for the trafﬁc light controller: on the left, T1 that enforces p = true once. On the right, T2 that enforces p = true inﬁnitely often ¬c c f ¬f ¬f f ¬c c ¬c f ¬f ¬f f true Fig. 3 Two adaptive test strategies for the trafﬁc light controller: on the left, T1 that enforces p = true on On the right, T2 that enforces p = true inﬁnitely often Fig. 3 Two adaptive test strategies for the trafﬁc light controller: on the left, T1 that enforces p = true once. On the right, T2 that enforces p = true inﬁnitely often makes no difference. We can force the farmroad’s trafﬁc light to turn green (f=true) by relying on a correct implementation of Property 2 and setting c=true. Depending on how the system is implemented, f=true might also be achieved by setting c=false all the time, but this is not guaranteed. Adaptive test strategies Certain test goals may not be enforceable with a static input sequence. For our example, for p to be true, a car must do a fast start. Yet, the speciﬁcation does not prescribe the exact point in time when the trafﬁc light turns to green. We thus synthesize adaptive test strategies that guide the controller’s inputs based on the previous inputs and outputs and, therefore, can take advantage of situational possibilities by exploiting previous system behavior. Figure 3 shows a test strategy T1 (on the left) to reach p=true, illustrated as a state machine. States are labeled by the value of controller input c (which is an output of the test strategy T1). Edges represent transitions and are labeled with conditions on observed output values (since the SUT’s outputs are inputs for the test strategy). First, c is set to false to provoke h=true via Property 3, implying f=false via Property 1. 2 Motivating example As soon as this happens, the strategy traverses to the middle state, setting c=true in order to have f=true eventually (Property 2). As soon as f switches from false to true, T1 sets c=false in the rightmost state to trigger a picture (Property 4). A system with a permanent stuck-at-0 fault at signal p is unable to satisfy the speciﬁcation and the resulting violation can be detected by a runtime veriﬁcation technique. Coverage objectives We follow a fault-centered approach to deﬁne the test objectives to enforce. The user deﬁnes a class of (potentially transient) faults. Our approach then computes adaptive test strategies (in form of state machines) that detect these faults. For a permanent stuck-at-0 fault at signal p, our approach could produce the test strategy T1 from the previous paragraph: for any correct implementation of ϕ, the strategy enforces p becoming true at least once. Thus, a faulty version where p is always false necessarily violates the speciﬁcation, which can be detected [6] during test strategy execution. The test strategy T2, as shown on the right of Fig. 3, is even more powerful since it also reveals stuck-at-0 faults for p that occur not always but only from some point in time onwards. The difference to T1 is mainly in the bold transition, which makes T2 enforce p=true inﬁnitely often rather than only once. Our approach distinguishes four fault occurrence frequencies (a fault occurs at least once, inﬁnitely often, from some point on, or always) and synthesizes test strategies for the lowest one for which this is possible. 3 Background and related work Fault-based testing Fault-based test case generation methods that use the concept of muta- tion testing [29] seed simple faults into a system implementation (or model) and compute 123 Formal Methods in System Design (2019) 55:103–135 108 tests that uncover these faults. Two hypotheses support the value of such tests. The Compe- tent Programmer Hypothesis [1,16] states that implementations are mostly close to correct. The Coupling Effect [16,41] states that tests that detect simple faults are also sensitive to more complex faults. Our approach also relies on these hypotheses. However, in contrast to most existing work that considers permanent faults and deterministic system descriptions that deﬁne behavior unambiguously, our approach can deal with transient faults and focuses on uncovering faults in every implementation of a given LTL [46] speciﬁcation (and all behaviors of the uncontrollable part of the system’s environment). Adaptive tests If the behavior of the system or the uncontrollable part of the environment is not fully speciﬁed, tests may have to react to observed behavior at runtime to achieve their goals. Many testing theories and test case generation algorithms from speciﬁcations of labelled transition systems have been developed. Tretmans [49], for instance, proposed a test- ing theory analogous to the theory of testing equivalence and preorder for labelled transition systems under the assumption that an implementation communicates with its environment via inputs and outputs. Adaptive tests have been studied by Hierons [28] from a theoretical perspective, relying on fairness assumptions (every non-deterministic behavior is exhibited when trying often enough) or probabilities. Petrenko et al. compute adaptive tests for trace inclusion [43–45] or equivalence [35,42,44] from a speciﬁcation given as non-deterministic ﬁnite state machine, also relying on fairness assumptions. Our work makes no such assump- tions but considers the SUT to be fully antagonistic. Aichernig et al. [2] present a method to compute adaptive tests from (non-deterministic) UML state machines. Starting from an initial state, a trace to a goal state, the state that shall be covered by the resulting test case, is searched for every possible system behavior, issuing inconclusive verdicts only if the goal state is not reachable any more. Our approach uses reactive synthesis to enforce reaching the testing goal for all implementations if this is possible. 3 Background and related work Testing as a game Yannakakis [52] points out that testing reactive systems can be seen as a game between two players: the tester providing inputs and trying to reveal faults, and the SUT providing outputs and trying to hide faults. The tester can only observe outputs and has thus partial information about the SUT. The goal is to ﬁnd a strategy for the tester that wins against every SUT. The underlying complexities are studied by Alur et al. [3] in detail. Our work builds upon reactive synthesis [47] (with partial information [33]), which can also be seen as a game. However, we go far beyond the basic idea. We combine the game concept with user-deﬁned fault models, work out the underlying theory, optimize the faults sensitivity in the temporal domain, and present a realization and experiments for LTL [46]. Nachmanson et al. [40] synthesize game strategies as tests for non-deterministic software models, but their approach is not fault-based and focuses on simple reachability goals. A variant of their approach considers the SUT to behave probabilistically with known probabilities [40]. The same model is also used in [8]. Test strategies for reachability goals are also considered by David et al. [13] for timed automata. Vacuity detection Several approaches [5,7,34] aim at ﬁnding cases where a temporal spec- iﬁcation is trivially satisﬁed (e.g., because the left side of an implication is false). Good tests avoid such vacuities to challenge the SUT. The method by Beer et al. [7] can produce wit- nesses that satisfy the speciﬁcation non-vacuously, which can serve as tests. Our approach avoids vacuities by requiring that certain faulty SUTs violate the speciﬁcation. 123 Formal Methods in System Design (2019) 55:103–135 109 Testing with a model checker Model checkers can be utilized to compute tests from temporal speciﬁcations [25]. The method by Fraser and Ammann [22] ensures that properties are not vacuously satisﬁed and that faults propagate to observable property violations (using ﬁnite-trace semantics for LTL). Tan et al. [50] also deﬁne and apply a coverage metric based on vacuity for LTL. Ammann et al. [4] create tests from CTL [12] speciﬁcations using model mutations. All these methods assume that a deterministic system model is available in addition to the speciﬁcation. Fraser and Wotawa [23] also consider non-deterministic models, but issue inconclusive verdicts if the system deviates from the behavior foreseen in the test case. 3 Background and related work In contrast, we search for test strategies that achieve their goal for every realization of the speciﬁcation. Boroday et al. [11] aim for a similar guarantee (calling it strong test cases) using a model checker, but do not consider adaptive test cases, and use a ﬁnite state machine as a speciﬁcation. Synthesis of test strategies Bounded synthesis [21] aims for ﬁnding a system implementa- tion of minimal size in the number of states. Symbolic procedures based on binary decision diagrams [18] and satisﬁability solving [31] exist. In our setting, we do not synthesize an implementation of the system, but an adaptive test strategy, i.e., a controller that mimics the system’s environment to enforce a certain test goal. In contrast to a complete implementation of the controller, we strive for ﬁnding a partial implementation that assigns values only to those signals that necessarily contribute to reach the test goal. Other signals can be kept non- deterministic and either chosen during execution of the test strategy or randomized. We use a post-processing procedure that eliminates assignments from the test strategy and invokes a model-checker to verify that the test goal is still enforced. This post-processing step is con- ceptually similar to procedures that aim for counterexample simpliﬁcation [30] and don’t care identiﬁcation in test patterns [38]. Jin et al. [30] separate a counterexample trace into forced segments that unavoidably progress towards the speciﬁcation violation and free segments that, if avoided, may have prevented the speciﬁcation violation. Our post-processing step is similar, but instead of counterexamples, adaptive test strategies are post-processed. Miyase and Kajihara [38] present an approach to identify don’t cares in test patterns of combina- tional circuits. In contrast to combinational circuits, we deal with reactive systems. Instead of post-processing a complete test strategy, a partial test strategy can be directly synthesized by modifying a synthesis procedure to compute minimum satisfying assignments [17]. Although feasible, modifying a synthesis procedure requires a lot of work. Our post-processing pro- cedure uses the synthesis procedure in a plug-and-play fashion and does not require manual changes in the synthesis procedure. 4 Preliminaries and notation Mealy machines We use Mealy machines to model the reactive system under test. A Mealy machine is a tuple S = (Q, q0, I , O, δ, λ), where Q is a ﬁnite set of states, q0 ∈Q is the initial state, δ : Q × I →Q is a total transition function, and λ : Q × I →O is a total output function. Given the input trace σI = x0x1 . . . ∈ω I , S produces the output trace σO = S(σI ) = λ(q0, x0)λ(q1, x1) . . . ∈ω O, where qi+1 = δ(qi, xi) for all i ≥0. That is, in every time step i, the Mealy machine reads the input letter xi ∈I , responds with an output letter λ(qi, xi) ∈O, and updates its state to qi+1 = δ(qi, xi). A Mealy machine can directly model synchronous hardware designs, but also other systems with inputs and outputs evolving in discrete time steps. We write Mealy(I, O) for the set of all Mealy machines with inputs I and outputs O. Moore machines We use Moore machines to describe test strategies. A Moore machine is a special Mealy machine with ∀q ∈Q . ∀x, x′ ∈I . λ(q, x) = λ(q, x′). That is, λ(q, x) is insensitive to x, i.e., becomes a function λ : Q →O. This means that the input xi at step i can affect the next state qi+1 and thus the next output λ(qi+1) but not the current output λ(qi). We write Moore(I, O) for the set of all Moore machines with inputs I and outputs O. Composition Given Mealy machines S1 = (Q1, q0,1, 2I , 2O1, δ1, λ1) ∈Mealy(I, O1) and S2 = (Q2, q0,2, 2I∪O1, 2O2, δ2, λ2) ∈Mealy(I ∪O1, O2), we write S = S1 ◦S2 for their sequential composition S = (Q1 × Q2, (q0,1, q0,2), 2I , 2O1∪O2, δ, λ), where S ∈Mealy(I, O1 ∪O2) with δ (q1, q2), x = δ1(q1, x), δ2(q2, x ∪λ1(q1, x)) and λ (q1, q2), x = λ1(q1, x) ∪λ2 q2, x ∪λ1(q1, x) . Note that x ∈2I . Systems and test strategies A reactive system S is a Mealy machine. An (adaptive) test strategy is a Moore machine T = (T , t0, O, I , , ) with input and output alphabet swapped. 4 Preliminaries and notation Traces We want to test reactive systems that have a ﬁnite set I = {i1, . . . , im} of Boolean inputs and a ﬁnite set O = {o1, . . . , on} of Boolean outputs. The input alphabet is I = 2I , the output alphabet is O = 2O, and = 2I∪O. An inﬁnite word σ over is an (execution) trace and the set ω is the set of all inﬁnite words over . Linear Temporal Logic We use Linear Temporal Logic (LTL) [46] as a speciﬁcation lan- guage for reactive systems. The syntax is deﬁned as follows: every input or output p ∈I ∪O is an LTL formula; and if ϕ1 and ϕ2 are LTL formulas, then so are ¬ϕ1, ϕ1 ∨ϕ2, X ϕ1 and ϕ1 U ϕ2. We write σ \| ϕ to denote that a trace σ = σ0σ1 . . . ∈ω satisﬁes LTL formula ϕ. This is deﬁned inductively as follows: Formal Methods in System Design (2019) 55:103–135 110 – σ0σ1σ2 . . . \| p iff p ∈σ0, – σ0σ1σ2 . . . \| p iff p ∈σ0, – σ \| ¬ϕ iff σ ̸\| ϕ, – σ \| ϕ1 ∨ϕ2 iff σ \| ϕ1 or σ \| ϕ2, – σ \| ϕ1 ∨ϕ2 iff σ \| ϕ1 or σ \| ϕ2, – σ0σ1σ2 . . . \| X ϕ iff σ1σ2 . . . \| ϕ, and σ0σ1 . . . \| ϕ1 U ϕ2 iff ∃j ≥0 . σ jσ j+1 . . . \| ϕ2 ∧∀0 ≤k < j . σkσk+1 . . . \| ϕ1. – σ0σ1 . . . \| ϕ1 U ϕ2 iff ∃j ≥0 . σ jσ j+1 . . . \| ϕ2 ∧∀0 ≤k < j . σkσk+1 . . . \| ϕ1 That is, X ϕ requires ϕ to hold in the next step, and ϕ1 U ϕ2 means that ϕ1 must hold until ϕ2 holds (and ϕ2 must hold eventually). We also use the usual abbreviations ϕ1 ∧ϕ2 = ¬(¬ϕ1 ∨¬ϕ2), ϕ1 →ϕ2 = ¬ϕ1 ∨ϕ2, F ϕ = trueUϕ (meaning that ϕ must hold eventually), and G ϕ = ¬ F ¬ϕ (ϕ must hold always). By ϕ[x ←y] we denote the LTL formula ϕ where all occurrences of x have been textually replaced by y. 4 Preliminaries and notation That is, T produces values for input signals and reacts to values of output signals. A test strategy T can be run on a system S as follows. In every time stepi (starting withi = 0), T ﬁrstcomputes the nextinput xi = (ti). Then, the system computes the output yi = λ(qi, xi). Finally, both machines compute their next state ti+1 = (ti, yi) and qi+1 = δ(qi, xi). We write σ(T , S) = (x0 ∪y0)(x1 ∪y1) . . . ∈ω for the resulting execution trace. If T = (T , t0, 2O′, I , , ) ∈Moore(O′, I) can observe only a subset O′ ⊆O of the outputs, we deﬁne σ(T , S) with ti+1 = (ti, yi ∩O′). A test suite is a set TS ⊆Moore(O, I) of adaptive test strategies. Realizability A Mealy machine S ∈Mealy(I, O) realizes an LTL formula ϕ, written S \|\| ϕ, if ∀M ∈Moore(O, I) . σ(M, S) \| ϕ. An LTL formula ϕ is Mealy-realizable if there exists a Mealy machine that realizes it. A Moore machine M ∈Moore(I, O) realizes 123 Formal Methods in System Design (2019) 55:103–135 111 ϕ, written M \|\| ϕ, if ∀S ∈Mealy(O, I) . σ(M, S) \| ϕ. A model checking procedure checks if a given Mealy (Moore) machine S (M) realizes an LTL speciﬁcation ϕ and returns true iff S \|\| ϕ (M \|\| ϕ) holds. We denote the call of a model checking procedure by modelcheck S, ϕ (modelcheck M, ϕ ). ϕ, written M \|\| ϕ, if ∀S ∈Mealy(O, I) . σ(M, S) \| ϕ. A model checking procedure checks if a given Mealy (Moore) machine S (M) realizes an LTL speciﬁcation ϕ and returns true iff S \|\| ϕ (M \|\| ϕ) holds. We denote the call of a model checking procedure by modelcheck S, ϕ (modelcheck M, ϕ ). Reactive synthesis We use reactive synthesis [47] to compute test strategies. A reactive (Moore, LTL) synthesis procedure takes as input a set I of Boolean inputs, a set O of Boolean outputs, and an LTL speciﬁcation ϕ over these signals. It produces a Moore machine M ∈ Moore(I, O) that realizes ϕ, or the message unrealizable if no such Moore machine exists. We denote this computation by M = synt(I, O, ϕ). 4 Preliminaries and notation Synthesis with partial information [33] is deﬁned similarly, but this problem takes a subset I ′ ⊆I of the inputs as an additional input. As output, the synthesis procedure produces a Moore machine M′ = syntp(I, O, ϕ, I ′) with M′ ∈Moore(I ′, O) that realizes ϕ while only observing the inputs I ′, or the message unrealizable if no such Moore machine exists. We assume that both synthesis procedure, synt and syntp, can be called incrementally with an additional parameter , where denotes a set of Moore machines. The incremental synthesis procedures M = synt(I, O, ϕ, ) and M′ = syntp(I, O, ϕ, I ′, ) compute Moore machines M and M′, respectively, as before but with the additional constraints that M, M′ /∈ . In synthesis, we often use assumptions A and guarantees G. The assumptions are meant to state the requirements on the environment under which the guarantees should be met by the synthesized system. Technically, we synthesize a system M that fulﬁlls the speciﬁcation A →G. Obviously, whenever the environment violates the assumptions, the implication is trivially satisﬁed and the behavior of the system is irrelevant. For the purposes of this paper, we take synthesis as a black box. We will not describe the technical details of synthesis here but rather refer the interested reader to [9] for details. Fault versus failure A Mealy machine S ∈Mealy(I, O) is faulty with respect to LTL formula ϕ (speciﬁcation) iff S ̸\|\| ϕ, i.e., ∃M ∈Moore(O, I) . σ(M, S) ̸\| ϕ. We call a trace σ(M, S) that uncovers a faulty behavior of S a failure and a deviation between S and any correct realization S′, i.e., S′ \|\|ϕ, a fault. For a ﬁxed faulty S, there are multiple correct S′ that realize ϕ and thus a fault in S can be characterized by multiple, different ways. As a simpliﬁcation, we assume that in practice every faulty S is close to a correct S′ and only deviates in a simple fault. In the next section, we will show how this idea can be leveraged to determine test suites independent of the implementation and the concrete fault manifestation. 5 Synthesis of adaptive test strategies This section presents our black-box testing approach for synthesizing adaptive test strategies for reactive systems speciﬁed in LTL. First, we elaborate on the coverage objective we aim to achieve. Then we present our strategy synthesis algorithm. Finally, we discuss extensions and variants of the algorithm. 5.1 Coverage objective for test strategy computation Many coverage metrics [37] exist to assess the quality of a test suite. Since the goal in testing is to detect bugs, we follow a fault-centered approach: a test suite has high quality if it reveals 123 Formal Methods in System Design (2019) 55:103–135 112 S′ \|= ϕ F \|= δ o′ i I oi o1 . . . on . . . SUT S Fig. 4 Coverage goal illustration for fault Fig. 4 Coverage goal illustration for fault certain kinds of faults in a system. In contrast to existing approaches such as mutation testing which model potential faults in a concrete implementation, we provide a novel fault model that models faults on the speciﬁcation-level, agnostic of the concrete implementation. We assume that the SUT is “almost correct” and contains only simple faults that propagate to at most one output.3 As illustrated in Fig. 4, we formalize this assumption on speciﬁcation-level and model the SUT as composed of a correct implementation S′ of the speciﬁcation ϕ and a fault F that affects one output. In order to make our approach ﬂexible, we allow the user to deﬁne the considered faults as an LTL formula δ. Through δ, the user can deﬁne both permanent and transient faults of various types. For instance, δ = F(oi ↔¬o′ i) describes a bit-ﬂip that occurs at least once, GF ¬oi models a stuck-at-0 fault that occurs inﬁnitely often, and G(X(oi) ↔o′ i) models a permanent shift by one time step. We strive for a test suite that reveals every fault that satisﬁes δ for every realization of ϕ. This renders the test suite independent of the implementation and the concrete fault manifestation. The following deﬁnition formalizes this intuition into a coverage objective. Deﬁnition 1 A test suite TS ⊆Moore(O, I) for a system with inputs I, outputs O, and speciﬁcation ϕ is universally complete4 with respect to a given fault model δ iff ∀oi ∈O . ∀S′ ∈Mealy(I, O ∪{o′ i}\{oi}) . ∀F ∈Mealy(I ∪O ∪{o′ i}\{oi}, {oi}) . ∃T ∈TS . S′ \|\|ϕ[oi ←o′ i] ∧F \|\|δ → σ(T , S′ ◦F) ̸\| ϕ . (5) (5) That is, for every output oi, system S′ \|\|ϕ[oi ←o′ i], and fault F \|\|δ, TS must contain a test strategy T that reveals the fault by causing a speciﬁcation violation (Fig. 4). 4 The word “complete” indicates that every considered fault is revealed at every output. The word “universal” indicates that this is achieved for every (otherwise correct) system. 3 This fault model is different from the standard fault model in mutation testing, which considers simple faults in a concrete implementation that can affect multiple outputs. Proof Equation 6 implies Equation 6 implies oi ∈O . ∀S ∈Mealy(I, O ∪{o′ i}) . ∃T ∈Moore(O, I) . S \|\|ϕ[oi ←o′ i] ∧δ → σ(T , S) ̸\| ϕ (7) ∀oi ∈O . ∀S ∈Mealy(I, O ∪{o′ i}) . ∃T ∈Moore(O, I) . S \|\|ϕ[oi ←o′ i] ∧δ → σ(T , S) ̸\| ϕ (7) ∀oi ∈O . ∀S ∈Mealy(I, O ∪{o′ i}) . ∃T ∈Moore(O, I) . S \|\|ϕ[oi ←o′ i] ∧δ → σ(T , S) ̸\| ϕ (7) S \|\|ϕ[oi ←o′ i] ∧δ → σ(T , S) ̸\| ϕ (7) because (a) going from ∃T ∀S to ∀S∃T can only make the formula weaker, and (b) S \|\| ϕ[oi ←o′ i] ∧δ implies σ(T , S) \| ϕ[oi ←o′ i] ∧δ for all T , which can only make the left side of the implication stronger. In turn, Eq. 7 is equivalent to ∀oi ∈O . ∀S′ ∈Mealy(I, O ∪{o′ i}\{oi}) . ∀F ∈Mealy(I ∪O ∪{o′ i}\{oi}, {oi}) . ∃T ∈Moore(O, I) . S′ \|\|ϕ[oi ←o′ i] ∧F \|\|δ → σ(T , S′ ◦F) ̸\| ϕ (8) (8) because for a given S′ \|\| ϕ[oi ←o′ i] and F \|\| δ from Eq. 8 we can deﬁne an equivalent system S = (S′ ◦F) ∈Mealy(I, O ∪{o′ i}) for Eq. 7 such that S \|\| ϕ[oi ←o′ i] ∧δ is satisﬁed. Also, for a given S \|\| ϕ[oi ←o′ i] ∧δ from Eq. 7 we can deﬁne a corresponding S′ \|\|ϕ[oi ←o′ i] and F \|\|δ by stripping off different outputs. Theorem 1 states that Eq. 6 is a sufﬁcient condition for a universally complete test suite to exist. If it were also a necessary condition, then computing one test strategy per output signal would be enough. Unfortunately, this is not the case in general. Example 1 Consider a system with input I = {i}, output O = {o}, and speciﬁcation ϕ = G(i →G i) ∧F i → G(o →G o) ∧F o ∧G(i ∨¬o) . The left side of the implication assumes that the input i is set to true at some point, after which i remains true. The right side requires the same for the output o. In addition, o must not be raised while i is still false. 5.1 Coverage objective for test strategy computation Note that the test strategies T ∈TS ⊆Moore(O, I) cannot observe the signal o′ i. The reason is that this signal o′ i does not exist in the real system implementation(s) on which we run our tests—it was only introduced to deﬁne our coverage objective. There can be an unbounded number of system realizations S′ \|\| ϕ[oi ←o′ i] and faults F \|\|δ. Computing a separate test strategy for each combination is thus not a viable option. We rather strive for computing only one test strategy per output variable. Theorem 1 A universally complete test suite TS ⊆Moore(O, I) with respect to fault model δ exists for a system with inputs I, outputs O, and speciﬁcation ϕ if 123 113 Formal Methods in System Design (2019) 55:103–135 ¬i i ¬o true o Fig. 5 Test strategy T5 true o Fig. 5 Test strategy T5 Fig. 5 Test strategy T5 ∀oi ∈O . ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O ∪{o′ i}) . σ(T , S) \| (ϕ[oi ←o′ i] ∧δ) →¬ϕ . (6) (6) 5 This is (at least partially) conﬁrmed by our test strategy synthesis tool: it reports that no test strategy with less than 12 states can satisfy Eq. 6. Proof Equation 6 implies Lemma 1 For every LTL speciﬁcation ψ over some inputs I and outputs O, we have that ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O) . σ(T , S) \| ψ holds if and only if ∀S ∈ Mealy(I, O) . ∃T ∈Moore(O, I) . σ(T , S) \| ψ holds. Lemma 1 For every LTL speciﬁcation ψ over some inputs I and outputs O, we have that ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O) . σ(T , S) \| ψ holds if and only if ∀S ∈ Mealy(I, O) . ∃T ∈Moore(O, I) . σ(T , S) \| ψ holds. Proof Synthesis from LTL speciﬁcations under complete information is (ﬁnite memory) determined [36], which means that either ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O) . σ(T , S) \| ψ or ∃S ∈Mealy(I, O) . ∀T ∈Moore(O, I) . σ(T , S) \| ¬ψ holds, but not both. Less formally we can say that either there exists a test strategy T that satisﬁes ψ for all systems S, or there exists a system S that can violate ψ for all test strategies T . From that, it follows that ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O) . σ(T , S) \| ψ iff ¬∃S ∈Mealy(I, O) . ∀T ∈Moore(O, I) . σ(T , S) \| ¬ψ iff ∀S ∈Mealy(I, O) . ∃T ∈Moore(O, I) . σ(T , S) \| ψ. The second lemma is again limited to perfect information. It states that the following two conditions are equivalent: (1) for any system that fulﬁlls an assumption A, there is a test strategy that elicits behavior satisfying a guarantee G and (2) for any system there is a test strategy that elicits behavior satisfying the LTL property A →G. This lemma implies that in the case of complete information, an LTL synthesis tool sufﬁces. Lemma 2 For all LTL speciﬁcations A, G over inputs I and outputs O, we have that mma 2 For all LTL speciﬁcations A, G over inputs I and outputs O, we have that ∀S ∈Mealy(I, O) . ∃T ∈Moore(O, I) . (S \|\| A) → σ(T , S) \| G (9) iff ∀S ∈Mealy(I, O) . ∃T ∈Moore(O, I) . σ(T , S) \| A →G. (10) (9) (10) Proof Direction ⇒: We show that Eq. 10 being false contradicts with Eq. 9 being true. Proof Direction ⇒: We show that Eq. Proof Equation 6 implies This speciﬁcation is realizable (e.g., by always setting o = i). The test suite TS = {T5} with T5 shown in Fig. 5 is universally complete with respect to fault model δ = F(o ↔¬o′), which requires the output to ﬂip at least once: as long as i is false, any correct system implementation S′ ∈Mealy({i}, {o′}) \|\| ϕ[oi ←o′ i] must keep the output o′ = false. Eventually, F \|\| δ must ﬂip the output o to true. When this happens, i is set to true by T5 so that the resulting trace σ(T , S′ ◦F) violates ϕ. Still, Eq. 6 is false.5 Strategy T5 does not satisfy Eq. 6 because for the system S ∈Mealy({i}, {o, o′}) that sets o′ = true and o = false in all time steps, we have σ(T5, S) \| ϕ[oi ←o′ i] ∧δ ∧ϕ . The reason is that i stays false, so ϕ[oi ←o′ i] and ϕ are vacuously satisﬁed by σ(T5, S). The formula δ is satisﬁed because o ↔¬o′ holds in all time steps. Thus, S is a counterexample to T5 satisfying Eq. 6. Similar counterstrategies exist for all other test strategies. 123 123 12 114 Formal Methods in System Design (2019) 55:103–135 The fact that Eq. 6 is not a necessary condition for a universally complete test suite to exist is somewhat surprising, especially in the light of the following two lemmas. Based on these lemmas, the subsequent propositions will show that Eq. 6 is both sufﬁcient and necessary (i.e., one test per output is enough) for many interesting cases. The fact that Eq. 6 is not a necessary condition for a universally complete test suite to exist is somewhat surprising, especially in the light of the following two lemmas. Based on these lemmas, the subsequent propositions will show that Eq. 6 is both sufﬁcient and necessary (i.e., one test per output is enough) for many interesting cases. The following lemma, which is based on the determinacy of complete-information games, states that the following two conditions are equivalent: (1) there is a single test strategy that shows a fault in any implementation and (2) for any implementation there is a strategy that shows the fault. This means that in certain settings, a single test strategy sufﬁces to ﬁnd a fault. Proof Equation 6 implies Proof ϕ[oi ←o′ i] ∧G(o′ i ↔ψ) is equivalent to ϕ[oi ←ψ] ∧G(o′ i ↔ψ). Thus, Eq. 6 becomes ∀oi ∈O . ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O ∪{o′ i}) . σ(T , S) \| (ϕ[oi ←ψ] ∧G(o′ i ↔ψ)) →¬ϕ, ∀oi ∈O . ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O ∪{o′ i}) . σ(T , S) \| (ϕ[oi ←ψ] ∧G(o′ i ↔ψ)) →¬ϕ, Proof Equation 6 implies 10 being false contradicts with Eq. 9 being true ¬∀S ∈Mealy(I, O) . ∃T ∈Moore(O, I) . σ(T , S) \| A →G ¬∀S ∈Mealy(I, O) . ∃T ∈Moore(O, I) . σ(T , S) \| A →G ¬∀S ∈Mealy(I, O) . ∃T ∈Moore(O, I) . σ(T , S) \| A →G iff ∃S ∈Mealy(I, O) . ∀T ∈Moore(O, I) . σ(T , S) \| A ∧¬G iff ∃S ∈Mealy(I, O) . S \|\|(A ∧¬G), which implies ∃S ∈Mealy(I, O) . ∀T ∈Moore(O, I) . (S \|\| A) ∧ σ(T , S) \| ¬G . iff ∃S ∈Mealy(I, O) . ∀T ∈Moore(O, I) . σ(T , S) \| A ∧¬G iff ∃S ∈Mealy(I, O) . S \|\|(A ∧¬G), which implies ∃S ∈Mealy(I, O) . ∀T ∈Moore(O, I) . (S \|\| A) ∧ σ(T , S) \| ¬G . 123 Formal Methods in System Design (2019) 55:103–135 115 Direction ⇐: Using the LTL semantics, we can rewrite σ(T , S) \| A →G in Eq. 10 as σ(T , S) \| A → σ(T , S) \| G . Since S \|\| A implies σ(T ′, S) \| A for every T ′ ∈Moore(I, O), the assumption in Eq. 9 is not weaker, so Eq. 9 is not stronger. Yet, in our setting, test strategies T ∈Moore(O, I) have incomplete information about the system S ∈Mealy(I, O ∪{o′ i}) because they cannot observe o′ i. Still, T must enforce (ϕ[oi ←o′ i] ∧δ) →¬ϕ, which refers to this hidden signal. Thus, Lemma 1 and 2 cannot be applied to Eq. 6 in general. However, in cases where there is (effectively) no hidden information, the lemmas can be used to prove that Eq. 6 is both a necessary and a sufﬁcient condition for a universally complete test suite to exist. The following propositions show that this holds for many cases of practical interest. The intuitive reason is that ϕ[oi ←o′ i] can be rewritten to ϕ[oi ←ψ] in Eq. 6, which eliminates the hidden signal such that Lemmas 1 and 2 can be applied. Proposition 1 Given a fault model of the form δ = G(o′ i ↔ψ), where ψ is an LTL formula over I and O, a universally complete test suite TS ⊆Moore(O, I) with respect to δ, I, O, and ϕ exists if and only if Eq. 6 holds. which is equivalent to ∀oi ∈O . ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O) . σ(T , S) \| ϕ[oi ←ψ] →¬ϕ. ∀oi ∈O . ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O) . ∀oi ∈O . ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O) . σ(T , S) \| ϕ[oi ←ψ] →¬ϕ. Because of the G operator, a unique value for o′ i exist in all time steps and thus, o′ i is just an abbreviation for ψ. Whether this abbreviation o′ i is available as output of S or not is irrelevant, because T cannot observe o′ i anyway. Since o′ i no longer occurs, Lemmas 1 and 2 can be applied to prove equivalence between Eq. 6 and ∀oi ∈O . ∀S ∈Mealy(I, O) . ∃T ∈Moore(O, I) . (S \|\|ϕ[oi ←ψ]) →(σ(T , S) ∀oi ∈O . ∀S ∈Mealy(I, O) . ∃T ∈Moore(O, I) . (S \|\|ϕ[oi ←ψ]) →(σ(T , S) ̸\| ϕ). As T cannot observe o′ i, it is irrelevant whether the truth value of ψ is available as additional output o′ i of S or not. Hence, the above formula is equivalent to ∀oi ∈O . ∀S ∈Mealy(I, O ∪{o′ i}) . ∃T ∈Moore(O, I) . (S \|\|(ϕ[oi ←ψ] ∧G(o′ i ↔ψ)) →(σ(T , S) ̸\| ϕ) ∀oi ∈O . ∀S ∈Mealy(I, O ∪{o′ i}) . ∃T ∈Moore(O, I) . and and ∀oi ∈O . ∀S ∈Mealy(I, O ∪{o′ i}) . ∃T ∈Moore(O, I) . (S \|\|(ϕ[oi ←o′ i] ∧δ) →(σ(T , S) ̸\| ϕ), i.e., to Eq. 7. The remaining steps can be taken from the proof of Theorem 1. i.e., to Eq. 7. The remaining steps can be taken from the proof of Theorem 1. Proposition 1 entails that computing one test strategy per output oi ∈O is enough for fault models such as permanent bit ﬂips (deﬁned by δ = G(o′ i ↔¬oi)). 123 116 Formal Methods in System Design (2019) 55:103–135 Proposition 2 If the fault model δ does not reference o′ i, a universally complete test suite TS ⊆Moore(O, I) with respect to δ, I, O, and ϕ exists iff Eq. 6 holds. Proof We show that Eq. 6 holds if and only if Eq. 7 holds. The remaining steps have already been proven for Theorem 1. Lemma 3 Equation 6 holds if and only if Lemma 3 Equation 6 holds if and only if ∀oi ∈O . ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O) . σ(T , S) \| δ →¬ϕ. (11) (11) Proof Direction ⇐is obvious because Eq. 6 contains stronger assumptions (and ∀S ∈ Mealy(I, O) can be changed to ∀S ∈Mealy(I, O ∪{o′ i}) in Eq. 11 because δ →¬ϕ does not contain o′ i). i Direction ⇒: We show that Eq. 11 being false contradicts with Eq. 6 being true. ¬∀oi ∈O . ∃T ∈Moore(O, I) . ∀S ∈Mealy(I, O) . σ(T , S) \| δ →¬ϕ (12) (12) ∀S ∈Mealy(I, O) . σ(T , S) \| δ →¬ϕ (12) iff ∃oi ∈O . ∀T ∈Moore(O, I) . iff ∃oi ∈O . ∀T ∈Moore(O, I) . ∃S ∈Mealy(I, O) . σ(T , S) \| δ ∧ϕ (13) (13) iff ∃oi ∈O . ∃S ∈Mealy(I, O) . ∀T ∈Moore(O, I) . σ(T , S) \| (δ ∧ϕ) (14) ∀T ∈Moore(O, I) . σ(T , S) \| (δ ∧ϕ) (14) iff ∃oi ∈O . ∃S ∈Mealy(I, O) . S \|\|δ ∧ϕ (15) iff ∃oi ∈O . ∃S ∈Mealy(I, O) . S \|\|δ ∧ϕ (15) (15) iff ∃oi ∈O . ∃S ∈Mealy(I, O) . S \|\|δ ∧ϕ (15) iff ∃oi ∈O . ∃S′ ∈Mealy(I, O ∪{o′ i}) . iff ∃oi ∈O . ∃S′ ∈Mealy(I, O ∪{o′ i}) . ∀T ∈Moore(O ∪{o′ i}, I) . σ(T , S) \| ϕ[oi ←o′ i] ∧δ ∧ϕ, (17) iff ∃oi ∈O . ∀T ∈Moore(O ∪{o′ i}, I) . ∃S′ ∈Mealy(I, O ∪{o′ i}) . σ(T , S) \| ϕ[oi ←o′ i] ∧δ ∧ϕ, (18) ⇒∃oi ∈O . ∀T ∈Moore(O, I) . ∃S′ ∈Mealy(I, O ∪{o′ i}) . σ(T , S) \| ϕ[oi ←o′ i] ∧δ ∧ϕ, (19) (18) σ(T , S) \| ϕ[oi ←o′ i] ∧δ ∧ϕ, (19) (19) which contradicts Eq. 6. (13)⇔(14) holds because of Lemma 1 and (Eq. 15)⇔(Eq. 16) holds because δ ∧ϕ does not contain o′ i, so S′ can be S with o′ i ↔oi. (Eq. 17)⇔(Eq. 18) holds because of Lemma 1. Finally, (Eq. 18) implies (Eq. 19) because T has less information in (Eq. 19). Lemma 4 Equation 11 holds if and only if Eq. 7 holds. Lemma 4 Equation 11 holds if and only if Eq. 7 holds. Proof Direction ⇒: is obvious because Eq. 11 is equivalent to Eq. 6 (Lemma 3) and Eq. 6 implies Eq. 7 (see proof for Theorem 1). Proof Direction ⇒: is obvious because Eq. 11 is equivalent to Eq. 6 (Lemma 3) and Eq. 6 implies Eq. 7 (see proof for Theorem 1). p q p Direction ⇐: we show that Eq. 11 being false contradicts Eq. 7 being true. Equation 11 being false implies Eq. 16 (see above). As S′ \|\| (ϕ[oi ←o′ i] ∧δ ∧ϕ) implies (S′ \|\| Direction ⇐: we show that Eq. 11 being false contradicts Eq. 7 being true. Equation 11 being false implies Eq. 16 (see above). As S′ \|\| (ϕ[oi ←o′ i] ∧δ ∧ϕ) implies (S′ \|\| 123 Formal Methods in System Design (2019) 55:103–135 117 ϕ[oi ←o′ i] ∧δ) ∧ σ(T , S) \| ϕ for all T ∈Moore(O ∪{o′ i}, I) and thus also for all T ∈Moore(O, I), Eq. 7 cannot hold. ϕ[oi ←o′ i] ∧δ) ∧ σ(T , S) \| ϕ for all T ∈Moore(O ∪{o′ i}, I) and thus also for all T ∈Moore(O, I), Eq. 7 cannot hold. Thus, the assumption S′ \|\| ϕ[oi ←o′ i] can be dropped from Eq. 5 if the fault model does not reference o′ i. Correspondingly, σ(T , S) \| (ϕ[oi ←o′ i] ∧δ) →¬ϕ simpliﬁes to σ(T , S) \| (δ →¬ϕ) in Eq. 6. Since o′ i is now gone, Lemmas 1 and 2 apply. In general, the assumption S′ \|\| ϕ[oi ←o′ i] is needed to prevent a faulty system S′ ̸\|\| ϕ[oi ←o′ i] from compensating the fault F \|\|δ such that S′ ◦F \|\|ϕ. E.g., for I = ∅, O = {o}, ϕ = G o and δ = G(o ↔¬o′), Eq. 5 would be false without S′ \|\|ϕ[oi ←o′ i] because there exists an S′ that always sets o′ = false, in which case S′ ◦F has o correctly set to true. However, if δ does not reference o′, such a fault compensation is not possible. Proposition 2 applies to permanent or transient stuck-at-0 or stuck-at-1 faults (e.g., δ = F ¬oi orδ = GF oi),butalsotofaultswhereoi keepsitspreviousvalue(e.g.,δ = F(oi ↔X oi) or takes the value of a different input or output (e.g., δ = GF(oi ←i3)). Lemma 4 Equation 11 holds if and only if Eq. 7 holds. Together with Proposition 1, it shows that computing one test strategy per output is enough for many interesting fault models. Finally, even if neither Propositions 1 nor 2 applies, computing one test strategy per output may still sufﬁce for the concrete ϕ and δ at hand. In the next section, we thus rely on Eq. 6 to compute one test strategy per output in order to obtain universally complete test suites. 5.2 Test strategy computation 6 Examples of different coverage objectives Algorithm 1 SyntLtlTest: Synthesizes a universally complete test suite from an LTL speciﬁcation for all outputs in O 1: procedure SyntLtlTest(I, O, ϕ, κ), returns: A set TS of test strategies 2: TS := ∅ 3: for each oi ∈O do 4: TS := TS ∪SyntLtlIterate(I, O, ϕ, oi, κ, ∅); 5: return TS Algorithm 1 SyntLtlTest: Synthesizes a universally complete test suite from an LTL speciﬁcation for all outputs in O 1: procedure SyntLtlTest(I, O, ϕ, κ), returns: A set TS of test strategies 2: TS := ∅ 3: for each oi ∈O do 4: TS := TS ∪SyntLtlIterate(I, O, ϕ, oi, κ, ∅); 5: return TS Algorithm 1 SyntLtlTest: Synthesizes a universally complete test suite from an LTL speciﬁcation for all outputs in O 1: procedure SyntLtlTest(I, O, ϕ, κ), returns: A set TS of test strategies 2: TS := ∅ 3: for each oi ∈O do 4: TS := TS ∪SyntLtlIterate(I, O, ϕ, oi, κ, ∅); 5: return TS – Frequency FG means that the fault occurs from some time step i on permanently. Yet, we do not make any assumptions about the precise value of i. – Frequency FG means that the fault occurs from some time step i on permanently. Yet, we do not make any assumptions about the precise value of i. – Frequency FG means that the fault occurs from some time step i on permanently. Yet, we do not make any assumptions about the precise value of i. y p p Frequency GF states that the fault strikes inﬁnitely often, but not when exactly. – Frequency GF states that the fault strikes inﬁnitely often, but not when exactly. – Frequency F means that the fault occurs at least once. – Frequency F means that the fault occurs at least once. The fault model δ is then deﬁned as δ = frq(κ). Note that there is a natural order among our 4 fault frequencies: a fault of kind κ that occurs permanently (frequency G) is just a special case of the same fault κ occurring from some point onwards (frequency FG), which is in turn a special case of κ occurring inﬁnitely often (frequency GF), which is a special case of κ occurring at least once. 5.2 Test strategy computation Basic idea Our test case generation approach builds upon Theorem 1: for every output oi ∈O, we want to ﬁnd a test strategy Ti ∈Moore(O, I) such that ∀S ∈Mealy(I, O ∪ {o′ i}) . σ(Ti, S) \| (ϕ[oi ←o′ i] ∧δ) →¬ϕ holds. Recall from Sect. 4 that a synthesis procedure M = syntp(I, O, ψ, I ′, ) with partial information computes a Moore machine M ∈Moore(I ′, O)\ with I ′ ⊆I such that a certain LTL objective ψ is enforced in all environments, i.e., ∀S ∈Mealy(O, I) . σ(M, S) \| ψ. If no such M exists, syntp returns unrealizable. Also recall that a test strategy is a Moore machine with input and output signals swapped. We can thus call Ti := syntp O ∪{o′ i}, I, (ϕ[oi ←o′ i] ∧δ) →¬ϕ, O, for every output oi ∈O in order to obtain a universally complete test suite with respect to fault model δ for a system with inputs I, outputs O, and speciﬁcation ϕ. If syntp succeeds (does not return unrealizable) for all oi ∈O, the resulting test suite TS = {Ti \| oi ∈O} is guaranteed to be universally complete. However, since Theorem 1 only gives a sufﬁcient but not a necessary condition, this procedure may fail to ﬁnd a universally complete test suite, even if one exists, in general. In cases where Propositions 1 or 2 applies, it is both sound and complete, though. Fault models In order to simplify the user input, we split the fault model δ in our coverage objective from Deﬁnition 1 into two parts: the fault kind κ and the fault frequency frq (Fig. 6). The fault kind κ is an LTL formula that is given by the user and deﬁnes which faults we consider. For instance, κ = ¬oi describes a stuck-at-0 fault, κ = oi ↔¬o′ i deﬁnes a bit-ﬂip, and κ = o′ i ↔X oi describes a delay by one time step. The fault frequency frq describes how often a fault of the speciﬁed kind occurs, and is chosen by our algorithm, unless it is speciﬁed by the user. We distinguish 4 fault frequencies, which we describe using temporal LTL operators. – Fault frequency G means that the fault is permanent. 5.2 Test strategy computation 123 123 Formal Methods in System Design (2019) 55:103–135 118 Fault frequency Fault kind Globally (permanent fault) Eventually (fault occurs only once) Arbitrary intermediate stage… … Stuck-at-0/1 faults Bit-ﬂip Delay fault ( ↔¬ ′) ( ↔¬ ′) ( = 0/1) ( = 0/1) ( ( ) ↔ ′) ( ( ) ↔ ′) ( ( ↔¬ ′)) … … ⋮ ⋮ Fig. 6 Examples of different coverage objectives Fault frequency Fault kind Globally (permanent fault) Eventually (fault occurs only once) Arbitrary intermediate stage… … Stuck-at-0/1 faults Bit-ﬂip Delay fault ( ↔¬ ′) ( ↔¬ ′) ( = 0/1) ( = 0/1) ( ( ) ↔ ′) ( ( ) ↔ ′) ( ( ↔¬ ′)) … … ⋮ ⋮ Fig. 6 Examples of different coverage objectives Algorithm 1 SyntLtlTest: Synthesizes a universally complete test suite from an LTL speciﬁcation for all outputs in O 1: procedure SyntLtlTest(I, O, ϕ, κ), returns: A set TS of test strategies 2: TS := ∅ 3: for each oi ∈O do 4: TS := TS ∪SyntLtlIterate(I, O, ϕ, oi, κ, ∅); 5: return TS Fault frequency Fault kind Globally (permanent fault) Eventually (fault occurs only once) Arbitrary intermediate stage… … Stuck-at-0/1 faults Bit-ﬂip Delay fault ( ↔¬ ′) ( ↔¬ ′) ( = 0/1) ( = 0/1) ( ( ) ↔ ′) ( ( ) ↔ ′) ( ( ↔¬ ′)) … … ⋮ ⋮ Fig. 6 Examples of different coverage objectives … ( ( ) ↔ ′) ( ↔¬ ′) Fault kind … Fig. 5.2 Test strategy computation If such a strategy 123 119 Formal Methods in System Design (2019) 55:103–135 Algorithm 2 SyntLtlIterate: Synthesize an adaptive test strategy from an LTL speciﬁca- tion with the lowest fault occurrence frequency 1: procedure SyntLtlIterate(I, O, ϕ, oi, κ, ), returns: A singleton {T } with a test strategy T on success or ∅ 2: for each frq from (F, GF, FG, G) in this order do 3: T := syntp O ∪{o′ i}, I, ϕ[oi ←o′ i] ∧frq(κ) →¬ϕ, O, 4: if T ̸= unrealizable then 5: return {T }; 6: return ∅ Algorithm 2 SyntLtlIterate: Synthesize an adaptive test strategy from an LTL speciﬁ tion with the lowest fault occurrence frequency Algorithm 2 SyntLtlIterate: Synthesize an adaptive test strategy from an LTL speciﬁca- tion with the lowest fault occurrence frequency 2: for each frq from (F, GF, FG, G) in this order do 2: for each frq from (F, GF, FG, G) in this order do 3: T := syntp O ∪{o′ i}, I, ϕ[oi ←o′ i] ∧frq(κ) →¬ϕ, O, 4: if T ̸= unrealizable then 5: return {T }; 6: return ∅ q ( , , , ) 3: T := syntp O ∪{o′ i}, I, ϕ[oi ←o′ i] ∧frq(κ) →¬ϕ, O, 4: if T ̸= unrealizable then 5: return {T }; 6: return ∅ exists, it is returned to Algorithm 1 and added to TS. Otherwise, the procedures proceeds with the next higher fault frequency. exists, it is returned to Algorithm 1 and added to TS. Otherwise, the procedures proceeds with the next higher fault frequency. Sanity checks Note that our coverage goal in Eq. 5 is vacuously satisﬁed by any test suite if ϕ or δ is unrealizable. The reason is that the test suite must reveal every fault F realizing δ for every system S′ realizing ϕ. If there is no such fault or system, this is trivial. As a sanity check, we thus test the (Mealy) realizability of ϕ and G κ before starting Algorithm 1 (because if G κ is realizable, then so are FG κ, GF κ and F κ). Handling unrealizability If, for some output, Line 3 of Algorithm 2 returns unrealizable for the highest fault frequency frq = G, we print a warning and suggest that the user examines these cases manually. 5.2 Test strategy computation There are two possible reasons for unrealizability. First, due to limited observability, we do not ﬁnd a test strategy although one exists (see Example 1). Second, no test strategy exists because there is some S′ \|\| ϕ[oi ←o′ i] and F \|\| δ such that the composition S = S′ ◦F (see Fig. 4) is correct, i.e., S′ ◦F \|\| ϕ. In other words, for some realization, adding the fault may result in an equivalent mutant in the sense that the speciﬁcation is still satisﬁed. For example, in case of a stuck-at-0 fault model, there may exist a realization of the speciﬁcation that has the considered output oi ∈O ﬁxed to false. Such a high degree of underspeciﬁcation is at least suspicious and may indicate unintended vacuities [7] in the speciﬁcation ϕ, which should be investigated manually. If Proposition 1 or 2 applies, or if synt O ∪{o′ i}, I, ϕ[oi ←o′ i]∧G(κ) →¬ϕ, returns unrealizable, we can be sure that the second reason applies. Then, we can even compute additional diagnostic information in the form of two Mealy machines S′ \|\|ϕ[oi ←o′ i] and F \|\|δ (by synthesizing some Mealy machine S \|\|(ϕ[oi ←o′ i]∧G(κ)∧ϕ) and splitting it into S′ and F by stripping off different outputs). The user can then try to ﬁnd inputs for S′ ◦F such that the resulting trace violates the speciﬁcation. Failing to do so, the user will understand why no test strategy exists (see also [32]). If the speciﬁcation is as intended but no test strategy exists, we could use “collaborative” strategies. Among such strategies, we can choose one that requires as little collaboration from the adversary as necessary [19,20]. In our setting, this means that we weaken the requirement that we ﬁnd the fault regardless of the implementation of the system but rather require that we ﬁnd it for maximal classes of implementations. This is not unusual in testing, which is typically explorative and does not make the guarantees that we attempt to give. For instance, if the speciﬁcation is G(r →F g) with input r and output g and the fault model is GF ¬g, then there is no test strategy that ﬁnds this fault for all implementations. 5.2 Test strategy computation Thus, a test strategy that reveals a fault that occurs at least once (without knowing when) will also reveal a fault that occurs inﬁnitely often, etc. We say that F is the lowest and G is the highest fault frequency. In our approach, we thus compute test strategies to detect faults at the lowest frequency for which a test strategy can be found. Figure 6 presents different examples of the fault model. Algorithm The procedure SyntLtlTest in Algorithm 1 formalizes our approach using the procedure SyntLtlIterate in Algorithm 2 as a helper. The input consists of (1) the inputs I of the SUT, (2) the outputs O of the SUT, (3) an LTL speciﬁcation ϕ of the SUT, and (4) a fault kind κ. The result of SyntLtlTest is a test suite TS. The algorithm iterates over all outputs oi ∈O (Line 3) and invokes the procedure SyntLTLIterate (Line 4). The procedure SyntLTLIterate then iterates over the 4 fault frequencies (Line 2), starting with the lowest one, and attempts to compute a strategy to reveal a fault (Line 3). 5.2 Test strategy computation Yet, an input sequence in which r is always true is a better test sequence than one in which r is always false, because the former strategy will ﬁnd the fault in some implementations, whereas the latter will not ﬁnd the fault in any implementation. We leave the extension to collaborative strategies to future work. 123 120 Formal Methods in System Design (2019) 55:103–135 Complexity Both syntp(O, I, ψ, O′, ) and synt(O, I, ψ, ) are 2EXPTIME complete in \|ψ\| [33], so the execution time of Algorithm 2, and consequently also Algorithm 1, are at most doubly exponential in \|ϕ\| + \|κ\|. Complexity Both syntp(O, I, ψ, O′, ) and synt(O, I, ψ, ) are 2EXPTIME complete in \|ψ\| [33], so the execution time of Algorithm 2, and consequently also Algorithm 1, are at most doubly exponential in \|ϕ\| + \|κ\|. Theorem 2 For a system with inputs I, outputs O, and LTL speciﬁcation ϕ over I ∪O, if the fault kind κ is of the form κ = ψ or κ = (o′ i ↔ψ), where ψ is an LTL formula over I and O, SyntLtlTest(I, O, ϕ, κ) will return a universally complete test suite with respect to the fault model δ = G(κ) if such a test suite exists. Theorem 2 For a system with inputs I, outputs O, and LTL speciﬁcation ϕ over I ∪O, if the fault kind κ is of the form κ = ψ or κ = (o′ i ↔ψ), where ψ is an LTL formula over I and O, SyntLtlTest(I, O, ϕ, κ) will return a universally complete test suite with respect to the fault model δ = G(κ) if such a test suite exists. Proof Since G(κ) implies frq(κ) for all frq ∈{F, GF, FG, G}, Theorem 1 and the guarantees of syntp entail that the resulting test suite TS is universally complete with respect to δ = G(κ) if \|TS\| = \|O\|, i.e., if SyntLtlTest found a strategy for every output. It remains to be shown that \|TS\| = \|O\| for κ = ψ or κ = (o′ i ↔ψ) if a universally complete test suite for δ = G(κ) exists: either Propositions 1 or 2 states that Eq. 6 holds with δ = G(κ). Thus, syntp cannot return unrealizable in SyntLtlIterate with frq = G, so \|TS\| must be equal to \|O\| in this case. 5.2 Test strategy computation Theorem 2 states that SyntLtlTest is not only sound but also complete for many inter- esting fault models such as stuck-at faults or permanent bit-ﬂips. For κ = ψ, Theorem 2 can even be strengthened to hold for all δ = frq(κ) with frq ∈{F, GF, FG, G}. 5.3 Extensions and variants 8 Test strategy T7 on the left, T8 in the middle and T9 on the right Example 2 Consider a system with input I = {i}, output O = {o}, and speciﬁcation ϕ = G((i ↔X ¬i) →X o). The speciﬁcation enforces o to be set to true whenever input i alternates between true and false in consecutive time steps. Consider a stuck-at-0 fault δ = GF ¬o at the output o. The test suite TS = {T6} with the test strategy T6 illustrated in Fig. 7(on the left) is universally complete with respect to δ. The test strategy T6 ﬂips input i in every time step and thus forces the system to set o = true in the second time step. Now consider the concrete and faulty system implementation in Fig. 7(on the right) of ϕ. The test strategy T6, when executed, ﬁrst follows the bold edge and then remains forever in the same state. As a consequence, the fault in the system implementation, i.e., o stuck-at-0, is not uncovered. To uncover the fault, i has to be set to false in the initial state. Faults within a system implementation can be considered by computing more than one test strategy for a given test objective. We extend Algorithm 1 to generate a bounded number b of test strategies by setting = TS in Line 4 and enclosing the line by a while-loop that uses an additional integer variable c to count the number of test strategies generated per output oi. The while-loop terminates if no new test strategy could be generated or if c becomes equal to b. Note that this approach is correct in the sense that all computed test strategies are universally complete with respect to the fault model frq(κ); however, in many cases it is more efﬁcient to determine the lowest fault frequency ﬁrst in Line 4 of Algorithm 2 and then generate multiple test strategies with the same (or higher) frequency by enclosing Line 3 with the while-loop. Test strategy generalization A synthesis procedure usually assigns concrete values to all variables in every state of the generated test strategy. In many cases, however, not all assignments are necessary to enforce a test objective (see Example 3). 5.3 Extensions and variants A test suite computed by SyntLtlTest for speciﬁcation ϕ and fault model δ is universally complete and detects all faults with respect to ϕ and δ independent of the implementation and the concrete fault manifestation if the fault manifests at one of the observable outputs as illustrated in Fig. 4. In this section, we discuss some alternatives and extensions of our approach to improve fault coverage and performance. User-speciﬁed fault frequencies Besides the four fault frequencies (G, FG, GF, and F), other fault frequencies (with different precedences) may be of interest, e.g., if a speciﬁc time step is of special interest. Algorithm 2 supports full LTL and thus the procedure can be extended by replacing Line 2 by “for each frq from Frq in this order”, where Frq is an additional parameter provided by the user. Faults at inputs In the fault model in the previous section, we only consider faults at the outputs. However, considering SUTs that behave as if they would have read a faulty input is possible as well (by changing Line 3 in Algorithm 1 to “for each o ∈I ∪O do”). Multiple faults Faults that occur simultaneously at multiple (inputs or) outputs {o1, . . . , ok} ⊆O can be considered by computing a test strategy T := syntp O ∪{o′ 1, . . . , o′ k}, I, (ϕ[o1 ←o′ 1, . . . , ok ←o′ k] ∧ k i=1 δi) →¬ϕ, O, , where the fault model δi can be different for different outputs oi ∈{o1, . . . , ok}. where the fault model δi can be different for different outputs oi ∈{o1, . . . , ok}. Faults within a SUT If a fault manifests in a conditional fault in a system implementation, a universally complete TS may not be able to uncover the fault (see Example 2). 123 Formal Methods in System Design (2019) 55:103–135 121 Fig. 7 Test strategy T6 and a faulty system implementation of the speciﬁcation ϕ = G((i ↔X ¬i) →X o r1 ¬r2 ∗ r1 r2 ∗ r1 Fig. 8 Test strategy T7 on the left, T8 in the middle and T9 on the right r1 r2 ∗ Fig. 5.3 Extensions and variants 8) that sets r1 = true all the time but puts no restrictions on the value of r2, allows the tester to evaluate different paths in the SUT while still testing for the deﬁned fault class. The procedure in Algorithm 3 generalizes a given test strategy T by systematically remov- ing variable assignments from states and employing a model-checking procedure to ensure that the generalized test strategy still enforces the same test objective. The procedure loops in Line 2 over all states of T and in Line 3 over all inputs. In Line 4 the assignment to the input xi in a state is removed such that the corresponding variable becomes non-deterministic. If the resulting test strategy still enforce the test objective, then T is replaced by its generalization. Otherwise, the change is reverted. Algorithm 3 is integrated into Algorithm 2 and applied in Line 5 to generalize each generated test strategy. Note that generalizing a test strategy is a special way of computing multiple concrete test strategies, which was discussed in the previous section. However, generalization may fail when computing multiple strategies succeeds (by following different paths). Optimization for full observability If we restrict our perspective to the case with no partial information, i.e., all signals are fully observable, we can employ the optimization discussed in Proposition 2 to improve the performance of test strategy generation. In Line 3 of Algorithm 2 we drop a part of the assumption and simplify the synthesis step to Ti := synt O, I, frq(κ) → ¬ϕ, for cases in which κ does not refer to a hidden signal o′ i. Also, for a fault model δ that describes a fault of kind κ = (o′ i ↔ψ), where ψ is an LTL formula over I and O, we can drop the part of the assumption according to Proposition 1 if frq = G. This simpliﬁes Line 3 of Algorithm 2 to Ti := synt O, I, ϕ[oi ←ψ] →¬ϕ, . These simpliﬁcations, moreover, no longer require a synthesis procedure with partial information and thus, a larger set of synthesis tools is supported. Other speciﬁcation formalisms We worked out our approach for LTL, but it works for other languages if (1) the language is closed under Boolean connectives (∧, ¬), (2) the desired fault models are expressible, and (3) a synthesis procedure (with partial information) is available. 5.3 Extensions and variants Example 3 Consider a system with inputs I = {r1,r2} and outputs O = {g1, g2}, which implements the speciﬁcation of a two-input arbiter ϕ = G(r1 →F g1) ∧G(r2 →F g2) ∧ G(¬g1 ∨¬g2), i.e., every request ri shall eventually be granted by setting gi to true and there shall never be two grants at the same time. A valid test strategy T7 that tests for a stuck-at-0 fault of signal g1 from some point in time onwards may simply set r1 = true and r2 = false all the time (see Fig. 8). This forces the system in every time step to eventually grant this one request by setting g1 = true. Another valid test strategy T8 sets r1 = true and r2 = true all the time (see Fig. 8). Now the system has to grant both requests eventually. Both T7 and 123 Formal Methods in System Design (2019) 55:103–135 122 Algorithm 3 Generalize: Generalize a test strategy. Algorithm 3 Generalize: Generalize a test strategy. 1: procedure Generalize(I, O, ϕ, oi, frq, κ, T ), returns: A generalization of T 2: for each qi ∈T do 3: for each xi ∈I do 4: T ′ := remove assignment to xi from state qi in T 5: if modelcheckl(T ′, ϕ[oi ←o′ i] ∧frq(κ) →¬ϕ) then 6: T := T ′ 7: return T Algorithm 3 Generalize: Generalize a test strategy. 1: procedure Generalize(I, O, ϕ, oi, frq, κ, T ), returns: A generalization of T 2: for each qi ∈T do 3: for each xi ∈I do 4: T ′ := remove assignment to xi from state qi in T 5: if modelcheckl(T ′, ϕ[oi ←o′ i] ∧frq(κ) →¬ϕ) then 6: T := T ′ 7: return T T8 test for the deﬁned stuck-at-0 fault of signal g1 from some point in time onwards but will likely execute different paths in the SUT. Thus, considering the more general strategy T9 (see Fig. 8) that sets r1 = true all the time but puts no restrictions on the value of r2, allows the tester to evaluate different paths in the SUT while still testing for the deﬁned fault class. T8 test for the deﬁned stuck-at-0 fault of signal g1 from some point in time onwards but will likely execute different paths in the SUT. Thus, considering the more general strategy T9 (see Fig. 5.3 Extensions and variants These prerequisites do not only apply to many temporal logics but also to various kinds of automata over inﬁnite words. 123 Formal Methods in System Design (2019) 55:103–135 123 RXWSXW LQSXW Satellite soware &RPPDQGV 'DWD KRXVHNHHSLQJ Ground segment System FDIR Speciﬁcaon Fault model RXWSXW LQSXW Satellite soware &RPPDQGV 'DWD KRXVHNHHSLQJ Ground segment Adapve test strategy FDIR Synthesis Fig. 9 FDIR in practice (left) and the intended test setup (right) &RPPDQGV 'DWD KRXVHNHHSLQJ &RPPDQGV Fig. 9 FDIR in practice (left) and the intended test setup (right) Fig. 9 FDIR in practice (left) and the intended test setup (right) 6 Case study To evaluate our approach, we apply it in a case study on a real component of a satellite system that is currently under development. We ﬁrst present the system under test and specify a version of the respective component in LTL. Using this speciﬁcation, we compute a set of test strategies and evaluate them on a real implementation. Additional case studies can be found in [10]. 6.1 Eu:CROPIS FDIR specification An important task of each space and satellite system is to maintain its health state and react on failure. In modern space systems, this task is encapsulated in the Fault Detection, Isolation, and Recovery (FDIR) component, which collects information from all relevant sensors and on-board computers, analyzes and assesses the data in terms of correctness and health, and initiates recovery actions if necessary. The FDIR component is organized hierarchically in multiple levels [51] with the overall objective of maximizing the life-time and correct operation of the system. In this section, we focus on system-level FDIR and present a high-level abstraction of a part of the FDIR mechanisms used in the Eu:CROPIS satellite mission as a case study for adaptive test strategy generation. On the system-level, the FDIR mechanism deals with coarse-grained anomalies of the system behavior such as erroneous sensor data or impossible combinations of signals. Likewise the recovery actions are limited to restarting certain sub-systems, switching between redundant sub-systems if available, or switching into the satellite’s safe mode. The FDIR component is highly safety- and mission-critical. If recovery on this level fails, in many cases the mission has to be considered lost. Eu:CROPIS FDIR In Fig. 9 we illustrate where the FDIR component for the magnetic torquers of the Eu:CROPIS on-board computing system is placed in practice and in Fig. 10, we give a high-level overview of the FDIR component and its environment. The FDIR component regularly obtains housekeeping information from two redundantly-designed control units, S1 and S2, which control the magnetic torquers of the satellite, and interacts with them via the electronic power system, EP. The control units S1 and S2 have the same functionality, but 123 Formal Methods in System Design (2019) 55:103–135 124 EP FDIR System on1 off1 on2 off2 mode1 mode2 errnc errs reset safemode Fig. 10 High-level overview of the satellite software of Eu:CROPIS Fig. 10 High-level overview of the satellite software of Eu:CROPIS only one of them is active at any time. The other control unit serves as a backup that can be activated if necessary. The FDIR component signals the activation (or deactivation) of a control unit to the EP which regulates the power supply. only one of them is active at any time. The other control unit serves as a backup that can be activated if necessary. 6.1 Eu:CROPIS FDIR specification The FDIR component signals the activation (or deactivation) of a control unit to the EP which regulates the power supply. We distinguish two types of errors, called non-critical error and severe error, signaled to the FDIR component via housekeeping information. In case of a non-critical error, two recovery actions are possible. Either the erroneous control unit is disabled for a short time and enabled afterwards again or the erroneous control unit is disabled and the redundant control unit is activated to take over its task. In case of the severe error, however, only the latter recovery action is allowed, i.e., the erroneous control unit has to be disabled and the redundant control unit has to be activated. If this happens more than once and the redundant control unit as well shows erroneous behavior, the FDIR component initiates a switch of the satellite mode into safe mode. The safe mode is a fall-back satellite mode designed to give the operators on ground the maximum amount of time to analyze and ﬁx the problem. It is only invoked once a problem cannot be solved on-board and requires input from the operators to restore nominal operations. LTL speciﬁcation We model the speciﬁcation of the FDIR component in LTL. Let IFDI R = {mode1, mode2, errnc, errs, reset} and OFDI R = {on1, off1, on2, off2, safemode} be the Boolean variables corresponding to the input signals and the output signals of the FDIR component, respectively. These Boolean variables are abstractions of the real hardware/software implementation. The values of the Boolean variables are automatically extracted from the housekeeping information which is periodically collected from EP (mode1, mode2) and S1 or S2 (errnc, errs). The two error variables encompass multiple error conditions (e.g. communication timeouts, invalid responses, electrical errors like over-current or under-voltage, etc.) which are detected by the sub-system. The reset variable corresponds to a telecommand sent from ground to the FDIR component. For the output direction the values of the variables are used to generate commands which are sent to the EP or the satellite mode handling component. Additionally, we use the auxiliary Boolean variables O′ = {lastup, allowswitch} to model state information on speciﬁcation level. These auxiliary variables do not correspond to real signals in the system, but are used as unobservable outputs of the FDIR component. 6.1 Eu:CROPIS FDIR specification In Table 1, we present a summary of the Boolean variables involved in the speciﬁcation and describe their meaning. 123 123 Formal Methods in System Design (2019) 55:103–135 125 Table 1 Descriptions of inputs and outputs of the FDIR component Boolean variable Description mode1 true iff S1 is activated mode2 true iff S2 is activated errnc true iff a non-critical error is signaled by S1 or S2 errs true iff a severe error is signaled by S1 or S2 reset true iff the FDIR component is reset on1 true iff S1 shall be switched on off1 true iff S1 shall be switched off on2 true iff S2 shall be switched on off2 true iff S2 shall be switched off safemode true iff the FDIR component initiates the safemode of the satellite lastup true if the last active system was S1 and false if the last active system was S2 allowswitch true iff a switch of S1 to S2 or S2 to S1 is allowed The LTL speciﬁcation of the FDIR component is of form 6 i=1 Ai → 13 i=1 Gi 6 i=1 Ai → 13 i=1 Gi and consists of the six assumptions A1–A6 and the thirteen guarantees G1–G13. All properties are listed in Table 2, expressing the following intentions: and consists of the six assumptions A1–A6 and the thirteen guarantees G1–G13. All properties are listed in Table 2, expressing the following intentions: and consists of the six assumptions A1–A6 and the thirteen guarantees G1–G13. All properties are listed in Table 2, expressing the following intentions: A1 Whenever both systems are off, then there is no running system that can have an error. Thus, the error signals have to be low as well. A1 Whenever both systems are off, then there is no running system that can have an error. Thus, the error signals have to be low as well. A2 The error signals are mutual exclusive. If the environment enforces a reset then both error signals have to be low, because we assume that ground control has taken care of the errors. A3 After a reset enforced by the environment, one of the two systems has to be running and the other has to be off. 6.1 Eu:CROPIS FDIR specification A4 Whenever the FDIR component sends on1, we assume that in the next time step system number one is running (mode1) and the state of the second system (mode2) does not change. The same assumption applies analogously for on2. A5 Whenever the FDIR component sends off1, we assume that in the next time step system number one is off (¬mode1) and the state of the second system (mode2) does not change. The same assumption applies analogously for off2. A6 We assume that the environment, more speciﬁcally the electronic power unit, is not immediately free to change the state of the systems when there is no message from the FDIR component. It has to wait for one more time step (with no messages of the FDIR component). G1 This guarantee stores which system was last activated by the FDIR component. G2 We require the signals on1, off1, on2 and off2 to be mutually exclusively set to high. G3 Whenever both systems are off, then the FDIR component eventually requests to switch on one of the systems (on1, on2) or activates safemode or observes a reset. G4 We restrict the FDIR component to not enter safemode as long as the component can switch to the backup system. G4 We restrict the FDIR component to not enter safemode as long as the component can switch to the backup system. G4 We restrict the FDIR component to not enter safemode as long as the component can switch to the backup system. 6.2 Experimental results In this section, we present experimental results for generating test strategies for the LTL speciﬁcation of the Eu:CROPIS FDIR component. We ﬁrst analyze runtime and memory consumption of test strategy synthesis, and then evaluate the effectiveness of the generated test strategies on a concrete implementation of the FDIR component. The proposed test strategy synthesis approach, however, is a black-box testing technique, independent of the concrete implementation and can be applied even if no implementation is available. The synthesized test strategies do not contain the test oracle; for the experiments, we use a concrete implementation, that was manually veriﬁed, as test oracle. 6.1 Eu:CROPIS FDIR specification G12 Whenever the FDIR component observes a server error (errs), it must eventually switch to the backup system or activate safemode unless the environment performs a reset or the error disappears by itself (without restarting the system). G13 WhenevertheFDIRcomponentobservesanon-criticalerror(errnc),itmusteventually switch to the backup system or activate safemode or the error disappears (restarting the currently running system is allowed). G13 WhenevertheFDIRcomponentobservesanon-criticalerror(errnc),itmusteventually switch to the backup system or activate safemode or the error disappears (restarting the currently running system is allowed). 6 PARTYStrategy, https://www.iaik.tugraz.at/content/research/scos/tools/. 6.1 Eu:CROPIS FDIR specification 123 Formal Methods in System Design (2019) 55:103–135 126 Table 2 Temporal speciﬁcation of system-level FDIR component in LTL Assumptions A1–A6 A1 G((¬mode2 ∧¬mode1) →¬errnc ∧¬errs) A2 G(¬errnc ∨¬errs) ∧G(reset →¬errnc ∧¬errs) A3 G(reset →X(mode2 ⊕mode1)) A4 G(¬mode1 ∧on1 ∧¬off1 ∧¬on2 ∧¬off2 ∧¬reset ∧¬safemode) → X mode1 ∧(mode2 ↔X mode2)) G(¬mode2 ∧¬on1 ∧¬off1 ∧on2 ∧¬off2 ∧¬reset ∧¬safemode → X mode2 ∧(mode1 ↔X mode1)) A5 G(mode1 ∧¬on1 ∧off1 ∧¬on2 ∧¬off2 ∧¬reset ∧¬safemode → X ¬mode1 ∧(mode2 ↔X mode2)) G(mode2 ∧¬on1 ∧¬off1 ∧¬on2 ∧off2 ∧¬reset ∧¬safemode → X ¬mode2 ∧(mode1 ↔X mode1)) A6 G((¬(¬on2 ∧¬off1 ∧¬on1 ∧¬off2) ∧X(¬on2 ∧¬off1 ∧¬on1 ∧¬off2)∧ (¬reset ∧X ¬reset ∧¬safemode ∧X ¬safemode) → X((mode2 ↔X mode2) ∧(mode1 ↔X mode1)) Guarantees G1–G13 G1 G((on1 ∧¬on2) →(X lastup)) G((¬on1 ∧on2) →(X ¬lastup)) G((¬on1 ∧¬on2) →(lastup ↔X lastup)) G2 G(on1 →¬off1 ∧¬on2 ∧¬off2) G(off1 →¬on1 ∧¬on2 ∧¬off2) G(on2 →¬on1 ∧¬off1 ∧¬off2) G(off2 →¬on1 ∧¬on2 ∧¬off1) G3 G(¬mode2 ∧¬mode1 →F(reset ∨on2 ∨on1 ∨safemode)) G4 G(allowswitch →¬safemode) G5 G((mode2 ∨mode1) →¬on1 ∧¬on2) G6 G(¬allowswitch ∧lastup →¬on2) G(¬allowswitch ∧¬lastup →¬on1) G7 G(¬reset ∧allowswitch ∧lastup ∧on2 →X ¬allowswitch) G(¬reset ∧allowswitch ∧¬lastup ∧on1 →X ¬allowswitch) G8 G((allowswitch ∧¬(((lastup ∧on2) ∨(¬lastup ∧on1)))) →X allowswitch) G9 G(reset →X allowswitch) G10 G(safemode →(¬on1 ∧¬on2)) G11 G(¬allowswitch ∧¬reset →X ¬allowswitch) G12 G((errs ∧mode1 ∧¬reset) → F(reset ∨safemode ∨mode2 ∨(mode1 U (mode1 ∧¬errs)))) G((errs ∧mode2 ∧¬reset) → F(reset ∨safemode ∨mode1 ∨(mode2 U (mode2 ∧¬errs)))) G13 G((errnc ∧mode1 ∧¬reset) →F(reset ∨safemode ∨mode2 ∨(mode1 ∧¬errnc)) G((errnc ∧mode2 ∧¬reset) →F(reset ∨safemode ∨mode1 ∨(mode2 ∧¬errnc)) Table 2 Temporal speciﬁcation of system-level FDIR component in LTL Assumptions A1–A6 A1 G((¬mode2 ∧¬mode1) →¬errnc ∧¬errs) A2 G(¬errnc ∨¬errs) ∧G(reset →¬errnc ∧¬errs) A3 G(reset →X(mode2 ⊕mode1)) A4 G(¬mode1 ∧on1 ∧¬off1 ∧¬on2 ∧¬off2 ∧¬reset ∧¬safemode) → X mode1 ∧(mode2 ↔X mode2)) G(¬mode2 ∧¬on1 ∧¬off1 ∧on2 ∧¬off2 ∧¬reset ∧¬safemode → X mode2 ∧(mode1 ↔X mode1)) A5 G(mode1 ∧¬on1 ∧off1 ∧¬on2 ∧¬off2 ∧¬reset ∧¬safemode → X ¬mode1 ∧(mode2 ↔X mode2)) G(mode2 ∧¬on1 ∧¬off1 ∧¬on2 ∧off2 ∧¬reset ∧¬safemode → X ¬mode2 ∧(mode1 ↔X mode1)) A6 G((¬(¬on2 ∧¬off1 ∧¬on1 ∧¬off2) ∧X(¬on2 ∧¬off1 ∧¬on1 ∧¬off2)∧ (¬reset ∧X ¬reset ∧¬safemode ∧X ¬safemode) → X((mode2 ↔X mode2) ∧(mode1 ↔X mode1)) Guarantees G1–G13 G1 G((on1 ∧¬on2) →(X lastup)) G((¬on1 ∧on2) →(X ¬lastup)) G((¬on1 ∧¬on2) →(lastup ↔X lastup)) G2 G(on1 →¬off1 ∧¬on2 ∧¬off2) G(off1 →¬on1 ∧¬on2 ∧¬off2) G(on2 →¬on1 ∧¬off1 ∧¬off2) G(off2 →¬on1 ∧¬on2 ∧¬off1) G3 G(¬mode2 ∧¬mode1 →F(reset ∨on2 ∨on1 ∨safemode)) G4 G(allowswitch →¬safemode) G5 G((mode2 ∨mode1) →¬on1 ∧¬on2) G6 G(¬allowswitch ∧lastup →¬on2) G(¬allowswitch ∧¬lastup →¬on1) G7 G(¬reset ∧allowswitch ∧lastup ∧on2 →X ¬allowswitch) G(¬reset ∧allowswitch ∧¬lastup ∧on1 →X ¬allowswitch) G8 G((allowswitch ∧¬(((lastup ∧on2) ∨(¬lastup ∧on1)))) →X allowswitch) G9 G(reset →X allowswitch) G10 G(safemode →(¬on1 ∧¬on2)) G11 G(¬allowswitch ∧¬reset →X ¬allowswitch) G12 G((errs ∧mode1 ∧¬reset) → F(reset ∨safemode ∨mode2 ∨(mode1 U (mode1 ∧¬errs)))) G((errs ∧mode2 ∧¬reset) → F(reset ∨safemode ∨mode1 ∨(mode2 U (mode2 ∧¬errs)))) G13 G((errnc ∧mode1 ∧¬reset) →F(reset ∨safemode ∨mode2 ∨(mode1 ∧¬errnc G((errnc ∧mode2 ∧¬reset) →F(reset ∨safemode ∨mode1 ∨(mode2 ∧¬errnc Table 2 Temporal speciﬁcation of system-level FDIR component in LTL p p y p Assumptions A1–A6 A1 G((¬mode2 ∧¬mode1) →¬errnc ∧¬errs) A2 G(¬errnc ∨¬errs) ∧G(reset →¬errnc ∧¬errs) A3 G(reset →X(mode2 ⊕mode1)) A4 G(¬mode1 ∧on1 ∧¬off1 ∧¬on2 ∧¬off2 ∧¬reset ∧¬safemode) → X mode1 ∧(mode2 ↔X mode2)) G(¬mode2 ∧¬on1 ∧¬off1 ∧on2 ∧¬off2 ∧¬reset ∧¬safemode → X mode2 ∧(mode1 ↔X mode1)) A5 G(mode1 ∧¬on1 ∧off1 ∧¬on2 ∧¬off2 ∧¬reset ∧¬safemode → X ¬mode1 ∧(mode2 ↔X mode2)) G(mode2 ∧¬on1 ∧¬off1 ∧¬on2 ∧off2 ∧¬reset ∧¬safemode → X ¬mode2 ∧(mode1 ↔X mode1)) A6 G((¬(¬on2 ∧¬off1 ∧¬on1 ∧¬off2) ∧X(¬on2 ∧¬off1 ∧¬on1 ∧¬off2)∧ (¬reset ∧X ¬reset ∧¬safemode ∧X ¬safemode) → X((mode2 ↔X mode2) ∧(mode1 ↔X mode1)) Guarantees G1–G13 G1 G((on1 ∧¬on2) →(X lastup)) G((¬on1 ∧on2) →(X ¬lastup)) G((¬on1 ∧¬on2) →(lastup ↔X lastup)) G2 G(on1 →¬off1 ∧¬on2 ∧¬off2) G(off1 →¬on1 ∧¬on2 ∧¬off2) G(on2 →¬on1 ∧¬off1 ∧¬off2) G(off2 →¬on1 ∧¬on2 ∧¬off1) G3 G(¬mode2 ∧¬mode1 →F(reset ∨on2 ∨on1 ∨safemode)) G4 G(allowswitch →¬safemode) G5 G((mode2 ∨mode1) →¬on1 ∧¬on2) G6 G(¬allowswitch ∧lastup →¬on2) G(¬allowswitch ∧¬lastup →¬on1) G7 G(¬reset ∧allowswitch ∧lastup ∧on2 →X ¬allowswitch) G(¬reset ∧allowswitch ∧¬lastup ∧on1 →X ¬allowswitch) G8 G((allowswitch ∧¬(((lastup ∧on2) ∨(¬lastup ∧on1)))) →X allowswitch) G9 G(reset →X allowswitch) G10 G(safemode →(¬on1 ∧¬on2)) G11 G(¬allowswitch ∧¬reset →X ¬allowswitch) G12 G((errs ∧mode1 ∧¬reset) → F(reset ∨safemode ∨mode2 ∨(mode1 U (mode1 ∧¬errs)))) G((errs ∧mode2 ∧¬reset) → F(reset ∨safemode ∨mode1 ∨(mode2 U (mode2 ∧¬errs)))) G13 G((errnc ∧mode1 ∧¬reset) →F(reset ∨safemode ∨mode2 ∨(mode1 ∧¬errnc))) G((errnc ∧mode2 ∧¬reset) →F(reset ∨safemode ∨mode1 ∨(mode2 ∧¬errnc))) 123 Formal Methods in System Design (2019) 55:103–135 127 G5 The FDIR component must not request to switch on one of the systems (on1, on2) as long as one of the systems is running. 6.1 Eu:CROPIS FDIR specification G6 Whenever the FDIR component is not allowed anymore to switch to the backup system, then it must not request to switch the backup system on. G6 Whenever the FDIR component is not allowed anymore to switch to the backup system, then it must not request to switch the backup system on. G7 Once the FDIR component switches to the backup system it is not allowed anymore to switch again (unless the environment performs a reset, see G9). G7 Once the FDIR component switches to the backup system it is not allowed anymore to switch again (unless the environment performs a reset, see G9). G8 As long as the FDIR component only restarts the same system it is still allowed to switch in the future. G8 As long as the FDIR component only restarts the same system it is still allowed to switch in the future. G9 A reset by the environment allows the FDIR component again to switch to the backup system if required. G9 A reset by the environment allows the FDIR component again to switch to the backup system if required. G9 A reset by the environment allows the FDIR component again to switch to the backup system if required. G10 Whenever the FDIR component is in safemode it must not request to switch-on one of the systems (on1,on2). G10 Whenever the FDIR component is in safemode it must not request to switch-on one of the systems (on1,on2). G10 Whenever the FDIR component is in safemode it must not request to switch-on one of the systems (on1,on2). G11 Once a switch is not allowed anymore and the environment does not perform a reset, then the switch is also not allowed in the next time step. G11 Once a switch is not allowed anymore and the environment does not perform a reset, then the switch is also not allowed in the next time step. G12 Whenever the FDIR component observes a server error (errs), it must eventually switch to the backup system or activate safemode unless the environment performs a reset or the error disappears by itself (without restarting the system). G12 Whenever the FDIR component observes a server error (errs), it must eventually switch to the backup system or activate safemode unless the environment performs a reset or the error disappears by itself (without restarting the system). 6.2.1 Test strategy computation For synthesizing test strategies, both, the bound for the maximal number of states of a test strategy and the bound for the maximal number of test strategies, are set to four. We chose the bound to be four, because for this bound there exist strategies for all our chosen fault models and output signals. The size for the maximum number of strategies per variable and fault model is set arbitrarily to four and could also be set to a different value. In Table 3, we list the time and memory consumption for synthesizing the test strategies with our synthesis tool PARTYStrategy. The more freedom there is for implementations of the speciﬁcation, the harder it becomes to compute a strategy. The search for strategies that are capable of detecting a bit-ﬂip is the most difﬁcult one as we cannot make use of our optimization for full observability of the output signals. For all signals with a stuck-at-0 fault and for the off1 signal with one of the other two faults we are able to derive test strategies that can detect the fault if it is permanent from some point onwards. For the signals on1 and safemode we are able to derive strategies for stuck-at-1 faults and bit-ﬂips also at a lower frequency, i.e., we can detect those faults also if they occur at least inﬁnitely often. Illustration of a computed strategy We illustrate and explain one derived strategy in detail. The strategy derived for the signal safemode being stuck at 0 computed consists of four states. Figure 11 illustrates the strategy. In the ﬁrst state (state 0) we have the ﬁrst system running (mode1) and set the errnc ﬂag, i.e., we raise a non-critical error that requires the component to restart until the error is gone or to switch to the other system. We loop in this state until the FDIR component, if it behaves according to the speciﬁcation, switches off the running system. In the next state (state 1), we do not set any input and wait for the FDIR component to eventually switch on one of the systems. If the component switches on the same system, then we go back to the previous state (state 0), if it switches on the other system we go into the next state (state 3). 6.2.1 Test strategy computation Experimental setting All experiments for computing test strategies are conducted in a virtual machine with a 64 bit Linux system using a single core of an Intel i5 CPU running at 2.60GHz. We use the synthesis procedure PARTY [31] as black-box, which implements SMT-based bounded synthesis for full LTL and, thus, we call our tool PARTYStrategy.6 Test strategy computation From the previously described LTL speciﬁcation, we compute test strategies for the outputs on1, off1 and safemode of the FDIR component considering the fault models stuck-at-0, stuck-at-1, and bit-ﬂip with the lowest possible fault frequencies. These are general fault assumptions and cover faults where the speciﬁcation is violated with this signal being high (stuck-at-1), faults where the speciﬁcation is violated with this signal being low (stuck-at-0) and faults where the speciﬁcation is violated with this signal having the wrong polarity (bit-ﬂip). We do not synthesize test strategies for the outputs on2 and off2 because they behave identical to on1 and off1, respectively, if the role of S1 and S2 123 123 123 Formal Methods in System Design (2019) 55:103–135 128 Table 3 Results for the FDIR speciﬁcation. The sufﬁx “k” multiplies by 103 Fault oi frq \|T \| Time Peak memory (s) (MB) S-a-0 on1 FG 4 1.2k 400 off1 FG 3 517 396 safemode FG 4 934 324 S-a-1 on1 GF 4 438 222 off1 FG 4 753 378 safemode GF 3 169 192 Bit-Flip on1 GF 4 26k 3.6k off1 FG 4 98.9k 4.3k safemode GF 3 13.1k 4.3k Fault oi frq \|T \| Time Peak memory (s) (MB) S-a-0 on1 FG 4 1.2k 400 off1 FG 3 517 396 safemode FG 4 934 324 S-a-1 on1 GF 4 438 222 off1 FG 4 753 378 safemode GF 3 169 192 Bit-Flip on1 GF 4 26k 3.6k off1 FG 4 98.9k 4.3k safemode GF 3 13.1k 4.3k are mutually interchanged. For synthesizing test strategies, both, the bound for the maximal number of states of a test strategy and the bound for the maximal number of test strategies, are set to four. We chose the bound to be four, because for this bound there exist strategies for all our chosen fault models and output signals. The size for the maximum number of strategies per variable and fault model is set arbitrarily to four and could also be set to a different value. are mutually interchanged. 6.2.1 Test strategy computation In this state we have the second system running (mode2) and set again the errnc ﬂag, i.e., we again raise a non-critical error. We loop in this state until the FDIR component reacts and, if it conforms to the speciﬁcation, switches off the running system. Continuing according to the strategy we always raise a non-critical error whatever system the FDIR component activates. Eventually the FDIR component has to activate safemode or violate the speciﬁcation. State 2 is only entered when the FDIR violates G5. In this state, it is irrelevant how the test strategy behaves because the speciﬁcation has already been violated (which is easy to detect during test execution). 123 Formal Methods in System Design (2019) 55:103–135 129 0 errnc ¬errs mode1 ¬mode2 ¬reset 3 errnc ¬errs ¬mode1 mode2 ¬reset 2 errnc ¬errs mode1 mode2 ¬reset 1 ¬errnc ¬errs ¬mode1 ¬mode2 ¬reset else on2 off1 on1 off2 else else off1 off2 else on2 on1 Fig. 11 Test strategy that tests for a stuck-at-0 fault of signal safemode Fig. 11 Test strategy that tests for a stuck-at-0 fault of signal safemode 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 state 0 1 0 1 0 1 0 1 0 1 0 1 0 1 1 3 1 3 1 3 1 3 1 3 1 3 1 1 1 1 1 1 1 1 mode1 mode2 errnc errs reset on1 off1 on2 off2 safemode Fig. 12 Execution trace from a faulty system under the strategy that tests for a stuck-at-0 fault of signal safemode. Bold signals are controlled by the strategy Fig. 12 Execution trace from a faulty system under the strategy that tests for a stuck-at-0 fault of sig safemode. Bold signals are controlled by the strategy Fig. 12 Execution trace from a faulty system under the strategy that tests for a stuck-at-0 fault of signal safemode. Bold signals are controlled by the strategy 7 Given that the user has decided that we have waited long enough for safemode to become true. 6.2.2 Test strategy evaluation Test setting In the Eu:CROPIS satellite the FDIR component is implemented in software in the programming language C++. The implementation for the magnetic torquer FDIR handling is not an exact realization of the speciﬁcation in Table 2 but extends it by allowing commands to the EP to be lost (e.g. due to electrical faults). This is accommodated by adding timeouts for the execution of the switch-on/off commands and reissuing the commands if the timeout is triggered. 123 123 130 Formal Methods in System Design (2019) 55:103–135 Theimplementationisdesignedwithtestabilityandportabilityinmindandusesanabstract interface to access other sub-systems of the satellite. This allows engineers to exchange the used interface with a set of test adapters which connect to the signals generated by the test strategies. As we are only interested in the functional properties of the implementation, we can run the code on a normal Linux system, instead of the microprocessor which is used in the satellite. This gives access to all Linux based debugging and testing tools and allows us to use gcov to measure the line and branch coverage of the source code. A time step of a test run consists of the following operations: request values for the input variables IFDI R from the test strategy; feed the values to the test adapter from which they are read by the FDIR implementation; run the FDIR implementation for one cycle; extract the output values OFDI R from the test adapter and feed them back to the test strategy to get new input values. For each time step, the execution trace—the values assigned to the inputs IFDI R and outputs OFDI R of the FDIR component—is recorded. Mutationtesting Besideslineandbranchcoverage,weapplymutationanalysistoassessthe effectiveness, i.e., fault ﬁnding abilities, of a test suite. A test suite kills a mutant program M if it contains at least one test strategy that, when executed on M and the original program P, produces a trace where at least one output of M differs in at least one time step from the respective output of P (for the same input sequence). A mutant program M is equivalent to the original program P if M does not violate the speciﬁcation. For our evaluation we manually identify and remove equivalent mutants. We generate mutant programs of the FDIR component by systematically applying the following four mutations to each line of the C++ implementation: 1. Deletion of the line, 1. Deletion of the line, 1. 6.2.2 Test strategy evaluation Deletion of the line, 2. Replacement of true with false or false with true, 3. Replacement of == with != or != with ==, and 4. Replacement of && with \|\| or \|\| with && In total, 210 mutant programs are generated. Each having exactly one mutation. We use the GNU compiler gcc to remove all mutant programs, which do not compile and do not con- form to the C++ programming language. We analyzed the remaining 105 mutants manually and identiﬁed 5 equivalent mutant programs, i.e., they do not violate the speciﬁcation. We further correct this number by removing another 17 mutant programs related to un-speciﬁed implementation-speciﬁc behavior. Next, we executed all test strategies on the mutant pro- grams for 80 time steps each and log the corresponding execution traces. In Fig. 12 we illustrate the execution of the test strategy from Fig. 11 on a mutant program. This particular strategy aims at revealing a stuck-at-0 fault for signal safemode. The test strategy ﬁrst forces the FDIR component to eventually switch to the backup system. The switch happens in time step 14 after several restarts of the system. Then the strategy forces the FDIR component to eventually activate safemode. However, this mutant program is faulty and instead of activating safemode the system remains silent from time step 26 onwards. Thus, violating guarantee G3.7 From the 83 mutant programs that violate the speciﬁcation, the synthesized adaptive test strategies are able to kill 65 (78.31%). Since these test strategies are derived from require- ments, without any implementation-speciﬁc knowledge, they are applicable to any system that claims to implement the speciﬁcation. The mutation score of 78.31% motivate that the synthesized adaptive test strategies—although computed only for simple speciﬁc fault models—are sensitive to other faults. 123 Formal Methods in System Design (2019) 55:103–135 131 Table 4 Mutation coverage by fault models and signal when executing all four derived strategies Output Fault model S-a-0 S-a-1 Bit-ﬂip All (%) (%) (%) (%) on1 67.47 53.01 8.43 75.90 off1 13.25 3.61 16.87 16.87 safemode 61.45 13.25 13.25 16.87 All 71.08 55.42 16.87 78.31 Table 5 Overview of coverage and mutation score by testing approach Table 5 Overview of coverage and mutation score by testing approach Metric Random stimuli Adapt. 7 Conclusion We have presented a new approach to compute adaptive test strategies from temporal logic speciﬁcations using reactive synthesis with partial information. The computed test strategies reveal all instances of a user-deﬁned fault class for every realization of a given speciﬁcation. Thus, they do not rely on implementation details, which is important for products that are still under development or for standards that will be implemented by multiple vendors. Our approach is sound but incomplete in general, i.e., may fail to ﬁnd test strategies even if they exist. However, for many interesting cases, we showed that it is both sound and complete. The worst-case complexity is doubly exponential in the speciﬁcation size, but in our setting, the speciﬁcations are typically small. This also makes our approach an interesting application for reactive synthesis. Our experiments demonstrate that our approach can com- pute meaningful tests for speciﬁcations of industrial size and that the computed strategies are capable of detecting faults hidden in paths that are unlikely to be activated by random input sequences. We have applied our approach in a case study to the fault detection, isolation and recovery component of the satellite Eu:CROPIS. The computed test suite, based only on three different types of faults, achieves a line coverage, branch coverage, and mutation score of 83.0%, 70.8%, ad 78.3%, respectively, relying on information solely available from the speciﬁcation. The approach also allows us to detect faults that require complex input sequences and are unlikely detected by using random testing. y y g g Current directions for future work include improving scalability, success-rate, and usabil- ity of our approach. To this end, we are investigating using random testing for inputs in the strategies that are not ﬁxed to single values, and best-effort strategies [19,20] for the case that there are no test strategies that can guarantee triggering the fault. Another direction for future work is research on evaluating LTL properties speciﬁed on inﬁnite paths on ﬁnite traces to improve the evaluation process when executing the derived strategies. Acknowledgements Open access funding provided by Austrian Science Fund (FWF). This work was sup- ported in part by the European Commission through the Horizon2020 project IMMORTAL (grant no. 644905) funded under H2020- EU.2.1.1.1., the FP7 project eDAS (grant no. 608770) funded under FP7-ICT, the FP7 project STANCE (grant no. 6.2.2 Test strategy evaluation strategies R(0.1k) R(1k) R(10k) R(100k) S(80) S(80)+R(10k) (%) (%) (%) (%) (%) (%) Line 91.5 95.7 96.8 100.0 83.0 97.9 Branch 85.4 89.6 89.6 93.8 70.8 91.7 Mutation 88.0 92.8 94.0 98.0 78.3 97.6 In Table 4, we present the mutation scores for the three signals on1, off1, and safemode and the three fault models stuck-at-0 (S-a-0), stuck-at-1 (S-a-1), and bit-ﬂip (Bit-ﬂip). The last column and last row show the mutation scores when considering all three fault models and all three signal, respectively. Comparison with random testing We compare code coverage and mutation score of the synthesized adaptive test strategies and random testing when executed for 0.1k, 1k, 10k, 100k time steps. The sufﬁx “k” multiplies by 103. We use a uniform random distribution for choosing random values for all input signals, where reset is with 10% probability 1, and all other signals are with 50% probability 1. ThecoverageandmutationscoresarelistedinTable5.Coveragewasmeasuredwithgcov. The table is built as follows: the different testing approaches are shown in the columns. The columns R(0.1k), R(1k), R(10k), R(100k) refer to random testing with increasing numbers of input stimuli, and the columns S(80) and S(80)+R(10k) refer to the synthesized test strategies and the test strategies in combination with R(10k). Overall, random testing achieves high code and mutation scores when executed on the source code of the FDIR component, but can only be used if a concrete implementation of the system is available. The adaptive test strategies, on the other hand, are directly derived from the speciﬁcation and independent from a concrete implementation. They can be used to derive tests if the system is still under development. Parts of the implementation which reﬁne the speciﬁcation, or which are not speciﬁed at all are not necessarily covered. The last column S(80)+R(10k) also shows that the synthesized test strategies improve coverage and mutation scores over R(10k). Moreover, the test strategies are able to kill three mutants that are missed by all random test sequences. 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https://openalex.org/W4290379624	https://hal.archives-ouvertes.fr/hal-01963928/document	English	null	Estimating true species richness and the degree of hierarchical structuring of species abundances in eight frog communities from the North-Western Ghats of India.	HAL (Le Centre pour la Communication Scientifique Directe)	2,018	cc-by	13,958	Estimating true species richness and the degree of hierarchical structuring of species abundances in eight frog communities from the North-Western Ghats of India Jean Béguinot To cite this version: Jean Béguinot. Estimating true species richness and the degree of hierarchical structuring of species abundances in eight frog communities from the North-Western Ghats of India.. International Journal of Environment and Climate Change, 2018, 8 (2), pp.118-137. 10.9734/IJECC/2018/42067. hal- 01963928 HAL Id: hal-01963928 Submitted on 21 Dec 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. International Journal of Environment and Climate Change 8(2): 118-137, 2018; Article no.IJECC.2018.009 Previously known as British Journal of Environment & Climate Change ISSN: 2231–4784 Estimating True Species Richness and the Degree of Hierarchical Structuring of Species Abundances in Eight Frog Communities from the North-Western Ghats of India Jean Béguinot1* 1Biogéosciences, UMR 6282, CNRS, Université Bourgogne Franche-Comté, 6, Boulevard Gabriel, 21000 Dijon, France. Author’s contribution The sole author designed, analyzed and interpreted and prepared the manuscript. Article Information DOI: 10.9734/IJECC/2018/42067 Received 12th May 2018 Accepted 25th May 2018 Published 7th June 2018 ABSTRACT Method Article 8(2): 118-137, 2018; Article no.IJECC.2018.009 Previously known as British Journal of Environment & Climate Change ISSN: 2231–4784 Estimating True Species Richness and the Degree of Hierarchical Structuring of Species Abundances in Eight Frog Communities from the North-Western Ghats of India Jean Béguinot1* 1Biogéosciences, UMR 6282, CNRS, Université Bourgogne Franche-Comté, 6, Boulevard Gabriel, 21000 Dijon, France. Received 12th May 2018 Accepted 25th May 2018 Published 7th June 2018 Received 12th May 2018 Accepted 25th May 2018 Published 7th June 2018 Received 12th May 2018 Accepted 25th May 2018 Published 7th June 2018 Method Article Method Article Corresponding author: E-mail: jean-beguinot@orange.fr; ABSTRACT The Distribution of Species Abundances within natural communities – when properly analysed – can provide essential information regarding general aspects of the internal organisation of these communities. In particular, true species richness on the one hand and the intensity of the process of hierarchical structuring of species abundances on the other hand may be estimated independently and, thereby, can provide truly complementary information. In turn, specific issues may thereby be addressed. For example, whether one unique dominant factor or numerous combined factors are involved in the structuring process of a community can be tested contradictorily. Although these methods are not new conceptually, their implementation in common practice remains scarce. The reason is that the relevant implementation of these methods requires to be sure that virtually all member-species in the community have been sampled. As exhaustive samplings often reveal difficult to achieve in practice, an appropriate, least-biased procedure of numerical extrapolation of incomplete inventories is imperatively required. Considering the steadily increasing threats to the environment and biodiversity, especially facing the on-going climatic change, time has come now with ever greater urgency to go beyond the apparent limits of non-exhaustive sampling and make the most of what is available in terms of recorded field data, whatever the degree of incompleteness of species inventories. g p p As a modest and limited attempt to concretise this wish at the local level, I try, hereafter, to highlight the importance of additional information that may be unveiled through adequate post- analysis of a set of eight frog communities, recently inventoried by Katwate, Apte & Raut in an Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 amphibian hot-spot in the north-western Ghats of India. At last, the likely variations of both total species richness and the intensity of hierarchical structuring of species abundance are simulated as an answer to the steadily increasing influence of the ongoing climatic change. Keywords: Species diversity; species abundance; rank abundance distribution; amphibians; anurans; incomplete sampling; numerical extrapolation; climate change. 1. INTRODUCTION Considering together the five exhaustive Species Abundance Distributions and the three “numerically-completed” ones, I focused on the comparison between these eight frogs’ communities, regarding: (i) their respective levels of species richness and (ii) their respective patterns of abundances distribution. Beyond the mere description of abundance patterns, dealing with already complete or numerically extrapolated Species Abundance Distributions allows to relevantly address (i) the type and (ii) the strength of the process driving the hierarchical structuring of species abundances in each studied communities. Total species richness, taxonomic composition and hierarchical structuring of species abundance distribution are three main topics that together provide a good deal of information about species communities in the wild. The Species Abundance Distribution (especially under the form of the “Rank Abundance Distribution”) is a convenient tool for characterising species communities this way, but this requires, yet, that the sampling effort has been sufficient enough for the resulting abundance distribution being (quasi) exhaustive. This complementary, functional-type approach stands out by its particular interest in the context of the on-going climate change. Indeed, even before climatic change is expected to significantly affect the taxonomic composition within animal communities, it is the functional aspects of these communities – such as the hierarchical structuring of species abundances – that are likely to be impacted first. And frog communities, especially sensitive to climatic parameters, are among the priorities to be addressed in this respect [4,5]. Exhaustive samplings, however, are difficult to obtain and rarely reached in practice, especially when having to deal with species rich communities, such as most invertebrates’ assemblages. But even in some vertebrates’ communities, comprehensive species inventories may occasionally require very large sampling sizes, hard to implement in practice, when one or more of the less common member species happen to be excessively rare. Hopefully, even in such case, it remains possible to extract far much information than would be expected from substantially incomplete samplings, by implementing an appropriate numerical extrapolation procedure [1,2]. While the taxonomic identification of the still undetected species remains, of course, impossible, two other major descriptive features of communities (total species richness and the hierarchical structuring of species abundances) can be extrapolated fairly accurately, on the only basis of data extracted from substantially incomplete samples. 2.2 Numerical Extrapolation Procedures Applied to the Three Incomplete Inventories Extrapolation Procedures Three Incomplete - Not only provides an overview of both the true (total) species richness of the sampled community and the diversity of the respective abundances of member species, only provides an overview of both the species richness of the sampled community and the diversity of the respective abundances of member Total species richness: the least estimation of the number of still undetected species at the end of partial sampling and the resulting estimation of the total species richness of the partially community are derived according to the procedure defined in [9,10] and briefly summarised in Appendix 1. Estimates are based on the numbers fx observed x-times during partial sampling (x = 1 to 5: Figs. A1.1 to A1.3 in Appendix 1). : the least-biased estimation of the number of still undetected partial sampling and the resulting estimation of the total species partially sampled community are derived according to the e defined in [9,10] and briefly summarised in Appendix 1. Estimates are x of species times during partial sampling (x A1.1 to A1.3 in Appendix 1). * Total species richness: the least estimation of the number of still undetected species at the end of partial sampling and the resulting estimation of the total species richness of the partially community are derived according to the procedure defined in [9,10] and briefly summarised in Appendix 1. Estimates are based on the numbers fx observed x-times during partial sampling (x = 1 to 5: Figs. A1.1 to A1.3 in Appendix 1). : the least-biased estimation of the number of still undetected partial sampling and the resulting estimation of the total species partially sampled community are derived according to the e defined in [9,10] and briefly summarised in Appendix 1. Estimates are x of species times during partial sampling (x A1.1 to A1.3 in Appendix 1). p - But, also, can help addressing several important questions regarding the process driving the hierarchical structuration of the community (Fig , also, can help addressing several important questions regarding the kind of process driving the hierarchical of the community (Fig. 1). More precisely, these questions may relate to: More precisely, these questions may relate to: - The process of structuration community of species: for example, does only one (or very few) dominant factor is (are) at work to structure the community or, on the contrary, does many independent factors are contributing together. 2.2 Numerical Extrapolation Procedures Applied to the Three Incomplete Inventories Extrapolation Procedures Three Incomplete This may be tested by checking the conformity of the corresponding S.A.D. to either the series model or the log- respectively [12–16]; of structuration of a community of species: for example, does (or very few) dominant factor is (are) at work to structure the community or, many independent factors are contributing together. This may be tested by checking the conformity of the corresponding S.A.D. to either the log- -normal model g pp ) Species Abundance Distribution accurately exploit their full potential, the as-recorded Species Abundance Distributions (“S.A.D.s”) require [1,11]: pp ) Species Abundance Distribution: to eir full potential, the recorded Species Abundance Distributions (“S.A.D.s”) require [1,11]: - First, to be corrected for statistical sampling bias, resulting from the finite size of samplings and, for statistical sampling bias, resulting from the finite g - Second, and still more importantly, to be completed by numerical extrapolation to the extent that sampling is suspected to be incomplete, as revealed by the subsistence of singletons. , and still more importantly, to be by numerical extrapolation to the extent that sampling is suspected to be incomplete, as revealed by the p y [ ] - The degree of structuration of a community of species, which broadly refers to the level of unevenness between species abundances within the community. This may be appropriately tested by comparing the slope of the corresponding S.A.D. to either the “ideally even” model or the of a community species, which broadly refers to the level of unevenness between species abundances within the community. This may be appropriately tested by comparing the slope of the corresponding S.A.D. to the “ideally even” model or the p y [ ] - The degree of structuration of a community of species, which broadly refers to the level of unevenness between species abundances within the community. This may be appropriately tested by comparing the slope of the corresponding S.A.D. to either the “ideally even” model or the of a community species, which broadly refers to the level of unevenness between species abundances within the community. This may be appropriately tested by comparing the slope of the corresponding S.A.D. to the “ideally even” model or the The appropriate procedure of correction and least-biased numerical extrapolation of the as The appropriate procedure of correction and the numerical extrapolation of the as- Fig. 1. 2.1 Materials Katwate, Apte & Raut [3] reported on the inventories of eight frogs communities (Amphibians, anurans) from Phansad Wildlife Sanctuary, located in the Northern Western Ghats of India. Five of these inventories (labelled A, C, D, E, G) show no subsisting singletons (i.e. species sampled only once) and, accordingly, may be considered virtually exhaustive [6-8]. The other three inventories (labelled B, F, H) all retain, on the contrary, one or more singletons and, thus, likely remain more or less incomplete (as actually confirmed subsequently). Hereafter, I report on the analysis of the inventories of eight frogs communities originally sampled by Katwate, Apte & Raut [3] in northern- western Ghats of India. Among these eight inventories, five may be considered quasi exhaustive (since no singleton is actually subsisting in the samples), while the other three inventories remain more or less incomplete and thus require the implementation of numerical extrapolation to unveil the complete range of species abundance distribution. Details on the sites location where these frog communities were sampled, the local ecological conditions and constraints peculiar to these sites, 119 Béguinot; IJECC, 8(2): 118-137, 2018; Article no. , 2018; Article no.IJECC.2018.009 recorded S.A.D.s, described in details in [1], is briefly recalled in Appendix 2. described in details in [1], is the lists of species identities and the numbers of recorded individuals per species, are provided in the aforementioned reference [3]. the lists of species identities and the numbers of recorded individuals per species, are provided in After being corrected and accordingly, the S.A.D.: and extrapolated 2.2 Numerical Extrapolation Procedures Applied to the Three Incomplete Inventories Extrapolation Procedures Three Incomplete Schematic sketch showing how the combination of both historical and ecological contexts peculiar to a given community of species drive the relative “performance” latissimo - of each member species "i", thus generating the abundances in the community Schematic sketch showing how the combination of both historical and ecological contexts peculiar to a given community of species drive the relative “performance” species "i", thus generating the hierarchical structuring abundances in the community Schematic sketch showing how the combination of both historical and ecological contexts peculiar to a given community of species drive the relative “performance” - sensu hierarchical structuring of species Fig. 1. Schematic sketch showing how the combination of both historical and ecological contexts peculiar to a given community of species drive the relative “performance” latissimo - of each member species "i", thus generating the abundances in the community Schematic sketch showing how the combination of both historical and ecological contexts peculiar to a given community of species drive the relative “performance” species "i", thus generating the hierarchical structuring abundances in the community Schematic sketch showing how the combination of both historical and ecological contexts peculiar to a given community of species drive the relative “performance” - sensu hierarchical structuring of species 120 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 10f2 + 10f3 – 5f4 + f5) in all three cases: see the selective key in Appendix 1. “broken-stick” model. These two models provide two reference levels of structuration, namely the “ideally even” model characterises the zero level of structuration while the “broken-stick” model accounts for the degree of structuration that would be obtained by a random apportionment of relative abundances among all co-occurring species in the community [17]. Standardising the degree of structuration (the slope of the S.A.D.) to the “broken-stick” model is particularly relevant as this allows to set apart the “mechanistic”, trivial influence of species richness upon the level of unevenness of species abundances in the community [18 –20]. This “mechanistic” influence of species richness on the slope of the abundance distribution and, consequently, on the degree of community structuration, is demonstrated in Appendix 3). Thus standardised, the degree of structuration of a community becomes truly independent of the trivial influence of species richness. 2.2 Numerical Extrapolation Procedures Applied to the Three Incomplete Inventories Extrapolation Procedures Three Incomplete Accordingly, the corresponding least-biased estimations of the number Δ of species remaining undetected in the inventories of B, F, H, are given by Jackknife-5 and the resulting least-biased estimation is St = Ro + Δ, with Ro as the number of recorded species. For the five communities A, C, D, E, G, having no remaining singletons and thus considered to be comprehensively sampled, Δ thus equals 0 and, accordingly, the total species richness St is equal to the number Ro of already recorded species. All the results are summarised in Table 1. Note, in addition that, as might have been expected, the product of sample-size No times the relative abundance aSt of the rarest species in each sampled community is much less than 1 when dealing with incomplete samplings (communities B, F, H) and much greater than 1 when dealing with exhaustive samplings (communities A, C, D, E, G). 3.1 The Recorded or Estimated Total Species Richness of the Eight Frog Communities The ranked Species Abundance Distribution of community “F” (coarse grey dots and coarse solid line for the extrapolated part: ranks > 14) compared to two associated models: the “log-normal” distribution (dotted line) and the “log-series” distribution (double line) Fig. 3. The ranked Species Abundance Distribution of community “F” (coarse grey dots and coarse solid line for the extrapolated part: ranks > 14) compared to two associated models: the “log-normal” distribution (dotted line) and the “log-series” distribution (double line) These ranked Species Abundance Distributions highlight the detailed patterns of hierarchical structuring of species abundances which are specific to each studied community. In particular, the stronger the rate of abundance decrease (i.e. the steeper the slope of the ranked abundance distribution) and the more severe looks the hierarchical structuring. Yet, this descriptive approach does not relevantly account for the genuine strength of the structuring process at work in the community, because the slope of the 3.1 The Recorded or Estimated Total Species Richness of the Eight Frog Communities As a whole, abundance distributions best fit the corresponding “log-normal” model than the corresponding “log-series” model. Figs. 2 and 3 provide two typical examples of such close fitting of the Species Abundance Distribution to the corresponding “log-normal” distribution, for both an exhaustively sampled community (G) and an incompletely sampled community (F). As regards the three incompletely sampled communities (B, F, H) and considering the values of the numbers fx of species observed x-times in each of the three samples (see Figs. A1, A2, A3 in Appendix 1), it turns out that the least-biased nonparametric estimator of the number of undetected species is Jackknife-5 (JK-5 = 5f1 – Table 1. The sample-size No, the number of recorded species Ro, the selected least-biased estimator, the number of undetected species Δ, the total species richness St (either recorded for A, C, D, E, G, or extrapolated St = Ro+Δ for B, F, H), the relative abundance aSt of the rarest species (rank St). As expected, the sample-size multiplying the relative abundance of the rarest species (No.aSt) is << 1 in each of the three incomplete inventories (B, F, H) and >> 1 for each of the other five comprehensive inventories (A, C, D, E, G) Community No Ro Selected estimator Δ St aSt No.aSt B 468 11 JK-5 1.9 12.9 .0003 0.14 F 231 14 JK-5 2.8 16.8 .0006 0.14 H 417 10 JK-5 2.4 12.4 .0002 0.08 A 615 15 / 0 15 .0063 3.9 C 329 11 / 0 11 .0265 8.6 D 403 11 / 0 11 .0386 15.3 E 304 11 / 0 11 .0254 7.6 G 493 10 / 0 10 .0060 2.9 121 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 Fig. 2. The ranked Species Abundance Distribution of community “G” (coarse grey dots) compared to two associated models: the “log-normal” distribution (dotted line) and the “log- series” distribution (double line) Fig. 3. 3.1 The Recorded or Estimated Total Species Richness of the Eight Frog Communities The ranked Species Abundance Distribution of community “F” (coarse grey dots and coarse solid line for the extrapolated part: ranks > 14) compared to two associated models: the 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 species relative abundance species abundance rank G 0,0001 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 species relative abundance species abundance rank F 0,0001 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 species relative abundance species abundance rank F 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 species relative abundance species abundance rank G F G F ranked Species Abundance Distribution of community “G” (coarse grey two associated models: the “log-normal” distribution (dotted line) and t series” distribution (double line) nked Species Abundance Distribution of community “F” (coarse grey d ne for the extrapolated part: ranks > 14) compared to two associated mo mal” distribution (dotted line) and the “log-series” distribution (double l ng the Degree of Hierarchical g of Abundances in Species ties: From Pattern to the g Process ecies Abundance Distributions of ommunities are plotted in Figs. 4 vely, after all these distributions orrected and after the three ampled communities (B, F, H) extrapolated. These ranked Species Abundance Dis highlight the detailed patterns of hi structuring of species abundances w specific to each studied community. In the stronger the rate of abundance dec the steeper the slope of the ranked a distribution) and the more severe hierarchical structuring. Yet, this d approach does not relevantly accoun genuine strength of the structuring p work in the community, because the slo 3 4 5 6 7 8 9 10 11 species abundance rank 0,0001 0,001 0,01 0 1 2 3 4 5 6 7 8 9 10 11 12 13 species relative species abundance rank A 1 nce Fig. 2. The ranked Species Abundance Distribution of community “G” (coarse grey dots) compared to two associated models: the “log-normal” distribution (dotted line) and the “log- series” distribution (double line) series” distribution (double line) Fig. 3. 3.3 Quantifying the Degree of Hierarchical Structuring of Abundances in Species Communities: From Pattern to the Underlying Process The ranked Species Abundance Distribution of community “D” (coarse grey dots) and associated “broken-stick” distribution, computed for the same species richness (dashed line) line) Fig. 7. The ranked Species Abundance Distribution of community “E” (coarse grey dot the associated “broken-stick” distribution, computed for the same species richness (d line) Fig. 8. The ranked Species Abundance Distribution of community “G” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) Fig. 9. The ranked Species Abundance Distribution of community “B” (coarse grey dots and coarse solid line for the extrapolated part: ranks > 11) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 species relative abundance species abundance rank G 0,0001 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 species relative abundance species abundance rank B 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 species relative abundance species abundance rank G 0,0001 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 species relative abundance species abundance rank B G B Fig. 8. The ranked Species Abundance Distribution of community “G” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) ) Fig. 9. The ranked Species Abundance Distribution of community “B” (coarse grey dots and coarse solid line for the extrapolated part: ranks > 11) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) abundance distribution depends not only upon the structuring process itself but also depends on the level of species richness of the community. Indeed, all other things being equal, the rate (i.e. the slope) of abundance decrease is negatively dependent upon the level of species richness of the community, as highlighted in Appendix 3. Accordingly, the Species Abundance Distribution should relevantly be compared to the corresponding “broken-stick” model (i.e. the “broken-stick” model computed for the same level of species richness), in order to cancel the trivial influence of species richness level and, thus, unveil the genuine intensity of the structuring process. Thus, in Figs. 4 to 11, each complete (or completed) Species Abundance Distribution is plotted together with its corresponding “broken-stick” distribution. 3.3 Quantifying the Degree of Hierarchical Structuring of Abundances in Species Communities: From Pattern to the Underlying Process The ranked Species Abundance Distributions of the eight frog communities are plotted in Figs. 4 to 11 respectively, after all these distributions have been corrected and after the three incompletely sampled communities (B, F, H) have been duly extrapolated. Fig. 4. The ranked Species Abundance Distribution of community “A” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) Fig. 5. The ranked Species Abundance Distribution of community “C” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 species relative abundance species abundance rank A 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 species relative abundance species abundance rank C 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 species relative abundance species abundance rank C 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 species relative abundance species abundance rank A A C Fig. 4. The ranked Species Abundance Distribution of community “A” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) Fig. 5. The ranked Species Abundance Distribution of community “C” (coarse grey dots) and the associated “broken-stick” distribution computed for the same species richness (dashed Fig. 4. The ranked Species Abundance Distribution of community “A” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) line) Fig. 5. The ranked Species Abundance Distribution of community “C” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) 122 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 Fig. 6. The ranked Species Abundance Distribution of community “D” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) Fig. 7. The ranked Species Abundance Distribution of community “E” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) Fig. 8. The ranked Species Abundance Distribution of community “G” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) Fig. 9. 3.3 Quantifying the Degree of Hierarchical Structuring of Abundances in Species Communities: From Pattern to the Underlying Process The ranked Species Abundance Distribution of community “B” (coarse grey dots and coarse solid line for the extrapolated part: ranks > 11) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) abundance distribution depends not only upon the structuring process itself but also depends on the level of species richness of the community. Indeed, all other things being equal, the rate (i.e. the slope) of abundance decrease is negatively d d t th l l f i i h f level of species richness), in order to cancel the trivial influence of species richness level and, thus, unveil the genuine intensity of the structuring process. Thus, in Figs. 4 to 11, each complete (or completed) Species Abundance Di t ib ti i l tt d t th ith it 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 species relative abundance species abundance rank D 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 species relative abundance species abundance rank E 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 species relative abundance species abundance rank G 0,0001 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 species relative abundance species abundance rank B Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 species relative abundance species abundance rank E 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 species relative abundance species abundance rank D E D Fig. 6. The ranked Species Abundance Distribution of community “D” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) Fig. 7. The ranked Species Abundance Distribution of community “E” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) 1 1 Fig. 6. The ranked Species Abundance Distribution of community “D” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) Fig. 7. The ranked Species Abundance Distribution of community “E” (coarse grey dots) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) . 6. 4. DISCUSSION In a less detailed (and thus more reductionist) approach, the average slope of the Species Abundance Distribution provides a convenient, concise appreciation of the degree of hierarchical structuring. A “structuring index”, based on the average slope, can be defined accordingly. As aforementioned, to reliably reflect the genuine strength of the structuring process, this index must be stantardised to the average slope of the corresponding “broken-stick” distribution. Accordingly, an appropriate structuring index is relevantly defined as the ratio between the average slope of the actual abundance distribution and the average slope of the corresponding “broken-stick” distribution. To conform to the usual, conventional mode of plotting abundance distributions, the abundances will be classically log-transformed. Thus defined, the structuring index Istr is equal to: In a less detailed (and thus more reductionist) approach, the average slope of the Species Abundance Distribution provides a convenient, concise appreciation of the degree of hierarchical structuring. A “structuring index”, based on the average slope, can be defined accordingly. As aforementioned, to reliably reflect the genuine strength of the structuring process, this index must be stantardised to the average slope of the corresponding “broken-stick” distribution. Accordingly, an appropriate structuring index is relevantly defined as the ratio between the average slope of the actual abundance distribution and the average slope of the corresponding “broken-stick” distribution. To conform to the usual, conventional mode of plotting abundance distributions, the abundances will be classically log-transformed. Thus defined, the structuring index Istr is equal to: In a less detailed (and thus more reductionist) approach, the average slope of the Species Abundance Distribution provides a convenient, concise appreciation of the degree of hierarchical structuring. A “structuring index”, based on the average slope, can be defined accordingly. As aforementioned, to reliably reflect the genuine strength of the structuring process, this index must be stantardised to the average slope of the corresponding “broken-stick” distribution. Although the present study aims, first, at a general methodological purpose, rather than being focused toward a particular taxonomic target, a brief argumentation is provided however, supporting the choice of frogs as an appropriate illustrative taxonomic group for the application of the method. Amphibians in general, and frogs in particular, are among animal groups which are most sensitive to environmental changes, especially climatic modifications involving temperature and hygrometry [4,5]. 3.3 Quantifying the Degree of Hierarchical Structuring of Abundances in Species Communities: From Pattern to the Underlying Process This straightforwardly provides a reliable appreciation of the degree of hierarchical structuring of species abundances in each community. abundance distribution depends not only upon the structuring process itself but also depends on the level of species richness of the community. Indeed, all other things being equal, the rate (i.e. the slope) of abundance decrease is negatively dependent upon the level of species richness of the community, as highlighted in Appendix 3. Accordingly, the Species Abundance Distribution should relevantly be compared to the corresponding “broken-stick” model (i.e. the “broken-stick” model computed for the same 123 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 Fig. 10. The ranked Species Abundance Distribution of community “F” (coarse grey dots and coarse solid line for the extrapolated part: ranks > 14) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) Fig. 11. The ranked Species Abundance Distribution of community “H” (coarse grey dots and coarse solid line for the extrapolated part: ranks > 10) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) 0,0001 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 species relative abundance species abundance rank F 0,0001 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 species relative abundance species abundance rank H 0,0001 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 species relative abundance species abundance rank H 0,0001 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 species relative abundance species abundance rank F H Fig. 10. The ranked Species Abundance Distribution of community “F” (coarse grey dots and coarse solid line for the extrapolated part: ranks > 14) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) Fig. 11. The ranked Species Abundance Distribution of community “H” (coarse grey dots and coarse solid line for the extrapolated part: ranks > 10) and the associated “broken-stick” distribution, computed for the same species richness (dashed line) 4. DISCUSSION More specifically, amphibians in general and frogs in particular are typically stenothermic species, as such directly affected by the on-going climate changes, via the increase of temperature and resulting imposed shifts in local distributions, especially altitudinal increase when possible and, otherwise, local extinction [21–23]. Often connected to temperature evolution are more or less drastic changes in precipitation with resulting risks of shortage of water availability which is so important for most amphibians’ survival. Severe declines of various kinds of amphibians are already reported for this reason [24–26]. And both temperature and hygrometry are major drivers of reproductive activities which may thus be strongly affected by global climate change [27–29]. The issue is all the more acute when considering amphibians “hot-spots” having a high proportion of particularly fragile endemic species, Istr = [log(a1) – log(aSt)]/[log(a’1) – log(a’St)] that is: 4.2 The Hierarchical Structuring of Species Abundances: Only One Dominant Causal Factor or Many Independent Ones Jointly Involved? 4.2 The Hierarchical Structuring of Species Abundances: Only One Dominant Causal Factor or Many Independent Ones Jointly Involved? that is: Thus, it is in this sense that should be understood the following warning by noted that the three incompletely sampled communities B, F, H, are precisely those ones that are the most strongly structured, i.e. having the least even distribution of species abundances, as shown later in Fig. 12. Correlatively, in each of communities B, F, H, the rarest species have, by far, the smallest relative abundance levels: see Table 1. As a result, the product No.aSt of the abundance aSt of the rarest species times the sample-size No remains far less than unity in the incomplete samplings of communities B, F, H, while this product largely exceeds unity in the exhaustive samplings of the five other communities (Table 1). This, indeed, is the reason for the sampling incompleteness of as is the case in the western-Ghats of India [3]. Hence, the importance of assessing, and further analysing in detail, the current state of frog communities in this region. This implies not only drawing up species-lists as complete as possible but also to record (and if necessary to extrapolate numerically) the Species Abundance Distributions in each of these frog communities [16,30–35]. Species Abundance Distributions are not only of descriptive interest as a pattern, but also ought to be considered as a mean to address the process governing the hierarchical structuring of species abundances within communities. Thus, it is in this sense that should be understood the following warning by Southwood & Henderson [36]: "A great deal of time and expertise has been expended on the compilation of faunal lists for particular habitats, but the consequent increase in our understanding [...] is still meagre." noted that the three incompletely sampled communities B, F, H, are precisely those ones that are the most strongly structured, i.e. having the least even distribution of species abundances, as shown later in Fig. 12. Correlatively, in each of communities B, F, H, the rarest species have, by far, the smallest relative abundance levels: see Table 1. As a result, the product No.aSt of the abundance aSt of the rarest species times the sample-size No remains far less than unity in the incomplete samplings of communities B, F, H, while this product largely exceeds unity in the exhaustive samplings of the five other communities (Table 1). that is: This, indeed, is the reason for the sampling incompleteness of communities B, F, H (rather than a lesser sampling effort, as compared to the five exhaustively sampled communities). that is: Istr = log(a1/aSt)/log(a’1/a’St) (1) (1) Istr = log(a1/aSt)/log(a’1/a’St) (1 Istr = log(a1/aSt)/log(a’1/a’St) where a1 and aSt stand for the highest and the lowest abundances in the studied community and a’1 and a’St stand for the highest and the lowest abundances in the corresponding “broken-stick” distribution (i.e. computed for the same level of species richness St). Results are given in Table 2. 124 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 Table 2. The degree of hierarchical structuration (true unevenness) of species abundances, relevantly quantified by the structuring index ‘Istr’ defined as the average slope of the Species Abundance Distribution standardised to the average slope of the corresponding “broken- stick” distribution (data from Figs 4 to 11) Table 2. The degree of hierarchical structuration (true unevenness) of species abundances, relevantly quantified by the structuring index ‘Istr’ defined as the average slope of the Species Abundance Distribution standardised to the average slope of the corresponding “broken- stick” distribution (data from Figs. 4 to 11) Table 2. The degree of hierarchical structuration (true unevenness) of species abundances, relevantly quantified by the structuring index ‘Istr’ defined as the average slope of the Species Abundance Distribution standardised to the average slope of the corresponding “broken- stick” distribution (data from Figs. 4 to 11) Community St Sp. Abund. Distr. “broken-stick” structuring index log[a1/aSt] / log[a’1/a’St] a1 aSt a'1 a'St A 15 .1810 .0063 .2212 .0044 0.86 C 11 .2676 .0265 .2745 .0083 0.66 D 11 .2391 .0386 .2745 .0083 0.52 E 11 .2190 .0254 .2745 .0083 0.62 G 10 .2048 .0060 .2929 .0100 1.05 B 12.9 .3146 .00033 .2448 .0080 2.01 F 16.8 .2266 .00062 .2024 .0050 1.59 H 12.4 .2360 .00018 .2586 .0050 1.82 as is the case in the western-Ghats of India [3]. Hence, the importance of assessing, and further analysing in detail, the current state of frog communities in this region. This implies not only drawing up species-lists as complete as possible but also to record (and if necessary to extrapolate numerically) the Species Abundance Distributions in each of these frog communities [16,30–35]. Species Abundance Distributions are not only of descriptive interest as a pattern, but also ought to be considered as a mean to address the process governing the hierarchical structuring of species abundances within communities. 4.1 True Species Richness of Communities While the inventories of most invertebrate communities are often doomed to remain incomplete in practice - due to their usually high species richness including numerous rare species - the exhaustive sampling of vertebrates and, here of frog communities, is usually less out of reach. Thus, among the eight frog communities sampled by Katwate, Apte & Raut [3] in northern-western Ghats of India, five may be considered as already virtually exhaustive while the other three prove being only moderately incomplete (from 80% to 85% completeness: Table 1) but nevertheless require numerical extrapolation. Total species richness levels, either recorded (communities A, C, D, E, G) or extrapolated (communities B, F, H), range from 10 to 17 species. Interestingly, it should be Schematically, the structuration of species abundances within natural communities may result either from the influence of one dominant factor or from the interplay of numerous independent factors. As a result, the corresponding Species Abundance Distribution would fit more closely the “log-series” model or the “log-normal” model respectively [12–16]. Reliably testing each hypothesis requires, however, to consider the whole range of the Species Abundance Distribution [15,37–40]. Accordingly, if it cannot be recorded exhaustively, the Species Abundance Distribution must be numerically extrapolated. When considered along their whole range, the Species Abundance Distributions of the eight 125 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 species abundances, leaving aside the influence of the level of species richness, devoid of biological sense (Table 2). frog communities most often fit the “log-normal” model best (Figs. 2 & 3). This suggests that the hierarchical structuring of species abundances in these communities is generally driven by the combined influences of numerous, independent factors (related to ecological and/or historical constraints), rather than by the sole influence of one major, strongly determinant factor [15,39– 43]. frog communities most often fit the “log-normal” model best (Figs. 2 & 3). This suggests that the hierarchical structuring of species abundances in these communities is generally driven by the combined influences of numerous, independent factors (related to ecological and/or historical constraints), rather than by the sole influence of one major, strongly determinant factor [15,39– 43]. Thus removing the trivial influence of species richness St in the definition of the structuring index, Istr, warrants the independence a priori between Istr and St. Accordingly, an empirically observed dependence or, on the contrary, independence, between Istr and St will gain true biological significance. 4.3 The Degree of Hierarchical Structuring of Species Abundances in Each Community The degree of unevenness of the distribution of species abundances in a community mainly depends, of course, on the intensity of the hierarchical structuring. But, as aforementioned, the total species richness also influences “mechanically” the degree of unevenness, at the risk of providing, thus, a biased appreciation of the intensity of hierarchical structuring. This is because, the degree of species dominance unavoidably tends to decrease with increasing total species richness, all other things being equal: the dominance tends to be somewhat “diluted” by the increasing number of co- occurring species [1,18–20]. This trend – and its essentially numerical rather than biological origin – is clearly demonstrated theoretically by considering a constant process of abundance structuring (such as the random apportionment of abundances among species in the “broken-stick” model) applied to communities of varying species richness. The average steepness of the “broken- stick” distribution consistently decreases with increasing species richness: see Appendix 3. The intensity of the hierarchical structuring process, relevantly quantified by Istr, varies to a large extent, ranging from 0.52 to 2.01, according to communities (Fig. 12 and Table 2). No correlation is empirically highlighted between Istr and St (Fig. 12), which means that, here, the true intensity of the hierarchical structuring process, driving the species abundance pattern, develops independently of species richness in the community. This is an original and important finding that was by no means obvious a priori. Looking further in the detail of structuring process (Figs. 4 to 11, Fig. 12 and Table 3), it is worth noting that the hierarchical structuration may be either: - “regular”, i.e. with a gently varying rate in the abundance decrease, as observed in communities C, D, E, F, G, or - “regular”, i.e. with a gently varying rate in the abundance decrease, as observed in communities C, D, E, F, G, or - “irregular”, i.e. suddenly exhibiting a sharp acceleration of the decreasing rate of species abundances and, thus, a brutal and statistically significant recess of the abundance level for the few rarest species, as in communities A, B, H (Figs. 4, 9, 11). - “irregular”, i.e. suddenly exhibiting a sharp acceleration of the decreasing rate of species abundances and, thus, a brutal and statistically significant recess of the abundance level for the few rarest species, as in communities A, B, H (Figs. 4, 9, 11). 4.1 True Species Richness of Communities Hence, the interest of plotting Istr against St, as proposed in Figs. 12 & 13. In addition, this representation has the advantage of providing a synthetic overview of the main results derived from this study. 4.3 The Degree of Hierarchical Structuring of Species Abundances in Each Community 4.3 The Degree of Hierarchical Structuring of Species Abundances in Each Community Accordingly, a relevant appreciation of the intensity of the hierarchical structuring process requires to separate and leave out the purely “mechanical” influence of the level of species richness. This is appropriately achieved: Being limited to three communities out of eight, the “irregular” pattern invites to seek for some species-specific rather than generic causes. - Graphically, by plotting simultaneously the Species Abundance Distribution under study and the corresponding “broken-stick” model, computed for the same level of species richness (Figs. 4 to 11), - Graphically, by plotting simultaneously the Species Abundance Distribution under study and the corresponding “broken-stick” model, computed for the same level of species richness (Figs. 4 to 11), In this respect, the species list provided in Table 3 of the paper by Katwate, Apte & Raut [3] leads to the following remarks: - Quantitatively, by standardising the average slope of the Species Abundance Distribution under study to the average slope of the corresponding “broken-stick” model, leading to the definition of a structuring index, Istr (equation (1)), which thereby reflects the genuine contribution of the hierarchical structuring process driving - Fejervarya caperata Kuramoto et al. (in community A), Fejervarya cf. keralensis Dubois (in community B), Ramanella marmorata Jerdon (in comm. A & B), Uperodon globulosus Gunther (in community A), Sphaerotheca dobsonii 126 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 Gunther (in community F), Hylarana malabarica Tschudi (in community F) are by no means affected the same, although these species occur at similar or even lower levels of relative abundance. Boulenger (in communities A & H) are strongly affected by the recess of abundances, sharply disconnecting from the “broken-stick” model; while, on the contrary, y, Raorchestes bombayensis Annandale (in community C), Polypedates maculatus Gray (in community F), Indirana leithii Boulenger (in community F), Pseudophilautus cf. amboli Biju & Bossuyt (in community F), Indirana beddomii Now, what makes Fejervarya caperata, Fejervarya cf. keralensis, Ramanella marmorata, Uperodon globulosus, Sphaerotheca dobsonii specifically affected (as compared to the other six species cited above) remains conjectural. Fig. 12. A synthetic presentation of the situation of the eight frog communities with respect to two major quantitative features describing species communities: (i) the true (total) species richness St and (ii) the genuine intensity of the hierarchical structuring of species abundances, quantified by the structuring index Istr. White figures: exhaustively sampled communities; grey figures: communities requiring numerical extrapolations. The hierarchical structuring may be “regular” (i.e. 4.3 The Degree of Hierarchical Structuring of Species Abundances in Each Community roughly constant among species: discs) or “irregular” (i.e. with the sharp recess of abundance for the 3 or 4 rarest species: diamonds) 0,0 0,5 1,0 1,5 2,0 8 9 10 11 12 13 14 15 16 17 hierarchical structuring index Istr true species richness St G C E D F A H B less even more even 0,0 0,5 1,0 1,5 2,0 8 9 10 11 12 13 14 15 16 17 hierarchical structuring index Istr true species richness St G C E D F A H B less even more even Fig. 12. A synthetic presentation of the situation of the eight frog communities with Fig. 12. A synthetic presentation of the situation of the eight frog communities with respect to two major quantitative features describing species communities: (i) the true (total) species Fig. 12. A synthetic presentation of the situation of the eight frog communities with respect to two major quantitative features describing species communities: (i) the true (total) species richness St and (ii) the genuine intensity of the hierarchical structuring of species abundances, quantified by the structuring index Istr. White figures: exhaustively sampled communities; grey figures: communities requiring numerical extrapolations. The hierarchical structuring may be “regular” (i.e. roughly constant among species: discs) or “irregular” (i.e. with the sharp recess of abundance for the 3 or 4 rarest species: diamonds) Fig. 12. A synthetic presentation of the situation of the eight frog communities with respect to two major quantitative features describing species communities: (i) the true (total) species richness St and (ii) the genuine intensity of the hierarchical structuring of species abundances, quantified by the structuring index Istr. White figures: exhaustively sampled communities; grey figures: communities requiring numerical extrapolations. The hierarchical structuring may be “regular” (i.e. roughly constant among species: discs) or “irregular” (i.e. with the sharp recess of abundance for the 3 or 4 rarest species: diamonds) Table 3. Contrasting features of the Species Abundance Distributions between communities B, H, (and to a lesser extent A) and the other five communities: under a threshold abundance value ≈ 0.04, the decreasing rate of species abundances communities abruptly accelerates for communities A, B and H : see Figs. 4, 9, 11. 4.3 The Degree of Hierarchical Structuring of Species Abundances in Each Community The resulting sudden recess of species abundances below the “broken-stick” distribution, as a consequence of this sharp acceleration, is statistically significant (statistical test based on Bayesian inference: p < 0.05) What When B Sharp acceleration of decreasing rate For ai < 0.030 (i.e. ai < a9) H Sharp acceleration of decreasing rate For ai < 0.030 (i.e. ai < a9) A Less sharp acceleration of decreasing rate For ai < 0.023 (i.e. ai < a11) D No such acceleration Even down to ai = aSt = 0.038 C No such acceleration Even down to ai = aSt = 0.027 E No such acceleration Even down to ai = aSt = 0.015 F No such acceleration Even down to ai = aSt = 0.008 G No such acceleration Even down to ai = aSt = 0.006 127 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 4.5 Tentatively Speculating About the Effect of Climatic Change on the Features of the Species Abundance Distribution Yet, species-specific reasons for the brutal acceleration of the decreasing rate under a given threshold of relative abundance may be speculated and attributed to: (i) an intrinsic rarity of species being very rare all across their respective ranges of repartition ; Let consider a pejoration of environmental conditions assumed to occur around a given frog community – say community G, actually having 10 species with its Species Abundance Distribution shown in Figs. 2 and 8. This pejoration may be due, for example, to a steadily increasing climate change. It is assumed, here, that pejoration affects species abundances all the more than these abundances are already low, thus making the slope of the species abundances distribution becoming steadily steeper, as pejoration progressively increases. In addition, there inevitably exists some threshold level of absolute abundance below which the survival of a species becomes no longer possible, for example because of too low probability of finding mates for reproduction. Let this minimum survival threshold be fixed, for example, as half the absolute abundance of the rarest species (rank 10) as presently recorded, i.e. before pejoration goes on increasing. (ii) a local rarity of species, approaching, here, the limits of their respective ranges of repartition ; (iii) an occasional rarity of “vagrant” species, poorly adapted to the local ecological conditions prevailing in communities B, H, (A) and, accordingly, only present by more or less brief incursions; (iv) a rarity resulting from some negatively density-dependent detrimental factor, applying to those species specifically (such as the increasing difficulty of finding mates to reproduce, below some threshold level of abundance); ) (v) a rarity related to the stochastic character of colonisation events along time with, for example, exceptionally recent establishments assumed for those species in communities B, H, (A). ) (v) a rarity related to the stochastic character of colonisation events along time with, for example, exceptionally recent establishments assumed for those species in communities B, H, (A). Fig. 14 highlights graphically what is expected to happen with such increasing pejoration, in terms of (i) species richness St and (ii) hierarchical structuring intensity, Istr. Here, numerical extrapolations reach the limits of their explanatory capacities and going on any further would require specific biological knowledge regarding each of these species. 4.5 Tentatively Speculating About the Effect of Climatic Change on the Features of the Species Abundance Distribution In a first step (0  1), the hierarchical structuring intensity, Istr, will increase (see equation (1)) since a1/aSt steadily increases while a’1/a’St remains of course unchanged, as long as St remains equal to 10. 4.4 The Role of Forest Degradation on the Total Species Richness and the Intensity of the Hierarchical Structuring of Species Abundances Pristine forest (E, D): white figures ; intermediate degree of forest degradation (A, B, C, F): grey figures ; strong degrees of forest degradation (G, H): black figures 0,0 0,5 1,0 1,5 2,0 8 9 10 11 12 13 14 15 16 17 hierarchical structuring index Istr true species richness St G C E D F A H B less even more even 2 1 And so on, in a saw-tooth pattern, as shown in Fig. 14. (i) The steadily decrease of species richness St, consequence of the thinning effect due to the minimum abundance threshold for survival and (i) The steadily decrease of species richness St, consequence of the thinning effect due to the minimum abundance threshold for survival and g Let consider now the overall trend, behind the detail of the sequential, saw-tooth variations. As might have been expected, this overall trend mainly consist in: t, q g to the minimum abundance threshold for survival and (ii) A global increase of the genuine intensity of hierarchical structuring, Istr (note, yet, Fig. 13. Seeking for the possible influence of the degree of forest degradation on (i) the true species richness St and (ii) the intensity of the hierarchical structuring of species abundances Istr. Pristine forest (E, D): white figures ; intermediate degree of forest degradation (A, B, C, F): grey figures ; strong degrees of forest degradation (G, H): black figures Fig. 14. Simulation of the consequences of an increasing environmental pejoration (for example related to climate change,) on (i) the total species richness “St” and (ii) the genuine intensity of the hierarchical structuring “Istr” of species abundances for the frog community “G”. See text for the influence of this pejoration on the respective abundances of species 0,0 0,5 1,0 1,5 2,0 8 9 10 11 12 13 14 15 16 17 hierarchical structuring index Istr true species richness St G C E D F A H B less even more even 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 4 5 6 7 8 9 10 11 structuring intensity Istr species richness St 1 2 0 5 4 3 6 7 9 8 Let consider now the overall trend, behind the detail of the sequential, saw-tooth variations. 4.4 The Role of Forest Degradation on the Total Species Richness and the Intensity of the Hierarchical Structuring of Species Abundances As might have been expected, this overall trend mainly consist in: (ii) A global increase of the genuine intensity of hierarchical structuring, Istr (note, yet, 0,0 0,5 1,0 1,5 2,0 8 9 10 11 12 13 14 15 16 17 hierarchical structuring index Istr true species richness St G C E D F A H B less even more even 129 Fig. 13. Seeking for the possible influence of the degree of forest degradation on (i) the true species richness St and (ii) the intensity of the hierarchical structuring of species abundances Istr. Pristine forest (E, D): white figures ; intermediate degree of forest degradation (A, B, C, F): grey figures ; strong degrees of forest degradation (G, H): black figures Fig. 14. Simulation of the consequences of an increasing environmental pejoration (for example related to climate change,) on (i) the total species richness “St” and (ii) the genuine intensity of the hierarchical structuring “Istr” of species abundances for the frog community “G”. See text for the influence of this pejoration on the respective abundances of species 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 4 5 6 7 8 9 10 11 structuring intensity Istr species richness St 1 2 0 5 4 3 6 7 9 8 Fig. 13. Seeking for the possible influence of the degree of forest degradation on (i) the true species richness St and (ii) the intensity of the hierarchical structuring of species abundances Istr. Pristine forest (E, D): white figures ; intermediate degree of forest degradation (A, B, C, F): grey figures ; strong degrees of forest degradation (G, H): black figures Fig. 14. Simulation of the consequences of an increasing environmental pejoration (for example related to climate change,) on (i) the total species richness “St” and (ii) the genuine intensity of the hierarchical structuring “Istr” of species abundances for the frog community “G” S t t f th i fl f thi j ti th ti b d f i 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 4 5 6 7 8 9 10 11 structuring intensity Istr species richness St 1 2 0 5 4 3 6 7 9 8 Fig. 13. Seeking for the possible influence of the degree of forest degradation on (i) the true species richness St and (ii) the intensity of the hierarchical structuring of species abundances Istr. 4.4 The Role of Forest Degradation on the Total Species Richness and the Intensity of the Hierarchical Structuring of Species Abundances No consistent trend emerges in this respect that could yet have been expected (Fig. 13). Thus, pristine forests are by no means host to the most species-rich frog communities. And the hierarchical structuring of abundances fails, as well, to clearly correlate with the level of forest degradation. This suggests, among other possibilities, that historical aspects (such as the more or less stochastic succession of species colonisation events at a given site) may partially obliterate the more deterministic influence of environmental parameters, including the level of forest degradation. Alternatively, this may also signify that expectations on the subject (such as the compelling decrease of species richness with increasing disturbance) are simply irrelevant, here. Then, when the absolute abundance of the rarest species (rank 10) finally falls below the survival threshold, it disappears (step 1  2) and, consequently, the hierarchical structuring intensity, Istr, abruptly decreases because species ranked 9 – now becoming the rarest species – is appreciably more abundant than was species ranked 10 at the moment of its disappearance. Thus, at stage 2, Istr = log(a1/a9)/log(a’1/a’9) is lower than log(a1/a10)/log(a’1/a’10) at stage 1 (namely: Istr = 0.82 and Istr = 1.30, respectively). Then, with now St = 9, the same happen as for the first step at St = 10: the hierarchical structuring intensity Istr increases, as long as St remains equal to 9: (step 2  3). 128 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 And so on, in a saw-tooth pattern, as shown in Fig. 14. Let consider now the overall trend, behind the detail of the sequential, saw-tooth variations. As might have been expected, this overall trend mainly consist in: (i) The steadily decrease of species richness St, consequence of the thinning effect due to the minimum abundance threshold for survival and (ii) A global increase of the genuine intensity of hierarchical structuring, Istr (note, yet, Fig. 13. Seeking for the possible influence of the degree of forest degradation on (i) the true species richness St and (ii) the intensity of the hierarchical structuring of species abundances Istr. 4.4 The Role of Forest Degradation on the Total Species Richness and the Intensity of the Hierarchical Structuring of Species Abundances Pristine forest (E, D): white figures ; intermediate degree of forest degradation (A, B, C, F): grey figures ; strong degrees of forest degradation (G, H): black figures Fig. 14. Simulation of the consequences of an increasing environmental pejoration (for example related to climate change,) on (i) the total species richness “St” and (ii) the genuine intensity of the hierarchical structuring “Istr” of species abundances for the frog community “G” See text for the influence of this pejoration on the respective abundances of species Fig. 14. Simulation of the consequences of an increasing environmental pejoration (for example related to climate change,) on (i) the total species richness “St” and (ii) the genuine intensity of the hierarchical structuring “Istr” of species abundances for the frog community “G”. See text for the influence of this pejoration on the respective abundances of species 129 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 Fig. 15. The new Species Abundance Distribution when community “G” has reached stage 2 (St = 9 ; Istr = 0.83) as a consequence of the environmental pejoration (see Fig. 14). The arrow highlights the beginning separation from the log-normal model (dotted line) towards the “log- series” model (double line). Initial situation (step 0) of community “G” is provided at Fig. 2 0,001 0,01 0,1 1 0 1 2 3 4 5 6 7 8 9 10 species relative abundance species abundance rank FG FG FG Fig. 15. The new Species Abundance Distribution when community “G” has reached stage 2 (St = 9 ; Istr = 0.83) as a consequence of the environmental pejoration (see Fig. 14). The arrow highlights the beginning separation from the log-normal model (dotted line) towards the “log- series” model (double line). Initial situation (step 0) of community “G” is provided at Fig. 2 that, without standardisation to the “broken-stick”, the apparent unevenness level would, on the contrary, seem to decrease, instead of increase; see [35]). from the metapopulation context; that is implicitly out of reach from external inputs of new species that would possibly be better adapted to the currently evolving local environment. Such colonisation by “appropriate” new species would likely more or less compensate for both the reduction of abundances and the ultimate disappearance of the successively rarest species and, thus, would tend to more or less buffer the consequences of environmental pejoration on the shape of the Species Abundance Distribution. 5. CONCLUSION Estimating the level of total species richness in animal communities, as well as getting insights on the genuine causes and intensity of the hierarchical structuring of species abundances, are major topics that likely contribute to a more comprehensive understanding of these communities. Acquiring such knowledge also provides a basic reference for the future monitoring of the consequences of the on-going climatic change on living communities. However, this program imperatively requires performing exhaustive species inventories or, if impractical, impose to extrapolate numerically the incomplete samplings with minimum bias. An appropriate methodological approach in this respect is provided above and its implementation is exemplified by the treatment of a series of communities of tropical frogs, a particularly exposed and endangered group of animals, potentially under threat of excessive heat and drought. 4. Pounds JA, Crump ML. Amphibian declines and climatic disturbance: The case of the Golden Toad and the Harlequin Frog. Conservation Biology. 1994;8:72-85. g gy 5. Mc Callum ML. Future climate change spells catastrophe for Blanchard’s cricket frog, Acris blanchardi (Amphibia: Anura: Hylidae). Acta Herpetologica. 2010;5(1): 119-130. 6. Coddington JA, Agnarsson I, Miller JA, Kuntner M, Hormiga G. Undersampling bias: the null hypothesis for singleton species in tropical arthropod surveys. Journal of Animal Ecology. 2009;78:573- 584. 7. Gotelli NJ, Colwell RK. Estimating species richness. In: Biological Diversity: Frontiers in Measurement and Assessment. A.E. Magurran and B.J. McGill (Eds.). 2010;39- 54. Oxford University Press, Oxford. 345. 8. 8. Gotelli NJ, Chao A. Measuring and estimating species richness, species diversity, and biotic similarity from sampling data. In: Levin S.A. (ed.) Encyclopedia of Biodiversity. Second edition. Waltham, MA: Academic Press. 2013;5:195-211. ACKNOWLEDGEMENTS A fruitful advice from the Editor, on a previous version of the manuscript, is gratefully acknowledged. 9. ; Béguinot J. Theoretical derivation of a bias-reduced expression for the extrapolation of the Species Accumulation Curve and the associated estimation of total species richness. Advances in Research. 2016;7(3):1-16. DOI: 10.9734/AIR/2016/26387; <hal- 01367803> 4.4 The Role of Forest Degradation on the Total Species Richness and the Intensity of the Hierarchical Structuring of Species Abundances After having considered the consequences of environmental pejoration on species richness and the intensity of hierarchical structuring, let move now to the expected effect on the shape of the Species Abundance Distribution and its related functional significance. One might expect that, as the pejoration goes on increasing, the role of this detrimental factor would progressively become more and more predominant on the other ecological factors that drive the distribution of species abundances. Accordingly, it is expected that the Species Abundance Distribution progressively shifts from its original compliance with the “log-normal” model (see Figs. 2 and 3) towards a progressively better compliance with the “log-series” model [12–16]. This, indeed, is what is demonstrated by the simulation. Thus, as soon as stage 2 is reached (Fig. 15), the Species Abundance Distribution already clearly disconnects from the “log-normal” model, and is already halfway towards the “log- series” model. To now conclude this speculative section, I would like to draw attention on the practical interest of considering Species Abundance Distributions in the context of evolving environmental conditions. Devoting attention at each of the three main aspects that shape Species Abundance Distributions (i.e. species richness, hierarchical structuring intensity and selective fitting to either reference models), will offer a corresponding set of typically diagnostic features, able to reliably highlight the consequences of an increasing degree of environmental pejoration. 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APPENDIX 1 Each solution Rx (N) is appropriate for a given range of values of f1 compared to the other numbers fx (according to [9]): * for f1 up to f2  R1 (N) = (R(N0) + f1) – f1.N0/N * for f1 up to f2  R1 (N) = (R(N0) + f1) – f1.N0/N ( ) ( ( ) ) * for larger f1 up to 2f2 – f3  R2 (N) = (R(N0) + 2f1 – f2) – (3f1 – 2f2).N0/N – (f2 – f1).N0 2/N2 * for larger f1 up to 2f2 – f3  R2 (N) = (R(N0) + 2f1 – f2) – (3f1 – 2f2).N0/N – (f2 – f1).N0 2/N2 * for larger f1 up to 3f2 – 3f3 + f4  R3 (N) = (R(N0) + 3f1 – 3f2 + f3) – (6f1 – 8f2 + 3f3).N0/N – (– 4f1 + 7f2 – 3f3).N0 2/N2 – (f1 – 2f2 + f3).N0 3/N3 * for larger f1 up to 3f2 – 3f3 + f4  R3 (N) = (R(N0) + 3f1 – 3f2 + f3) – (6f1 – 8f2 + 3f3).N0/N – (– 4f1 + 7f2 – 3f3).N0 2/N2 – (f1 – 2f2 + f3).N0 3/N3 * for larger f1 up to 4f2 – 6f3 + 4f4 – f5  R4 (N) = (R(N0) + 4f1 – 6f2 + 4f3 – f4) – (10f1 – 20f2 + 15f3 – 4f4).N0/N – (– 10f1 + 25f2 – 21f3 + 6f4).N0 2/N2 – (5f1 – 14f2 + 13f3 – 4f4).N0 3/N3 – (– f1 + 3f2 – 3f3 + f4).N0 4/N4 * for f1 larger than 4f2 – 6f3 + 4f4 – f5  R5 (N) = (R(N0) + 5f1 – 10f2 + 10f3 – 5f4 + f5) – (15f1 – 40f2 + 45f3 – 24f4 + 5f5).N0/N – (– 20f1 + 65f2 – 81f3 + 46f4 – 10f5).N0 2/N2 – (15f1 – 54f2 + 73f3 – 44f4 + 10f5).N0 3/N3 – (– 6f1 + 23f2 – 33f3 + 21f4 – 5f5).N0 4/N4 – (f1 – 4f2 + 6f3 – 4f4 + f5).N0 5/N5 The associated non-parametric estimators of the number ΔJ of missing species in the sample [with ΔJ = R(N=∞) – R(N0) ] are derived immediately: The associated non-parametric estimators of the number ΔJ of missing species in the sample [with ΔJ = R(N=∞) – R(N0) ] are derived immediately: * f1 < f2  ΔJ1 = f1 ; R1 (N) * f2 < f1 < 2f2 – f3  ΔJ2 = 2f1 – f2 ; R2 (N) * 2f2 – f3 < f1 < 3f2 – 3f3 + f4  ΔJ3 = 3f1 – 3f2 + f3 ; R3 (N) * 3f2 – 3f3 + f4 < f1 < 4f2 – 6f3 + 4f4 – f5  ΔJ4 = 4f1 – 6f2 + 4f3 – f4 ; R4 (N) * f1 > 4f2 – 6f3 + 4f4 – f5  ΔJ5 = 5f1 – 10f2 + 10f3 – 5f4 + f5 ; R5 (N) N.B. 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Species diversity and community similarity in fluctuating environments: Parametric approaches using species abundance 133 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 APPENDIX 1 Bias-reduced extrapolation of the Species Accumulation Curve and associated bias-reduced estimation of the number of still unrecorded species, based on the recorded numbers of species occurring 1 to 5 times Consider the survey of an assemblage of species of size N0 (with sampling effort N0 typically identified either to the number of recorded individuals or to the number of sampled sites, according to the inventory being in terms of either species abundances or species incidences), including R(N0) species among which f1, f2, f3, f4, f5, of them are recorded 1, 2, 3, 4, 5 times respectively. The following procedure, designed to select the less-biased solution, results from a general mathematical relationship that constrains the theoretical expression of any theoretical Species Accumulation Curves R(N) (see [9,44,45]): ∂xR(N)/∂Nx = (-1)(x-1) fx(N) /CN, x ≈ (– 1)(x-1) (x!/Nx) fx(N) ( ≈ as N >> x) (A1.1) , x ≈ (– 1)(x-1) (x!/Nx) fx(N) ( ≈ as N >> x) (A1.1) (A1.1) Compliance with the mathematical constraint (equation (A.1)) warrants reduced-bias expression for the extrapolation of the Species Accumulation Curves R(N) (i.e. for N > N0). Below are provided, accordingly, the polynomial solutions Rx (N) that respectively satisfy the mathematical constraint [1], considering increasing orders x of derivation ∂xR(N)/∂Nx. APPENDIX 1 1: As indicated above (and demonstrated in details in [9]), this series of inequalities define the ranges that are best appropriate, respectively, to the use of each of the five estimators, JK-1 to JK-5. That is the respective ranges within which each estimator will benefit of minimal bias for the predicted number of missing species. 134 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 Besides, it is easy to verify that another consequence of these preferred ranges is that the selected estimator will always provide the highest estimate, as compared to the other estimators. Interestingly, this mathematical consequence, of general relevance, is in line with the already admitted opinion that all non-parametric estimators provide under-estimates of the true number of missing species [7,8,46– 48]. Also, this shows that the approach initially proposed by Brose et al. [49] – which has regrettably suffered from its somewhat difficult implementation in practice – might be advantageously reconsidered, now, in light of the very simple selection key above, of far much easier practical use. N.B. 2: In order to reduce the influence of drawing stochasticity on the values of the fx, the as- recorded distribution of the fx should preferably be smoothened: this may be obtained either by rarefaction processing or by regression of the as-recorded distribution of the fx versus x. N.B. 3: For f1 falling beneath 0.6 x f2 (that is when sampling completeness closely approaches exhaustivity), then Chao estimator may alternatively be selected: see reference [10]. Figs. APPENDIX 1 A1.1, A1.2, A1.3 – The recorded values of the numbers fx of species recorded x-times (grey discs) and the regressed values of fx (black discs) derived to reduce the consequence of stochastic dispersion for the three incomplete samplings of frog communities labelled B, F, H 0,0 0,2 0,4 0,6 0,8 1,0 1,2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 fx x B 0,0 0,5 1,0 1,5 2,0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 fx x F 0,0 0,2 0,4 0,6 0,8 1,0 1,2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 fx x H 0,0 0,2 0,4 0,6 0,8 1,0 1,2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 fx x H 0,0 0,2 0,4 0,6 0,8 1,0 1,2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 fx x B 0,0 0,5 1,0 1,5 2,0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 fx x F B Figs. A1.1, A1.2, A1.3 – The recorded values of the numbers fx of species recorded x-times (grey discs) and the regressed values of fx (black discs) derived to reduce the consequence of stochastic dispersion for the three incomplete samplings of frog communities labelled B, F, H 135 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 Correction and extrapolation of the as-recorded Species Abundance Distribution (S.A.D.) N.B.: details regarding the derivation of the following expressions are provided in [1]. 1) Correction for bias of the recorded part of the S.A.D. 1) Correction for bias of the recorded part of the S.A.D. The bias-corrected expression of the true abundance, ãi, of species of rank ‘i' in the S.A.D. is given by: (A2.1) (A2.1) ãi = pi.(1+1/ni).(1–f1/N0)/(1+R0/N0) where N0 is the actually achieved sample size, R0 (=R(N0)) the number of recorded species, among which a number f1 are singletons (species recorded only once), ni is the number of recorded individuals of species ‘i’, so that pi = ni/N0 is the recorded frequency of occurrence of species ‘i', in the sample. The crude recorded part of the “S.A.D.” – expressed in terms of the series of as-recorded frequencies pi = ni/N0 – should then be replaced by the corresponding series of expected true abundances, ãi, according to equation (A2.1). 2) Extrapolation of the recorded part of the S.A.D. accounting for the complementary abundance distribution of the set of unrecorded species The following expression stands for the estimated abundance, ai, of the unrecorded species of rank i (thus for i > R0): ai = (2/Ni).(1– [∂R(N)/∂N]Ni)/(1+ R(Ni)/Ni) (A2.2) which, in practice, comes down to: ai ≈ (2/Ni)/(1+ R(Ni)/Ni) (A2.3) as f1(N) already becomes quite negligible as compared to N for the extrapolated part. This equation provides the extrapolated distribution of the species abundances ai (for i > R(N0)) as a function of the least-biased expression for the extrapolation of the species accumulation curve R(N) (for N > N0), ‘i' being equal to R(Ni). The key to select the least-biased expression of R(N) is provided at Appendix 1. 136 Béguinot; IJECC, 8(2): 118-137, 2018; Article no.IJECC.2018.009 APPENDIX 3 The trivial (“mechanistic”) contribution of the level of species richness to the degree of structuring of species abundances The trivial (“mechanistic”) contribution of the level of species richness to the degree of structuring of species abundances All things equal otherwise, the larger the species richness, the weaker is the slope of the Species Abundance Distribution. This can be easily exemplified and quantified, on a theoretical basis, by considering a theoretically constant structuring process - such as the random distribution of the relative abundances that characterises the “broken-stick” distribution model. By applying this model successively to a series of communities with increasing species richness, a steadily decrease of the slope of abundance distributions is highlighted: Fig. A3. Fig. A3. The “broken-stick” distribution model applied to species communities with increasing species richness St = 10, 20, 30, 60. Although the theoretical structuring process involved in the “broken-stick” model remains unchanged (random apportionment of relative abundances among member species), the slope of the species abundance distribution strongly depends upon (and monotonously decreases with) the level of species richness St © 2018 Béguinot; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 0,0001 0,0010 0,0100 0,1000 1,0000 0 10 20 30 40 50 60 species relative abundance species abundance ranking St = 10 St = 20 St = 30 St = 60 Fig. A3. The “broken-stick” distribution model applied to species communities with increasing species richness St = 10, 20, 30, 60. Although the theoretical structuring process involved in the “broken-stick” model remains unchanged (random apportionment of relative abundances among member species), the slope of the species abundance distribution strongly depends upon (and monotonously decreases with) the level of species richness St © 2018 Béguinot; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 Béguinot; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 137
https://openalex.org/W2980676231	https://europepmc.org/articles/pmc6802107?pdf=render	English	null	Evaluation of left ventricular remodelling in young Afro-Caribbean athletes	Cardiovascular ultrasound	2,019	cc-by	6,895	Abstract Background: Cardiac adaptation to intense physical training is determined by many factors including age, gender, body size, load training and ethnicity. Despite the wide availability of ECG analysis, with a higher presence of abnormalities in different races, echocardiographic studies on young Afro-Caribean (AA) and Caucasian athletes (CA) are lacking in literature. We aimed to assess the effect in the secondary LV remodelling of load training in young AA players compared to matched CA players. Method: Seventy-seven AA and 53 CA matched soccer players (mean age 17.35 ± 0.50 and 18.25 ± 0.77 y) were enrolled. They were evaluated with echocardiography. A subgroup of 30 AA and 27 CA were followed up for a period of 4 years. The myocardial contractile function was evaluated by speckle-tracking echocardiographic global longitudinal strain (GLS). Results: No significant differences were found in weight and height and in blood pressure response to maximal ergometer test in either group. In AA a higher level of LV remodelling, consisting in higher LV wall thickness, higher interventricular septum (IVS) and posterior wall (PW) thickness were found (IVS: 10.04 ± 0.14 and 9.35 ± 0.10 in AA and CA respectively, p < 0.001. PW: 9.70 ± 0.20 and 9.19 ± 0.10 mm in AA and CA respectively, p < 0.05). Strain data showed no significant differences between the two groups (22.35 ± 0.48 and 23.38 ± 0.69 in AA (n = 27) and CA (n = 25), respectively). At the beginning of the follow-up study AA showed a significantly higher left ventricular remodelling (IVS = 9.29 ± 0.3 and 8.53 ± 0.12 mm in AA and CA respectively, p < 0.002. PW = 9.01 ± 0.2 and 8.40 ± 0.20 in AA and CA respectively, p = 0.1). During the next four years of follow-up we observed a regular parallel increase in LV wall thickness and chamber diameters in both groups, proportionally to the increase in body size and LV mass. (IVS = 10.52 ± 0.17 and 9.03 ± 0.22 mm in AA and CA respectively, p < 0.001. PW: 10.06 ± 0.17 and 8.26 ± 0.19 mm in AA and CA respectively, p < 0.001). Conclusion: The study shows that the ventricular remodelling observed in AA appears to be a specific phenotype already present in pre-adolescence. RESEARCH Open Access Evaluation of left ventricular remodelling in young Afro-Caribbean athletes Giorgio Galanti, Loira Toncelli, Benedetta Tosi, Melissa Orlandi, Chiara Giannelli, Laura Stefani, Gabriele Mascherini and Pietro A Modesti Abstract These data also suggest that genetic/ethnic factors play a central role in left ventricular remodelling during the first years of life in elite athletes. Keywords: Afro-Caribbean athletes (AA), Strain, Race Correspondence: giorgio.galanti@unifi.it Department of Experimental and Clinical Medicine-Sports Medicine and Exercise Unit, University of Florence, AOUC, Careggi, Florence, Italy * Correspondence: giorgio.galanti@unifi.it Department of Experimental and Clinical Medicine-Sports Medicine and Exercise Unit, University of Florence, AOUC, Careggi, Florence, Italy Galanti et al. Cardiovascular Ultrasound (2019) 17:20 https://doi.org/10.1186/s12947-019-0169-8 Galanti et al. Cardiovascular Ultrasound (2019) 17:20 https://doi.org/10.1186/s12947-019-0169-8 Galanti et al. Cardiovascular Ultrasound https://doi.org/10.1186/s12947-019-0169-8 © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Aim of the study The aim was to study cardiovascular and, specifically, left ventricular remodelling in young African Americans compared to young Caucasian soccer players. All the athletes belonged to the same team, trained with the same load and frequency, and had the same lifestyle. The role of a specific kind of sport on cardiovascular re- modelling (soccer) was studied. Furthermore, Strain ana- lysis, not commonly calculated in adolescent athletes, was used to evaluate myocardial contractile function in ultrasound imaging. Notably, a subgroup of the athletes enrolled in our study was evaluated for a 4-year follow-up, in order to assess how left ventricular remodelling changed in time. Introduction data are required to detect ethnic-specific cardiac adap- tations to exercise. Regular and intense physical exercise induces several morphological and functional heart modifications, char- acterizing the so-called “athlete’s heart”. This physio- logical “adaptive” myocardial hypertrophy is related to the intensity and kind of sport practiced. This ventricu- lar adaptive remodelling is reversible and characterized by preserved systolic and diastolic function, resulting in a more efficient cardiac pump [1, 2]. It has been widely demonstrated that cardiovascular adaptation to sports training is influenced by many factors including body size, age, gender, life style, load training and ethnicity [3, 4]. Ethnicity is considered a determining factor in the electrical, structural and functional remodelling of the athlete’s heart [4], although knowledge regarding cardiac adaptation to exercise largely derives from echocardio- graphic evaluation of adult CAs. The exponential in- crease in the number of multiethnic athletes competing at high level highlights the lack of studies on the role of ethnicity in myocardial adaptation to exercise and the need to define ethnicity-specific patterns. It is well estab- lished that ECGs recorded in elite athletes usually present characteristic changes reflecting structural and electrical remodelling of the heart [5]. However, these abnormalities of athletes’ ECGs may be difficult to differ- entiate from disease which carries a risk of exercise- related sudden cardiac death. The most common training-related ECG changes [6] are sinus bradycardia, first-degree AV block, incomplete right bundle branch block, early repolarization and isolated QRS voltage cri- teria for left ventricular hypertrophy [7]. “Uncommon” ECG changes have also been described in literature: these uncommon patterns, mostly derived from CA ECG studies, appear to be more frequent if we consider young AC athletes [8], in whom the application of current ECG criteria would result in an abnormal ECG. In fact T-wave inversion in 2 or more contiguous leads, ST-segment depression elevation and Brugada-like repo- larisation are some of the most frequently found changes in healthy AA athletes [4]. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Galanti et al. Cardiovascular Ultrasound (2019) 17:20 Galanti et al. Cardiovascular Ultrasound (2019) 17:20 Page 2 of 8 Echocardiographic study Trans-thoracic echocardiography was conducted by two experienced and certified cardiologists using a commer- cially available ultrasound system: iE33 Philips Medical System (Bothell, WA). These specialists work together and therefore the reproducibility of data is high and the inter-observer variability low (< 5%). Furthermore, at least 5 double blind echo tests were carried out after 3 days, in order to confirm the overlap of the data ob- tained. According to the ASE guidelines [17], all the systo-diastolic LV parameters were calculated at rest in parasternal long-axis view: interventricular septum (IVS), posterior wall thickness (PW), left ventricle end- Exercise stress testing The 2D images of four-chamber views were post- processed with X-Strain software (Philips Q-station) to provide an angle-independent tool for the evaluation of velocities and strain. This software allows automatic evaluation of both endocardial and sub-endocardial edges from 2D B-mode echocardiographic clips recorded at rest. Strain analysis by speckle tracking is independent of translational motion and of tethering effects of the nearby regions, allowing uniformity of measurements through the normal LV myocardium. The endocardial border is drawn by the operator in a four-chamber view on a single frame from one annulus to another; the first and last points delineate the mitral plane. Since the glo- bal longitudinal strain (GLS) has better reproducibility than other deformation indices [20], this evaluation was preferred. An upright treadmill stress test was performed with the modified Bruce protocol [15]. ECGs were recorded at 1- min intervals and blood pressure was measured in pre- test, at the apex of the exercise and at the 4th minute of recovery. Athletes exercised to volitional exhaustion and were assessed specifically for the development of abnor- mal or flat blood pressure response, ischemic changes, arrhythmias or symptoms [16]. ECG Left ventricular hypertrophy (LVH) was defined as a maximal LVMI of 102 g/m2, in agreement with current guidelines [19]. Standard 12-lead ECG was performed with the subject supine, after a few minutes of rest with normal breath- ing, and recorded at 25 mm/s. ECG analysis was per- formed according to the previously described criteria [12]. Specifically, we measured heart rate (beats/min), PR interval (ms), QRS duration (ms), QT interval cor- rected for the heart rate (s) [13], presence of Q waves (≥2 mm in depth in ≥2 leads), R/S-wave amplitude in precordial leads (S1 + R5) (mm), and Sokolow-Lyon cri- terion for LV hypertrophy (positive if ≥35 mm) [14], presence and shape (concave or domed) of ST-segment elevation (≥1 mm, in ≥2 contiguous leads), presence of J- wave (≥1 mm), or ST-segment slurring, T-wave inversion (≥2 mm in depth in ≥2 contiguous leads, with exclusion of III and a VR), and flat/biphasic T-wave pattern (in ≥2 contiguous leads). Right Ventricle (RV) chamber was studied morphologic- ally from long axis view (RV diameter) and investigated functionally by apex four-chamber view (contractile func- tion and TAPSE). Diastolic parameters were measured by Doppler ana- lysis in the presence of a stable RR interval and in three different but sequential measurements from the four- chamber view. Transmitral flow of E and A wave peak velocities, isovolumetric relaxation, deceleration times and E/A ratio were obtained. Eventual presence of valve insufficiency or stenosis was determined by continuous wave and colour Doppler analysis from the five-chamber view. The echocardiographic evaluation was also useful for excluding any cardiac abnormalities, i.e. interventric- ular or interatrial septal defects, or any pulmonary hypertension and other congenital heart disease. Materials and methods Subjects Approval for the whole procedure was obtained Page 3 of 8 Page 3 of 8 Galanti et al. Cardiovascular Ultrasound (2019) 17:20 diastolic diameter (LVEDd), end-systolic diameter (LVESd), right ventricle diameter (RVd), aortic root diameter (AOR) and left atrium antero-posterior dimen- sions. Assessment of left ventricular mass (LVM [g/m2) was obtained according to the formula of Devereux and it was also indexed to BSA (LVMI [g/m2]) [18]. Consid- ering the regularity of the athletes’ left ventricular cham- ber geometry, the ejection fraction (EF%) was calculated according to the formula (LVEDd - LVESd/ LVEDd), which assembles volumes to diameters. from the Human Research and Ethics Committee of the University of Florence. The studies conformed to the Declaration of Helsinki. from the Human Research and Ethics Committee of the University of Florence. The studies conformed to the Declaration of Helsinki. Materials and methods Subjects Our study enrolled 77 young sexually developed soccer players of Afro-Caribbean origin (Afro-Caribbean ath- letes: AA; mean age 17.34 ± 0.50 yrs) and 53 matched Caucasian soccer players (Caucasian athletes: CA; mean age18.35 ± 0.67 yr). In agreement with the common def- inition in literature, the AA (Afro–Caribbean athletes), despite the fact that not all of them came strictly from America, will be called “African-Americans”. Athletes with a previous personal or family history of cardiac or pulmonary disease, family history of premature (≤40 yr) sudden cardiac death or cardiomyopathy were excluded from the study. The athletes were members of the same soccer team, trained with the same load 6 times a week, each training session lasting ≥2 h, and they had a similar lifestyle. 30 AA and 27 CA were evaluated with echocardiography for a 4- year follow-up (mean age at first evaluation: 12.47 ± 0.60 and 13.60 ± 0.38 for AA and CA, respectively). Players who gave up football or changed team, as normally occurs in seasonal recruitment, were not included in the follow- up study (47 AA and 26 CA). Morphological and structural myocardial adaptations to exercise have been largely studied by echocardiog- raphy. The secondary LV remodelling to load training consists of an increased LV wall thickness and an aug- mented LV cavity dimension with preserved systolic and diastolic function [9]. Although the normal upper limits of the athlete’s heart have been established [9], there is still a grey zone where physiological heart adaptation overlaps with cardiac diseases implicated in exercise- related sudden cardiac death [10]. This is especially true in young AA athletes whose ECG and echocardiographic patterns could be markedly altered. Despite the wide availability of ECG and echocardiographic analysis in CA, less has been done in young elite AA athletes. More All athletes were evaluated yearly at the beginning of the season at the Sports Medicine Department of the University of Florence – Italy. They underwent assess- ment with a health questionnaire (to exclude any famil- iarity for chronic or metabolic diseases, use of illegal substances and any symptoms); physical examination, rest ECG, maximal ergometric test and 2-dimensional echocardiography as part of a pre-participation cardiac evaluation. We also evaluated contractile function using speckle-tracking echocardiographic global longitudinal strain (GLS). All athletes provided written consent for the evalu- ation. Clinical evaluation and anthropometric parameters Athletes underwent clinical evaluation, including weight, height and blood pressure measurement at rest. Body mass index (BMI) was calculated by the formula “weight [Kg]/height [m2]”. Body surface area (BSA in [m2]) was obtained using the formula of DuBois and DuBois for BSA (0.007184 x (Height (cm) 0.725 x Weight (kg) 0.425) [11]. LV Relative Wall Thickness (RWT) was determined as the ratio of wall thickness and end-diastolic diameter ac- cording to the formula: [2x(PWTd+IVSd)/LVDd] [19]. 5. Follow-up period Echocardiography showed parameters within normal limits in all athletes. Ultrasound analysis of the left ven- tricular remodeling showed normal and similar chamber diameters and volume values in both groups, with a pre- served EF, but a statistically significant increase of IVS thickness. PW thickness was present in AA compared to CA. IVS thickness was 10.1 ± 0.1 and 9.4 ± 0.1 mm in AA and CA respectively; PW thickness was 9.7 ± 0.1 and 9.2 ± 0.1 mm in AA and CA, respectively. Calculation of indexed left ventricular mass (iLVM) was within the nor- mal range and comparable in the two groups, as well as the LVEDd. End diastolic volume was significantly dif- ferent in the two groups, despite being potentially in agreement with the different morphology of the ethnic characteristics and slightly enhanced by the acceptable inter-operator variability. The differences found did not support any clinical implications. The systolic and Thirty AA and 27 CA were selected from the main group and were followed-up annually for 4 years with Table 2 Principal echocardiographic parameters in AA and CA Parameter AA (n = 77) CA (n = 53) P IVS(mm) 10,1 ± 0,1 9,4 ± 0,1 < 0,001 PW(mm) 9,7 ± 0,1 9,2 ± 0,1 < 0.001 RWT (%) 0,39 ± 0,1 0,38 ± 0,2 0,8 iLVM(g/m2) 99,5 ± 1,7 97,4 ± 1,5 0,09 LVEDd(mm) 50 ± 0,4 51 ± 0,6 0,06 LVESd(mm) 31 ± 0,5 32 ± 0,3 0,2 EDV (ml) 116 ± 2 124 ± 2 < 0,005 ESV (ml) 41 ± 2,8 41 ± 0,9 0,9 LA (mm) 33 ± 2,8 32,1 ± 3 0,2 RA (mm) 30,5 ± 1,2 29,8 ± 2,8 0,3 EF (%) 66 ± 0,4 66 ± 0,6 0,8 RVd (mm) 22 ± 0,2 23 ± 1,5 0,2 E (cm) 86,29 ± 2,1 85,24 ± 1,5 0,3 A (cm) 44,44 ± 2,1 44,78 ± 0,9 0,7 IVRT (t) 72,1 ± 1,2 73,8 ± 0,8 0,7 DT (cm) 188,9 ± 11,9 172,9 ± 2,5 0,07 E/A 1,96 ± 1 1,94 ± 1,2 0,08 Legend. PW: Posterior Wall; IVS: Inter Ventricular Septum; RWT: relative wall thickness; iLVM: Left Ventricular Mass index. Results No statistically significant differences in mean age and anthropometric parameters, as well as in lifestyle and training modalities, were present in either group (77 AA and 53 CA). Mean age, height, weight, BMI, resting heart rate and basal systolic and diastolic blood pressure (SBP, DBP) were similar in both groups (Table 1). 12 leads ECG Strain analysis was performed in a small and apparently homogeneous subgroup of players, in terms of age, training and anthropometrical parameters. It was calcu- lated in order to to have a deeper evaluation of the myo- cardial contractile function of the subjects enrolled. A basal and stress exercise 12-lead ECG was carried out with every athlete. Basal ECGs showed that altered repo- larization patterns such as inverted T waves in leads V2- V4 and early ventricular repolarization were more present and statistically significant in AA compared to CA, confirming data present in literature nowadays. Specifically, 55% AA and only 7% CA showed these al- terations [21]. The group included only 27 AA and 25 CA (mean age 18 yrs. for both groups) in order to find praecox impair- ment in systolic function. Results of GLS were within normal limits in both groups, without any significant dif- ference: −22.35 ± 0.48 and −23.38 ± 0.69 in AA and CA. Statistical analysis Statistical significance was considered at P < 0.05. Student’s t test was conducted post hoc and reported. Data are expressed as means±SD. Comparison of change scores was undertaken using T test. The correlation between posterior Page 4 of 8 Galanti et al. Cardiovascular Ultrasound (2019) 17:20 Page 4 of 8 wall and interventricular septum thicknesses and ECG al- terations was investigated with the Spearman non paramet- ric test. diastolic function was preserved and comparable in both groups. RV chambers showed normal diameter, function and morphology (Table 2). In the AA group we also looked for the possible pres- ence of a correlation between the alterations of ventricu- lar repolarization observed in baseline ECG and the values of wall thickness with the Spearman non- parametric test. The analysis showed no significant cor- relation between ECG changes and the thickening de- gree of the IVS and PW. 5. Follow-up period LVEDV: Left Ventricle End Diastolic Volume; LVESV: Left Ventricle End Systolic Volume; ESV: End Systolic Volume; LA: left atrium; RA: right atrium; EF: Ejection Fraction; RVd: Right Ventricle diameter; E wave; A wave; IVTR: isovolumic relaxation time; DT: Deceleration Time; A statistically significant difference in IVS thickness is present in AA compared to CA. Data in bold are significant Table 2 Principal echocardiographic parameters in AA and CA Parameter AA (n = 77) CA (n = 53) P Table 1 Principal characteristics of the two groups of athletes Parameter AA (n = 77) CA (n = 53) p Age (years±SE) 17 ± 0,5 18 ± 0,8 0.85 Height (cm ± SE) 173 ± 1,1 174 ± 0,9 0.93 Weight (Kg ± SE) 65 ± 1,3 66 ± 1 0.72 BMI (±SE) 21,4 ± 0,2 21,8 ± 0,2 0.56 Resting HR (AAm ± SE) 66,8 ± 0,8 64,1 ± 2 0.09 SBP (mmHg±SE) 115 ± 0,6 116 ± 1,3 0.52 DBP (mmHg±SE) 73 ± 0,5 70 ± 1,5 0.04 Legend: BMI: Body Mass Index; SBP: systolic blood pressure; DBP: diastolic blood pressure.HR:Heart Rate Table 1 Principal characteristics of the two groups of athletes Parameter AA (n = 77) CA (n = 53) p Galanti et al. Cardiovascular Ultrasound (2019) 17:20 Galanti et al. Cardiovascular Ultrasound (2019) 17:20 Page 5 of 8 Page 5 of 8 Page 5 of 8 African Americans and Caucasian athletes and this dif- ference is already present at an early age. the same clinical evaluation. At the first evaluation per- formed in our study, mean age, height, weight, BMI, resting heart rate and basal systolic and diastolic blood pressure were similar in both groups (Table 3). the same clinical evaluation. At the first evaluation per- formed in our study, mean age, height, weight, BMI, resting heart rate and basal systolic and diastolic blood pressure were similar in both groups (Table 3). In fact AA show a higher frequency of ECG repolariza- tion changes, particularly early repolarization patterns and inversed T waves, compared to CA. Since ethnicity was the principal differential characteristic between the groups, the presence of these ECG alterations could be attributed to genetic/ethnic factors. 5. Follow-up period The results of our study confirm the data present in literature: early repo- larization pattern, which is characteristic of athlete’s heart, has a higher frequency in African American ath- letes compared to Caucasian athletes in all the publica- tions (60% vs 30% in adult athletes, 11% vs 1% in adolescent athletes, in African American and Caucasian athletes, respectively). These alterations are more fre- quent in African American sedentary individuals as well, suggesting a correlation with genetic factors rather than with exercise [21]. Thirty AA and 27 CA were selected from the main group and were evaluated annually in a 4-year follow-up at the Sports Medicine Department of the University of Florence - Italy. The evaluation, which allowed for follow-up of the athletes during peripuberal develop- ment, consisted in a clinical examination, basal and stress exercise ECG and echocardiography. At the first evaluation (mean age around 12–13 yrs. in both groups), statistically significant differences in left ventricular re- modeling were observed between the two groups. Des- pite the heart chambers diameters were comparable, IVS thickness significantly increased in AA compared to CA, with a trend to an increase in PW thickness and also in iLVM. Our data showed that a significant increase in wall thickness appears to be present already in prepu- beral age. Data in literature also confirm the higher fre- quency of inversed T waves in precordial leads, which were found in 55% of AA and 7% of CA in our study. A 13% higher frequency of inversed T waves was described in African American athletes compared to Caucasian athletes, with a prevalence that varied between 12,7 and 23% [21]. The preva- lence of inverted T waves in our study was higher (55%), possibly due to the fact that all the individ- uals enrolled were elite athletes. The higher preva- lence of inverted T waves in individuals of African American descent could be caused by two mecha- nisms: ethnical and exercise-correlated. Data in lit- erature, in fact, show that 10,1% of sedentary African Americans present this pattern but with a lower fre- quency compared to African American athletes. Inversed T waves extend until V3-V4 in African American athletes, while Caucasian athletes show this pattern only in V1-V2 [22]. At first evaluation the statistically significant difference in IVS thickness and the trend in increase of PW thick- ness and iLVM in AA compared to CA can be noted. 5. Follow-up period During follow-up the statistically significant difference in IVS and PW thickness and the trend in increase of iLVM in AA compared to CA can be noted. At the 4-year follow-up evaluation, a comparable in- crease in echocardiographic parameters was observed in both groups. IVS and PW thickness and iLVM signifi- cantly increased in AA compared to CA, whereas EDd values were within normal range and showed no signifi- cant difference between the two groups. LVMI showed a mild increase in AA at the 4-year evaluation, but all the other parameters evaluated were within normal ranges and no contractile impairment was detected (Table 4). end. BMI: Body Mass Index; SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure After 4 years, mean age, height, weight, BMI, restin o-diastolic blood pressure continued without any statistically significant difference between the two groups SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure After 4 years, mean age, height, weight, BMI, resting heart rate and basal continued without any statistically significant difference between the two groups Legend. BMI: Body Mass Index; SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure After 4 years, mean age, heig systo-diastolic blood pressure continued without any statistically significant difference between the two groups Discussion and conclusions Early repolarization pattern and inversed T waves were only present in inferior precordial but not in The results of the study show that cardiovascular and, specifically, left ventricular remodelling is different in Table 3 Principal characteristics of the athletes at first evaluation and after the 4-year follow-up First Evaluation 4-year follow-up AA (n = 30) CA (n = 27) p AA(n = 30) CA (n = 27) p Age (years±SE) 12,5 ± 0,6 13,9 ± 0,5 0,06 Height (cm ± SE) 162 ± 9 167 ± 3 0,08 176,4 ± 2,6 172,9 ± 2 0,09 Weight (Kg ± SE) 57 ± 4,4 56 ± 3,1 0,2 69,2 ± 2,8 63,5 ± 2,2 0,2 BMI (kg/m2 ± SE) 19,3 ± 1,2 19,8 ± 0,5 0,3 22,6 ± 0,4 21,1 ± 0,3 < 0,05 Resting HR (AAm ± SE) 63,9 ± 3,7 66,6 ± 2,9 0,09 67,5 ± 2,2 61,5 ± 3,1 0,07 SBP (mmHg±SE) 110 ± 6 116 ± 2 0,07 116,6 ± 1,4 114,7 ± 6,1 0,09 DBP (mmHg±SE) 69 ± 4 71 ± 2 0,07 73,8 ± 1,6 69,1 ± 4,1 0,08 Legend. BMI: Body Mass Index; SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure After 4 years, mean age, height, weight, BMI, resting heart rate and basal systo-diastolic blood pressure continued without any statistically significant difference between the two groups Table 3 Principal characteristics of the athletes at first evaluation and after the 4-year follow-up Galanti et al. Discussion and conclusions PW: Posterior Wall; IVS: Inter Ventricular Septum; RWT: relative wall thickness; iLVM index; LVEDV: Left Ventricle End Diastolic Volume; LVESV: Left Ventricle End Systolic Volume; ESV: End Systolic Volume; LA: left atrium; RA: right atrium; EF: Ejection Fraction; RVd: Right Ventricle diameter; E wave; A wave; IVTR: isovolumic relaxation time; DT: Deceleration Time [24] and Sheikh et al. [21] studied African American adolescent athletes: left ventricular hypertrophy was present in 5–7% of them (IVS > 12 mm, some of them up to 15 mm), compared to 0,6% of Caucasian athletes, while no African American sedentary adolescent showed this pattern. This result would suggest an exercise- correlated etiology of ventricular hypertrophy rather than ethnicity, and the possibility of an early cardiovas- cular remodeling. Recently no study in literature has followed time-correlated trends of left ventricular re- modeling in adolescent athletes. Our study is unique from this point of view, showing that the different left ventricular remodeling between AA and CA, which is already present in pre-pubertal age, is maintained with development. inferolateral leads (V5-V6), according to literature [22]. This permitted excluding HCM and ARVD, where inverted T waves are present in 70% of the patients and extend to inferolateral leads [23]. Our results also confirm data from Sheikh [21], who studied adolescent African American athletes and found a higher prevalence of early repolarization (91% vs 56%), left ventricular hypertrophy (89% vs 42%), and deep inverted T waves (14% vs 3%) compared to Caucasian athletes. Echo data show significant differences in AA wall thickness (WT) compared to CA and that this pattern already appears in pre-pubertal age, (12–13 year-olds). Higher WT is also maintained after a 4-year follow-up. Besides, our study shows that left ventricular remodel- ling in African American athletes is a specific phenotype already present in adolescence, suggesting the role of ethnic and genetic factors in its development. Other dif- ferential elements (i.e. sex, age, kind of training) were not present. The IVS wall thickness is different in the two study groups, despite LVMI and RWT being similar. Firstly this aspect is difficult to interpret. It could be reasonable to think that the absence of change of the LVMI and RWT parameters could be related principally to the dif- ferent LV diameters found. The results of our study confirm data in literature: many publications report left ventricular WT 1–2 mm higher in African American athletes compared to Cauca- sians (i.e. Discussion and conclusions Cardiovascular Ultrasound (2019) 17:20 Page 6 of 8 Table 4 Values of parietal/wall thickness and chamber volumes in both groups at first evaluation and after 4 years First evaluation 4-year follow-up AA (n = 30) CA (n = 27) p AA (n = 30) CA (n = 27) p IVS(mm) 9,3 ± 0,3 8,5 ± 0,1 < 0,02 10,5 ± 0,2 9 ± 0,2 < 0,001 PW(mm) 9 ± 0,2 8,4 ± 0,2 0,1 10,1 ± 0,2 8,3 ± 0,2 < 0,001 RWT (%) 38,2 ± 0,9 38 ± 0,5 0,8 39,4 ± 0,2 39,1 ± 0,6 0,7 iLVM(g/m2) 96,7 ± 5,4 90,2 ± 3,3 0,1 110,1 ± 2,3 100,5 ± 4,4 < 0,05 LVEDd(mm) 46,5 ± 1,8 48,4 ± 1,1 0,5 50 ± 0,5 50 ± 1 0,7 LVESd(mm) 29,8 ± 0,8 30,4 ± 0,9 0,6 32 ± 0,6 31 ± 1 0,6 EDV (ml) 107,1 ± 7 110,8 ± 6 0,09 120 ± 4 117 ± 4 0,5 ESV (ml) 33,6 ± 2,8 37,1 ± 2,7 0,07 41 ± 2 39 ± 2 0,09 LA (mm) 35 ± 1,6 34,2 ± 1 0,8 37,8 ± 0,8 36,4 ± 0,4 0,6 RA (mm) 33,8 ± 3,5 34,2 ± 1,7 0,6 34,5 ± 2,6 34,1 ± 2,6 0,6 EF (%) 65,2 ± 1 66 ± 1 0,3 66,5 ± 1,2 65,1 ± 2,9 0,5 RVd(mm) 21,7 ± 0,6 19,4 ± 0,5 0,006 23,6 ± 0,6 21,4 ± 0,7 0,02 E (cm) 91,4 ± 6,4 90 ± 3,1 0,3 81,4 ± 3,4 82,6 ± 3,4 0,2 A (cm) 46,6 ± 3,4 47,7 ± 3,7 0,4 45,9 ± 3,3 42,8 ± 2,6 0,08 IVRT (t) 70,6 ± 4,8 74,4 ± 3 0,09 74,4 ± 2,8 76,1 ± 2,1 0,4 DT (cm) 164,4 ± 14,6 165 ± 6 0,5 179,3 ± 11,2 176,4 ± 6,2 0,07 E/A 1,9 ± 3,5 1,9 ± 1 0,9 1,8 ± 1,1 2 ± 1,2 0,3 Legend. PW: Posterior Wall; IVS: Inter Ventricular Septum; RWT: relative wall thickness; iLVM index; LVEDV: Left Ventricle End Diastolic Volume; LVESV: Left Ventricle End Systolic Volume; ESV: End Systolic Volume; LA: left atrium; RA: right atrium; EF: Ejection Fraction; RVd: Right Ventricle diameter; E wave; A wave; IVTR: isovolumic relaxation time; DT: Deceleration Time Legend. Discussion and conclusions 11.3 ± 1.6 vs 10.0 ± 1.5 mm), with 13% of Afri- can American athletes having a WT > 12 mm and 1, 1% > 16 mm, in the absence of HCM [23]. Rawlins et al. The mild increase of LVMI in AA at 4-year evaluation could be interpreted as a consequence of training, being part of the athlete’s heart manifestation. This is sug- gested by normal morphological and functional parame- ters with the absence of contractile impairment. Page 7 of 8 Page 7 of 8 Galanti et al. Cardiovascular Ultrasound (2019) 17:20 Galanti et al. Cardiovascular Ultrasound (2019) 17:20 Galanti et al. Cardiovascular Ultrasound (2019) 17:20 Nowadays, determining ethnic-specific morpho- logical and functional characteristics of left ventricular remodeling in elite athletes is a necessity. Since sports teams are multicultural, accurately recognizing ethnic- specific characteristics is an urgent need. Identifying specific echocardiographic criteria will help the Sports Physician to differentiate between physiological and pathological conditions, reducing the so-called “grey- zone”. Acknowledgements The Authors wish to thank Susan Seeley for her help in revising the English of the text. Received: 28 January 2019 Accepted: 3 September 2019 Received: 28 January 2019 Accepted: 3 September 2019 Some particular characteristics are present in our study. Some particular characteristics are present in our study. Firstly, all the athletes belonged to the same team, trained with the same load and frequency and had the same lifestyle. Ethnicity was the only differential element among the athletes who were matched for sex, mean age, anthropometrical and clinical characteristics, delet- ing possible differences in results related to these deter- minants. This made it possible to evaluate specifically the contribution of ethnicity to left ventricular remodel- ling in elite athletes. Consent for publication Not applicable. The mean age of the athletes enrolled was notable: they were evaluated before and during puberty, at ages that are well associated with important structural modi- fications. This allowed us to demonstrate that exercise- correlated left ventricular remodelling is already present early in life and that it is more marked in individuals of African American descent compared to Caucasians. Funding l Not applicable. Availability of data and materials The data sets used and analyzed during the study are available from the corresponding author on request. Limits of the study This study is a particular investigation of myocardial function evaluated by ethnic characteristics. Some limits are evident and among these the small sample investi- gated represents the principal aspect in the results ob- tained. This appears to be particularly important in terms of the interpretation of the results of myocardial size and thickness. In addition, many of the subjects enrolled were not followed for long because of normal seasonal team re- cruitment. Further studies will be necessary to confirm the present data. GLS has not been specifically determined in African American athletes yet. This is the reason why a com- parison with other data is not possible. The results of our study show no difference and normal range values of GLS in athletes of the two ethnicities. Russo et al. [25] compared GLS between sedentary individ- uals of different descent, and lower values of GLS were found in African Americans (−16,5 ± 3,5%) compared to Caucasians (−17,5 ± 3,0%), despite simi- lar values of ejection fraction. If these data and the results of our study were confirmed, a possible pro- tective role of physical exercise in individuals of Afri- can American descent would be suggested. Considering the aim of the study was to evaluate a homogeneous group of subjects, excluding as many con- fusing factors as possible, i.e. anthropometrics, lifestyle and load differences, the number of subjects that could be enrolled was consequently low. Authors’ contributions GG conceived the study; LT and CG collected the 2D Echo images and contributed to improving the message of the manuscript; BT, MO and GM wrote the paper and prepared the statistical analysis; LS and PAM revised and coordinated the final version of the manuscript. All authors read and approved the final manuscript. 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Exercise testing of patients with coronary heart disease: principles and normal standards for evaluation. Ann Clin Res. 1971;3:323–32. 16. Frenneaux MP, Counihan PJ, Caforio AL, Chikamori T, McKenna WJ. Publisher’s Note Abnormal blood pressure response during exercise in hypertrophic cardiomyopathy. Circulation. 1990;82:1995–2002. 17. Lang RM, Badano LP, Mor-Avi V, Afilalo J, Armstrong A, Ernande L, Flachskampf FA, Foster E, Goldstein SA, Kuznetsova T, Lancellotti P, Muraru D, Picard MH, Rietzschel ER, Rudski L, Spencer KT, Tsang W. Voight JU recommendations for cardiac chamber quantification by echocardiography in adults: an update from American Society of Echocardiography and European association of cardiovascular imaging. J Am SocEchocardiogr. 2015;28:1):1–39. e 14. https://doi.org/10.1016/j.echo.2014.10.003. 18. Galderisi M, Cosyns B, et al. Standardization of adult transthoracic echocardiography reporting in agreement with recent chamber quantification, diastolic function, and heart valve disease recommendations: an expert consensus document of the European Association of Cardiovascular Imaging. Eur Heart J - Cardiovascular Imaging. 2017;18:1301–10. https://doi.org/10.1093/ehjci/jex244. 19. Recommendations for Cardiac Chamber. Quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. J Am SocEchocardiogr. 2015;28:1–39. 20. Ternacle J, Bremont C, d'Humieres T, Faivre L, Doan HL, Gallet R, Oliver L, Dubois-Randé JL, Lim P. Left ventricular dyssynchrony and 2D and 3D global longitudinal strain for differentiating physiological and pathological left ventricular hypertrophy.Arch Cardiovasc Dis. 2017. https://doi.org/10. 1016/j.acvd.2016.11.003. 21. Sheikh N, Papadakis M, Carre F, Kervio G, Panoulas VF, Ghani S, Zaidi A, Gati S, Rawlins J, Wilson MG, Sharma S. Cardiac adaptation to exercise in adolescent athletes of African ethnicity: an emergent elite athletic population. Br J Sports Med. 2013;47(9):585–92. https://doi.org/10.1136/ bjsports-2012-091874. 22. Papadakis M, Carre F, Kervio G, Rawlins J, Panoulas VF, Chandra N, Basavarajaiah S, Carby L, Fonseca T, Sharma S. The prevalence, distribution, and clinical outcomes of electrocardiographic repolarization patterns in male athletes of African/afro-Caribbean origin. EurHeart J. 2011;32(18):2304– 13. https://doi.org/10.1093/eurheartj/ehr140. 23. Caselli S, Maron MS, Urbano-Moral JA, Pandian NG, Maron BJ, Pelliccia A. Differentiating left ventricular hypertrophy in athletes from that in patients with hypertrophic cardiomyopathy. Am J Cardiol. 2014;114(9):1383–9. https://doi.org/10.1016/j.amjcard.2014.07.070. 24. Rawlins J, Carre F, Kervio G, Papadakis M, Chandra N, Edwards C, Whyte GP, Sharma S. Ethnic differences in physiological cardiac adaptation to intense physical exercise in highly trained female athletes. Circulation. 2010;121(9): 1078–85. https://doi.org/10.1161/circulationaha.109.917211. 24. Rawlins J, Carre F, Kervio G, Papadakis M, Chandra N, Edwards C, Whyte GP, Sharma S. Ethnic differences in physiological cardiac adaptation to intense physical exercise in highly trained female athletes. Circulation. 2010;121(9): 1078–85. https://doi.org/10.1161/circulationaha.109.917211.
https://openalex.org/W2046854296	http://rcin.org.pl/Content/148116/PDF/WA35_178310_12807-7_Art67.pdf	English	null	483. On a pair of Differential Equations in the Lunar Theory	Cambridge University Press eBooks	2,009	public-domain	607	483j 483j 537 483. ON A PAIR OF DIFFERENTIAL EQUATIONS IN THE LUNAR THEORY. ON [From the Monthly Notices of the Royal Astronomical Society, vol. xxxιi. (1871—72), pp. 201—206.] [From the Monthly Notices of the Royal Astronomical Society, vol. xxxιi. (1871—72), pp. 201—206.] I CONSIDER the differential equations which when j = k = 1 give the following equations in the lunar theory {D = t- mt): or as far as wζ or as far as wζ 68 68 www.rcin.org.pl 538 ON A PAIR OF DIFFERENTIAL [483 f- is given by M. Delaunay only as far as the additional terms of - and expression ∖p p for ρ were kindly communicated to me by Prof. Adams^ ; and v = t f- is given by M. Delaunay only as far as the additional terms of - and expression ∖p p for ρ were kindly communicated to me by Prof. Adams^ ; and v = t v t (Delaunay, t. ιι. pp. 815, 83G, 845). (Delaunay, t. ιι. pp. 815, 83G, 845). (Delaunay, t. ιι. pp. 815, 83G, 845). (Delaunay, t. ιι. pp. 815, 83G, 845). To integrate the original equations write where the suffixes indicate the degrees in the coefficients k, j conjointly: the equations for p,ι, Vn take the form where Vn, Un, Pn, Qn do not contain pn or Vn. From the second equation we have where Ω,⅛ is a constant of integration, the integral ∣Pn dt containing only periodic terms; and then adding twice this to the first equation we have where Ω,⅛ is a constant of integration, the integral ∣Pn dt containing only periodic terms; and then adding twice this to the first equation we have where Ω,⅛ is a constant of integration, the integral ∣Pn dt containing only periodic terms; and then adding twice this to the first equation we have terms; and then adding twice this to the first equation we have dv which determines pn; and substituting its value in the other equation we have , and thence Vn; the constant is determined so that may contain no constant term. We have ∞c &c. ∞c. www.rcin.org.pl EQUATIONS IN THE LUNAR THEORY. 483] 539 & & &c. &c. In particular attending to the values of Pi, Qi the equations for p^, are in their original form whence in the transformed form they are whence in the transformed form they are and cZ?/ Thus the constant term of pi is 2Ωj + ⅛ W, giving in a constant term - 3∩ι - km^ this must vanish, or we have Ω, = — ∣ km?', and the equations thus become and then completing the integration and then completing the integration which are the accurate values of and Vj. which are the accurate values of and Vj. Expanding as far as m? have j ≈k is which for j ≈k is which for j ≈k is www.rcin.org.pl [48 3 ON A PAIR OF DIFFERENTIAL EQUATIONS IN THE LUNAR THEORY. 540 and and and which for j ≈k is and which for j ≈k is I have, not in general, but for the value j = k, calculated p.. and v., as far as m'": I have not made the calculation for and ¾, but their values may be deduced from the foregoing values of p, v, the final expressions (when j=k} of p, =1 pι +P2 +pa+∙∙. and = i + Wi + V2 + ⅝ ∙ ∙ ∙ ai'θ which for k=l agree with the foregoing formulae (verifying them as far as w®)> the present formulae exhibit the manner in which the expressions depend on the several powers of the disturbing force. www.rcin.org.pl
https://openalex.org/W2988930995	https://academic.oup.com/innovateage/article-pdf/3/Supplement_1/S754/33008472/igz038.2769.pdf	English	null	IS SOCIAL ENGAGEMENT RELATED TO FEWER HOSPITALIZATIONS IN COMMUNITY-DWELLING OLDER ADULTS?	Innovation in aging	2,019	cc-by	1,317	754 754 Innovation in Aging, 2019, Vol. 3, No. S1 IS SOCIAL ENGAGEMENT RELATED TO FEWER HOSPITALIZATIONS IN COMMUNITY-DWELLING OLDER ADULTS? National University of Singapore, Singapore, Singapore IS SOCIAL ENGAGEMENT RELATED TO FEWER HOSPITALIZATIONS IN COMMUNITY-DWELLING OLDER ADULTS? the context of driving cessation. Although some research has examined the association between driving cessation and certain kinds of social engagement activities, no research has specifically examined changes in social support, particu larly among older adults most vulnerable to social isolation – those who live alone . The present study addresses this gap, using data drawn from the 2006-2014 waves of the Health and Retirement Study (HRS) to examine how social support changes in the context of driving cessation among older adults who live alone (N=412). This study specific ally focuses on instrumental and emotional social support, and how different sources of the support (children, friends, and other family) are influenced by loss of driving. I use a series of ordinary least squares regression (OLS) to examine four-year changes in various forms of social support among those who live alone, comparing those who lose the ability to drive relative to their continuously driving counter parts. Preliminary results indicate that driving cessation is associated with decline in perceived instrumental support of friends (-0.984, p<01) for older adults who live alone. However, these effects did not extend to children or other family members. These results suggest that loss of driving may perpetuate vulnerabilities facing individuals who live alone by leading to lower levels of perceived support from non-family members. the context of driving cessation. Although some research has examined the association between driving cessation and certain kinds of social engagement activities, no research has specifically examined changes in social support, particu larly among older adults most vulnerable to social isolation – those who live alone . The present study addresses this gap, using data drawn from the 2006-2014 waves of the Health and Retirement Study (HRS) to examine how social support changes in the context of driving cessation among older adults who live alone (N=412). This study specific ally focuses on instrumental and emotional social support, and how different sources of the support (children, friends, and other family) are influenced by loss of driving. I use a series of ordinary least squares regression (OLS) to examine four-year changes in various forms of social support among those who live alone, comparing those who lose the ability to drive relative to their continuously driving counter parts. Preliminary results indicate that driving cessation is associated with decline in perceived instrumental support of friends (-0.984, p<01) for older adults who live alone. INVESTIGATING SOCIAL NETWORKS OF OLDER SINGAPOREAN LEARNERS: THE MIXED-METHODS SOCIAL NETWORK APPROACH Pei-Chun Ko1, 1. National University of Singapore, Singapore, Singapore Lifelong learning has been regarded as an important factor of promoting active engagement in later life for re searchers and policy makers. Most of the studies tend to illustrate old learners as a homogeneous and self-resilient group of people to engage in lifelong learning. Few studies address older learners’ social capital in affecting their de cision of engagement and in sustaining their motivation. The study documented the existing social networks of older Singaporeans in lifelong learning programs and illus trated how social networks contributed their participation in learning. The mixed methods consist of in-depth inter views and two network instruments (Name Generator and Position Generator) based on 30 older Singaporeans (be tween 50 and 79 years old) who attended lifelong learning courses between 2016 and 2018. Interviews are transcribed and analyzed. The network instruments of are quantified and visualized. The findings show that older learners’ net works included a mixture of social ties from family and friends. Learners’ closeness with network members and their living arrangement with them influenced learners’ involve ment in learning and future planning. Single respondents who had more non-kin members in the networks reported to be more active due to their weak ties. Overlapping net works among couple learners increase the spousal support for learning. Learners who had wider ranges of social re sources are associated with their interest in learning activ ities. The study suggests that advocating lifelong learning needs to take older adults’ networks into considerations as networks represent the social forces that influence their de cisions and motivations. SOCIAL ENGAGEMENT AND DEPRESSIVE SYMPTOMS IN OLDER AFRICAN AMERICANS: A 6-YEAR CROSS- LAGGED PANEL ANALYSIS James Muruthi,1 and J. Tina Savla2, 1. University of Oregon, Eugene, Oregon, United States, 2. Virginia Tech, Blacksburg, Virginia, United States IS SOCIAL ENGAGEMENT RELATED TO FEWER HOSPITALIZATIONS IN COMMUNITY-DWELLING OLDER ADULTS? However, these effects did not extend to children or other family members. These results suggest that loss of driving may perpetuate vulnerabilities facing individuals who live alone by leading to lower levels of perceived support from non-family members. Bryan D. James,1 Raj C. Shah,2 Melissa Lamar,3 Lisa L. Barnes,3 Robert S. Wilson,3 David A. Bennett,3 and Julie A. Schneider3, 1. Rush Alzheimer’s Disease Center, Chicago, Illinois, United States, 2. Rush University, Chicago, Illinois, United States, 3. Rush Alzheimer’s Disease Center, Chicago, Illinois, United States David A. Bennett, and Julie A. Schneider , 1. Rush Alzheimer’s Disease Center, Chicago, Illinois, United States, 2. Rush University, Chicago, Illinois, United States, 3. Rush Alzheimer’s Disease Center, Chicago, Illinois, United States We tested the hypothesis that more socially engaged older adults experience fewer hospitalizations. Data came from 1,153 older adults (72.4% female, mean age 80.8, 14.6 years education at baseline), enrolled in the Rush Memory and Aging Project, with survey data linked to Medicare claims records (mean 5.0 [SD=3.1] years of Medicare coverage after study baseline). Linear regression models were fit with annual rate of hospitalization as outcome with terms for age, sex, and education. Engaging in more social activities (est=-0.16, SE=0.05, p=0.002) and larger life space (est=-0.08, SE=0.03, p=0.005) were associated with a lower rate of hospitaliza tion, while a higher level of loneliness (est=0.18, SE=0.06, p=0.002) was associated with greater rate of hospitaliza tion; size of social networks (est=-0.01, SE=0.01, p=0.069) was not associated with hospitalization. When examined separately by admission type, the same significant associ ations were found for nonelective (emergency and urgent) hospitalizations, but not for elective hospitalizations. After further adjusting for marital status, baseline levels of depres sive symptoms, chronic medical conditions, physical activity, and ADL disabilities, only social activities were signifi cantly related to hospitalization rate (total and nonelective). Adjusting for disability attenuated these associations the most, indicating that functional status may confound the re lationship between social engagement and hospitalization. More research is necessary to determine if social engagement in older age can proactively help to keep older adults out of the hospital, or alternatively whether level of social engage ment is a marker for functional status or other health factor that is directly related to risk of hospitalization. Downloaded from https://academic.oup.com/innovateage/article/3/Supplement_1/S754/5616568 by guest on 24 October 2024 INVESTIGATING SOCIAL NETWORKS OF OLDER SINGAPOREAN LEARNERS: THE MIXED-METHODS SOCIAL NETWORK APPROACH Pei-Chun Ko1, 1. SOCIAL ENGAGEMENT AND DEPRESSIVE SYMPTOMS IN OLDER AFRICAN AMERICANS: A 6-YEAR CROSS- LAGGED PANEL ANALYSIS James Muruthi,1 and J. Tina Savla2, 1. University of Oregon, Eugene, Oregon, United States, 2. Virginia Tech, Blacksburg, Virginia, United States Although previous studies have extensively investigated the cross-sectional relationship between social engagement and depressive symptoms in late life, longitudinal studies have produced mixed results. Furthermore, studies on the as sociations between these two concepts among aging African Americans are few. Using a sample of 1688 older African Americans adults from waves 1 and 7 of the National Health and Aging Trends Study (60% women; Average age = 77 years), the present study investigates the longitu dinal associations between social engagement (an index from scores on visiting friends and family, attending religious services, attending religious services, participating in group activities, and going out for enjoyment) and depressive symp toms across seven years. Structural equation modeling was used to test cross-lagged relationships between the variables. GSA 2019 Annual Scientific Meeting
https://openalex.org/W2895905500	https://www.nature.com/articles/s41598-018-34135-x.pdf	English	null	Selection, hybridization, and the evolution of morphology in the Lake Malaŵi endemic cichlids of the genus Labeotropheus	Scientific reports	2,018	cc-by	8,259	Selection, hybridization, and the evolution of morphology in the Lake Malaŵi endemic cichlids of the genus Labeotropheus Michael J Pa ers1 2 3 Kelse R Fo 1 RobertA Hall2 4 & Kesha Patel2 Received: 16 March 2018 Accepted: 11 October 2018 Published: xx xx xxxx Michael J. Pauers1,2,3, Kelsey R. Fox1, Robert A. Hall2,4 & Kesha Patel2 The cichlid fishes of Lake Malaŵi are the paramount example of adaptive radiation in vertebrates. Evidence of their astounding diversity is perhaps most visible in their adaptations for obtaining food; the genus Labeotropheus, due to their prominent snouts, are an interesting example of an extreme adaptation for feeding. Two different body types are found in this genus: a deep-bodied form (e.g., L. fuelleborni) found most often in turbulent shallow water; and a slender bodied form (e.g., L. trewavasae) found in structurally-complex deep water habitats. Here we test the hypothesis that L. trewavasae should suffer a loss in fitness, measured as growth rate, if raised in turbulence; additionally, we examined growth and morphology of L. fuelleborni and L. fuelleborni x L. trewavasae hybrids under these conditions. We did find the predicted loss of fitness in turbulent-raised L. trewavasae, but found no loss of fitness for L. fuelleborni in either condition; hybrids, due to an unusual morphology, performed better in turbulent as opposed to control conditions. Fitness in turbulent conditions was dependent upon morphology, with deeper bodies and upturned neurocrania allowing a greater growth rate under these conditions. Directional selection on morphology was crucial in the evolution of morphology in the Labeotropheus. The cichlid fishes of Lake Malaŵi are one of the most fascinating examples of adaptive radiation and speciation; indeed, no other group of vertebrates can match either their sheer number of species or vast diversity of form and function. Nowhere is this diversity of form and function more prevalent than in the variety of adaptations these fishes have for acquiring food e.g.1,2. These fishes have evolved a striking number of ways to divide and subdivide what appear to be homogenous resources, yet the astounding array of craniofacial, oral, dental, and pharyngeal adaptations they have allow the various cichlid species to exploit very specific portions of the available food resources3–5, thus promoting the coexistence of so many disparate species. p g y p p Amongst the most unique and unusual feeding adaptations displayed by any of the cichlids of Lake Malaŵi is that of the genus Labeotropheus. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Section of Vertebrate Zoology, Milwaukee Public Museum, 800 W. Wells Street, Milwaukee, Wisconsin, 53233, USA. 2Department of Biological Sciences, University of Wisconsin-Milwaukee at Waukesha, 1500 N. University Drive, Waukesha, Wisconsin, 53188, USA. 3School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 E. Greenfield Avenue, Milwaukee, Wisconsin, 53204, USA. 4University of Wisconsin-Madison, Madison, WI, 53708, USA. Correspondence and requests for materials should be addressed to M.J.P. (email: mjpauers@gmail.com) Received: 16 March 2018 Accepted: 11 October 2018 Published: xx xx xxxx Selection, hybridization, and the evolution of morphology in the Lake Malaŵi endemic cichlids of the genus Labeotropheus Michael J Pa ers1 2 3 Kelse R Fo 1 RobertA Hall2 4 & Kesha Patel2 The diagnostic feature shared by all members of this small genus is an enlarged snout of fibrous connective tissue that hangs over an inferior, subterminal mouth, with which all of these species scrape filamentous, epilithic algae1,6–8. This snout has been the focus of many hypotheses regarding how the Labeotropheus feed, and it is generally assumed to act as a fulcrum with which the fish contacts the substrate, while the jaws work to remove the attached algae; this process is exquisitely described by Fryer1; see also3,8–10. The uniqueness of this feeding apparatus has made various species of Labeotropheus favorite subjects for studying the evolution and ecology of cichlid feeding8,11–13.f gy g Long overlooked in the study of Labeotropheus feeding is the role body shape may play in the different envi- ronments in which members of this genus are found. While Fryer1 was the first to note that its characteristic snout allowed species of Labeotropheus to feed almost parallel to the substrate, Ribbink et al.3 further suggested that this could be a significant advantage for L. fuelleborni, a deep-bodied species that primarily inhabits the shallow, wave-swept areas along the shore of Lake Malaŵi. These authors suggested that by keeping its stocky body parallel 1Section of Vertebrate Zoology, Milwaukee Public Museum, 800 W. Wells Street, Milwaukee, Wisconsin, 53233, USA. 2Department of Biological Sciences, University of Wisconsin-Milwaukee at Waukesha, 1500 N. University Drive, Waukesha, Wisconsin, 53188, USA. 3School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 E. Greenfield Avenue, Milwaukee, Wisconsin, 53204, USA. 4University of Wisconsin-Madison, Madison, WI, 53708, USA. Correspondence and requests for materials should be addressed to M.J.P. (email: mjpauers@gmail.com) Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x 1 1 www.nature.com/scientificreports/ Figure 1. The overall dimensionality of morphology among all three species. RW 1 explains 21.89% of the observed variation in body shape, and primarily accounts for the difference between a slender body with a downwardly-pointing head and a deep body with an upwardly-tilted head. RW 2 explains 14.41% of the variation in body shape, and accounts for the difference between a slender body with a dorsoventrally- compressed head and a deep body with an expanded craniofacial region. The deformation grids show the morphologies associated with the extremes of each axis, and are positioned accordingly. Figure 1. The overall dimensionality of morphology among all three species. Selection, hybridization, and the evolution of morphology in the Lake Malaŵi endemic cichlids of the genus Labeotropheus Michael J Pa ers1 2 3 Kelse R Fo 1 RobertA Hall2 4 & Kesha Patel2 RW 1 explains 21.89% of the observed variation in body shape, and primarily accounts for the difference between a slender body with a downwardly-pointing head and a deep body with an upwardly-tilted head. RW 2 explains 14.41% of the variation in body shape, and accounts for the difference between a slender body with a dorsoventrally- compressed head and a deep body with an expanded craniofacial region. The deformation grids show the morphologies associated with the extremes of each axis, and are positioned accordingly. to a horizontal rock surface, L. fuelleborni would be better able to maintain its position in this turbulent habitat3. Similarly, Konings14 observed that L. trewavasae, a slender-bodied species found at deeper depths than L. fuel- leborni, seems to use its slim body to penetrate into crevices among rocks, or to feed from the vertical surfaces of rocks, in order to avoid ingesting sediment. To date, neither of these observations has been experimentally confirmed. i If these divergent morphologies are adaptations to their respective habitats, then individuals forced to feed and grow in a mismatched habitat should suffer fitness-related consequences. Specifically, if L. trewavasae is grown in a turbulent habitat, we would expect that it should experience a reduced growth rate; growth rate being a commonly-used and reasonable proxy for fitness in fishes e.g.15,16. Alternatively, it might be possible that such individuals adapt to turbulence by adopting a morphology similar to that of L. fuelleborni; i.e., a deep, stocky, more massive body. Here, we test these hypotheses and predictions in L. fuelleborni, L. trewavasae, and their hybrids. Results Di i Dimensionality of morphology. Relative Warp (RW) 1 explains 21.89% of the observed variation in body shape amongst the experimental fishes. This axis primarily distinguishes between two opposing morphologies: a slender-bodied form with a dorsoventrally compressed, downturned head at the positive end; versus a deep- er-bodied form with an expanded, upturned head at the negative. RW 2 accounts for 14.41% of the variation in body shape, and distinguishes between a very slender body with a reduced snout; versus a distinctly deeper body that features a prominent, enlarged snout (Fig. 1). In this preliminary examination of morphology, it appears that the slender L. trewavasae specimens are clustered within the positive quadrant, while the robust L. fuelleborni individuals seem to be confined to the negative quadrant. Morphological differences between species and treatments. By the end of the experiment, while clear differences in body shape were evident among species, there appear to be some morphological commonal- ities among species between treatments. RW 1 distinguishes the species, as well as the experimental treatments within species (Table 1). The turbulent-reared individuals of both L. fuelleborni and L. trewavasae are situated negatively, with respect to the control fish, along RW 1. The hybrid individuals of the control and turbulent treat- ments are similarly distinguished, although their placements relative to each other are the reverse of the parental species; the turbulent individuals are situated positively relative to the control fish (Fig. 2). RW 2 separates the species from one another, with L. trewavasae located at the positive end of this axis, the hybrids at the negative end, and L. fuelleborni situated near the midpoint of RW 2 (Fig. 2; Table 1).f Surprisingly, body depth did not differ among treatments; L. trewavasae, L. fuelleborni, and their hybrids did not adopt a deeper body in the turbulent treatment as opposed to the control. Instead, the primary differences between the turbulent and control fish in both the parental species and the hybrids are an upwards-tilted neuro- cranium and a more prominent snout (Fig. 2). The tilt of the neurocranium can be measured by comparing the angle formed by landmarks 1, 18, and 3 (the “neurocranium angle”); as well as the angle formed by landmarks 1, 14, and 13 (the “jaw angle”); among treatment groups. Results Di i For all species, the neurocranium angle is greater in Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x 2 www.nature.com/scientificreports/ Analysis of effects on RW 1 (n = 202; Multiple R2 = 0.417) Variable Sum of Squares df Mean Square F p Species 0.029 2 0.014 54.714 ≤ 0.001 Treatment 0.000 1 0.000 0.249 0.618 SpeciesTreatment 0.007 2 0.003 12.924 ≤ 0.001 Error 0.051 196 0.000 Bonferroni-corrected pairwise differences in RW 1 among speciestreatment (LFC = L. fuelleborni Control; LHC = Labeotropheus hybrid Control; L. trewavasae Control; LFT = L. fuelleborni Turbulent; LHT = Labeotropheus hybrid Turbulent; LTT = L. trewavasae Turbulent) LFC LHC LTC LFT LHT LHC −0.017 LTC 0.018* 0.034*** LFT −0.016* 0.001 −0.033* LHT −0.001 0.016 −0.018*** 0.015* LTT 0.013* 0.030* −0.004 0.029* 0.014** Analysis of effects on RW 2 (n = 202; Multiple R2 = 0.433) Variable Sum of Squares df Mean Square F p Species 0.023 2 0.012 70.413 ≤ 0.001 Treatment 0.000 1 0.000 0.001 0.981 SpeciesTreatment 0.001 2 0.001 3.030 0.051 Error 0.033 196 0.000 Bonferroni-corrected pairwise differences in RW 2 among species L. fuelleborni Labeotropheus hybrid Labeotropheus hybrid −0.012 L. trewavasae 0.013* 0.025*** Table 1. Differences along each Relative Warp (RW) axis among species and treatments. p ≤ 0.05; p ≤ 0.01 *p ≤ 0.001. Analysis of effects on RW 1 (n = 202; Multiple R2 = 0.417) Table 1. Differences along each Relative Warp (RW) axis among species and treatments. p ≤ 0.05; p ≤ 0.01; p ≤ 0.001. Table 1. Differences along each Relative Warp (RW) axis among species and treatments. p ≤ 0.05; p ≤ 0.01; p ≤ 0.001. f g p g p p p p ≤ 0.001. Figure 2. Differences in body shape among treatment groups at Day 45. This is the same RW plot as shown in Fig. 1, but the treatment groups have been identified by symbol (circle = control treatment; triangle = turbulent treatment) and color (green = L. fuelleborni control; red = L. fuelleborni turbulent; pink = hybrid control; aqua = hybrid turbulent; blue = L. trewavasae control; orange = L. trewavasae turbulent); the colors of the deformation grids also correspond to these treatment groups. In the turbulent-raised L. fuelleborni andL. trewavasae, note the expanded and upturned heads, especially in comparison to their respective controls. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Jaw angle (n = 202; Multiple R2 = 0.302) Variable Sum of Squares df Mean Square F p Species 3056.101 2 1528.050 38.650 ≤0.001 Treatment 172.633 1 172.633 4.367 0.038 SpeciesTreatment 117.215 2 58.608 1.482 0.230 Error 7748.972 196 39.536 Neurocranium angle (n = 202; Multiple R2 = 0.098) Variable Sum of Squares df Mean Square F p Species 195.147 2 97.573 0.775 0.462 Treatment 2156.504 1 2156.504 17.132 ≤0.001 SpeciesTreatment 109.292 2 54.646 0.434 0.648 Error 24671.545 196 152.875 Table 2. Differences in jaw and neurocranium angles among species and treatments. Table 2. Differences in jaw and neurocranium angles among species and treatments. the turbulent group than in the control fishes, while the jaw angle is smaller for turbulent individuals than those raised in the control condition (Table 2; Fig. 3). Together, these two bodily angles conspire to increase the pre- sumed feeding angle of turbulent-reared individuals, regardless of species (Fig. 3). the turbulent group than in the control fishes, while the jaw angle is smaller for turbulent individuals than those raised in the control condition (Table 2; Fig. 3). Together, these two bodily angles conspire to increase the pre- sumed feeding angle of turbulent-reared individuals, regardless of species (Fig. 3). Selection. In the control condition, both L. fuelleborni and L. trewavasae, despite their radically different body shapes, have higher levels of fitness compared to the hybrids raised in this condition (Fig. 4a; Table 3). Maximal fitness in the control condition is primarily associated with a straight body, without any exaggerated curvature to the neurocranium (see deformation grid in Fig. 4b). Alternatively, by allowing young Labeotropheus to grow in the turbulent condition, there is a directional shift in the morphology that maximizes fitness (Fig. 4c). In the turbulent treatment, standardized daily growth is increased when the fishes adopt a morphology incorpo- rating the upward-tilted neurocranium described above (see deformation in Fig. 4d). Labeotropheus fuelleborni raised under these conditions have the highest fitness, and L. trewavasae the lowest; interestingly, the hybrid Labeotropheus have a fitness intermediate to that of the parental species (Fig. 4c; Table 3). Further, when compar- isons are made within species between treatments, there is no difference between treatments for L. fuelleborni, but both L. trewavasae (decrease in fitness from control to turbulent) and the Labeotropheus hybrids (increase in fitness from control to turbulent) do exhibit statistically significant differences in fitness (Table 3). Discussionh The results of this experiment support the hypothesis that L. trewavasae would suffer a loss of fitness if raised in a turbulent environment; young L. trewavasae have a lower growth rate when grown in turbulent conditions than when raised in calm conditions. This reduced growth and concomitant loss of fitness is largely due to the fact that L. trewavasae failed to effectively adapt its morphology to the turbulent treatment. Conversely, the fitness of L. fuelleborni was equally high in both the turbulent and control conditions, largely due to the adoption of an upturned neurocranium and enlarged snout when raised in turbulence. The Labeotropheus hybrids presented a more complicated situation. Surprisingly, they developed an upturned neurocranium and enlarged snout in the control treatment, which was not advantageous under these conditions, resulting in the lowest fitness of any of the species. In the turbulent treatment, their fitness increased significantly due to the adoption of a craniofacial morphology similar to that of L. fuelleborni under these conditions. Overall, exposure to turbulence resulted in directional selection against a slender body and straight or downturned head, instead favoring deeper bodies with an upturned neurocranium. p Although not directly measured in these experiments, the upturned neurocranium would actually increase the typical feeding angle of the fish13,17. As such, instead of feeding more or less parallel to the surface, gleaning algae as they swim, the turbulent-reared purebred fish, especially L. fuelleborni, would be feeding in a somewhat exaggerated “headstand,” similar to the feeding angles employed by species of Maylandia13,17. In this position, the prominent snout pad would likely be firmly pressed against the feeding surface while the fish swims forward, using the snout pad not as a fulcrum, but as a stabilizer, thus allowing it to maintain its position while feeding in the turbulence. While feeding angles among Lake Malaŵi cichlids vary depending upon the orientation of the substrate13, the feeding surface in this experiment was constant and consistent between treatments, strongly sug- gesting that turbulence was responsible for the changes to the neurocranium. g g p g Given L. trewavasae’s preference for deeper habitats, where they are less likely to encounter wave-generated turbulence, it is not necessarily surprising that they struggled to grow under laboratory conditions that mim- icked such turbulence. Additionally, although it was not tested in this experiment, the body shape typical of L. www.nature.com/scientificreports/ This directional selection is made apparent when the fitness curves are superimposed on the same set of axes (Fig. 4e). Results Di i In the hybrid fishes, note the deep body but exaggerated breadth of the craniofacial area in the control cohort, especially in comparison to the L. fuelleborni and L. trewavasae controls; conversely, the turbulent-raised hybrids are much more similar to the turbulent-raised cohorts of the purebred parental species. Figure 2. Differences in body shape among treatment groups at Day 45. This is the same RW plot as shown in Fig. 1, but the treatment groups have been identified by symbol (circle = control treatment; triangle = turbulent treatment) and color (green = L. fuelleborni control; red = L. fuelleborni turbulent; pink = hybrid control; aqua = hybrid turbulent; blue = L. trewavasae control; orange = L. trewavasae turbulent); the colors of the deformation grids also correspond to these treatment groups. In the turbulent-raised L. fuelleborni andL. trewavasae, note the expanded and upturned heads, especially in comparison to their respective controls. In the hybrid fishes, note the deep body but exaggerated breadth of the craniofacial area in the control cohort, especially in comparison to the L. fuelleborni and L. trewavasae controls; conversely, the turbulent-raised hybrids are much more similar to the turbulent-raised cohorts of the purebred parental species. Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x 3 Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x Discussionh Colors identify treatment groups, and are the same as in Fig. 2. From left to right: Panel (a) compares the jaw and neurocranium angles of L. fuelleborni control (green) and L. trewavasae control (blue), and further illustrates the landmarks that comprise each angle (jaw angle: landmarks 1, 14, 13; neurocranium angle: landmarks 1, 18, 3; see Fig. 5 in the Methods for a description of all landmarks). Panel (b) superimposes all landmarks of the control groups of L. fuelleborni and L. trewavasae as a heuristic to compare an approximate feeding position these fishes would have assumed in this experiment. Panel (c) comparison of jaw and neurocranium angles of L. fuelleborni control (green) and L. fuelleborni turbulent (red), including the average angle and standard error (SE) for both groups; Panel (d) superimposition of all landmarks of L. fuelleborni control and L. fuelleborni turbulent specimens. Panel (e) comparison of angles of hybrid control (pink) and hybrid turbulent (aqua); Panel (f) superimposition of all landmarks of hybrid control and hybrid turbulent. Panel (g) comparison of angles of L. trewavasae control (blue) and L. trewavasae turbulent (orange); Panel (h) superimposition of all landmarks of L. trewavasae control and L. trewavasae turbulent. Note the increased feeding angles in turbulent-raised L. fuelleborni and L. trewavasae, especially in comparison to their control-raised counterparts. See Table 2 for analyses of differences of angles among treatment groups. The differences in hybrid fitness and morphology between the treatments are puzzling. In the control condi- tion, the hybrids had an upwardly-tilted and expanded neurocranium, perhaps extremely so. This morphology is not found in either of the parental species raised in this condition; indeed, it is very different from that of either parental species’ control cohorts, which might explain the poor performance of the hybrids in this condition. As such, this appears to be a transgressive phenotype18,19, though one that is poorly matched to this environment. On the other hand, in the turbulent treatment, the hybrids adopt a morphology very similar to that of the parental species, which better met the demands of the turbulent condition, and likely explains the increased fitness of the hybrids raised in this environment.hi y The deeper bodies found in the fittest individuals in this experiment likely made them more massive, giving them more inertia with which they could resist turbulence, thus allowing them to forage more efficiently in the turbulent treatment sensu3. Discussionh trewavasae is hypothesized to be an adaptation for feeding on the vertical surfaces of rocks, or within the crevices among rocks3,9. As such, forcing this species to feed on a horizontal surface in turbulence could represent a signif- icant energetic challenge that could account for its impaired growth. Recent laboratory experiments demonstrate that, in conditions without turbulence, L. trewavasae both executes more bites and beats its fins more frequently when feeding on horizontal surfaces13, thus likely expending more energy. Conversely, it is not surprising that L. fuelleborni performed well and had high fitness under both conditions, given that these deeper bodied fish were not challenged to feed in an environment that might have been more favorable to L. trewavasae. Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x 4 www.nature.com/scientificreports/ Figure 3. Comparisons of neurocranium and jaw angles among treatment groups, and examinations of possible feeding angles resulting from these morphological angles. Colors identify treatment groups, and are the same as in Fig. 2. From left to right: Panel (a) compares the jaw and neurocranium angles of L. fuelleborni control (green) and L. trewavasae control (blue), and further illustrates the landmarks that comprise each angle (jaw angle: landmarks 1, 14, 13; neurocranium angle: landmarks 1, 18, 3; see Fig. 5 in the Methods for a description of all landmarks). Panel (b) superimposes all landmarks of the control groups of L. fuelleborni and L. trewavasae as a heuristic to compare an approximate feeding position these fishes would have assumed in this experiment. Panel (c) comparison of jaw and neurocranium angles of L. fuelleborni control (green) and L. fuelleborni turbulent (red), including the average angle and standard error (SE) for both groups; Panel (d) superimposition of all landmarks of L. fuelleborni control and L. fuelleborni turbulent specimens. Panel (e) comparison of angles of hybrid control (pink) and hybrid turbulent (aqua); Panel (f) superimposition of all landmarks of hybrid control and hybrid turbulent. Panel (g) comparison of angles of L. trewavasae control (blue) and L. trewavasae turbulent (orange); Panel (h) superimposition of all landmarks of L. trewavasae control and L. trewavasae turbulent. Note the increased feeding angles in turbulent-raised L. fuelleborni and L. trewavasae, especially in comparison to their control-raised counterparts. See Table 2 for analyses of differences of angles among treatment groups. Figure 3. Comparisons of neurocranium and jaw angles among treatment groups, and examinations of possible feeding angles resulting from these morphological angles. Discussionh Panel (a) depicts the fitness curve for body shape in the control condition; Panel (b) is the deformation grid that depicts the morphology with the highest fitness in this control treatment, which is a straight, somewhat deep body with no curvature to the neurocranium, similar to that found in L. fuelleborni. Panel (c) displays the fitness curve for the turbulent condition, and Panel (d) is a deformation grid depicting the morphology of maximum fitness depicted in the turbulent condition, which consists of a deep, somewhat curved body that results in an upturned neurocranium, which was found in L. fuelleborni and to a lesser degree in hybrids. The probability curves on the top and right sides of panels (a) and (c) show the distributions of RW1 scores and fitness, respectively. Panel (e) is a superimposition of the fitness curves, clearly displaying the directional selection against slender, straight bodies in the turbulent condition. Control (n = 105; Multiple R2 = 0.212) Variable Sum of Squares df Mean Square F p Species 15.050 2 7.525 13.733 ≤0.001 Error 55.894 102 0.548 Bonferroni-corrected pairwise differences in Ŷ among species in the Control treatment L. fuelleborni Labeotropheus hybrid Labeotropheus hybrid −0.804 L. trewavasae 0.037 0.841* Turbulent (n = 97; Multiple R2 = 0.262) Variable Sum of Squares df Mean Square F p Species 3.283 2 1.642 16.710 ≤0.001 Error 9.235 94 0.098 Bonferroni-corrected pairwise differences in Ŷ among species in the Turbulent treatment L. fuelleborni Labeotropheus hybrid Labeotropheus hybrid −0.143 L. trewavasae −0.430* −0.288* Table 3. Differences in fitness (Ŷ) among species within both treatments. p ≤ 0.05; p ≤ 0.01; *p ≤ 0.001. Table 3. Differences in fitness (Ŷ) among species within both treatments. p ≤ 0.05; p ≤ 0.01; p ≤ 0.001. Given that the parental specimens used to generate the experimental fishes were obtained from the same location in Lake Malaŵi, these results also provide insight on the likelihood of both the appearance and sur- vival of hybrid Labeotropheus in the wild. Any hybridization between L. fuelleborni (or other deeper-bodied Labeotropheus) and L. trewavasae (or other slender Labeotropheus) would require one or the other parental spe- cies to at least temporarily exploit a habitat for which it would be poorly adapted. In the case of L. fuelleborni, it would have to swim to a depth below which it is commonly found (≥8 m3,9), and to which it cannot acclimate3, while L. Discussionh Not coincidentally, this mimics the pattern found in Labeotropheus in Lake Malaŵi; deep-bodied Labeotropheus are found in wave-swept nearshore areas, while their slender congeners are found in deeper waters. This relationship between body depth and depth distribution becomes somewhat puzzling when considering the depths at which other rock-dwelling cichlids are found in Lake Malaŵi. Many other genera of these fishes, including Maylandia, Tropheops, and Petrotilapia, among others, are always deep-bodied, no matter the depths at which they live. Interestingly, the species within these genera employ much more elevated feeding angles than do the Labeotropheus; based upon in situ observations, Maylandia, Tropheops, and Petrotilapia achieve feeding angles of 58°–90°, while the Labeotropheus have the lowest feeding angle of all of the rock-dwelling cich- lids of Lake Malaŵi, ranging between 35° and 45°17. As we hypothesize above, these elevated feeding angles likely offer greater stability to these species, and would be useful in any habitat, but especially in turbulent areas. The Labeotropheus, on the other hand, are restricted to much more acute feeding angles by their characteristic snout; as such, the only way for them to achieve higher angles, and the greater stability offered by such positions, would be via an upturned neurocranium. Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x 5 www.nature.com/scientificreports/ Figure 4. Fitness curves of control- and turbulent-raised specimens. Panel (a) depicts the fitness curve for body shape in the control condition; Panel (b) is the deformation grid that depicts the morphology with the highest fitness in this control treatment, which is a straight, somewhat deep body with no curvature to the neurocranium, similar to that found in L. fuelleborni. Panel (c) displays the fitness curve for the turbulent condition, and Panel (d) is a deformation grid depicting the morphology of maximum fitness depicted in the turbulent condition, which consists of a deep, somewhat curved body that results in an upturned neurocranium, which was found in L. fuelleborni and to a lesser degree in hybrids. The probability curves on the top and right sides of panels (a) and (c) show the distributions of RW1 scores and fitness, respectively. Panel (e) is a superimposition of the fitness curves, clearly displaying the directional selection against slender, straight bodies in the turbulent condition. Figure 4. Fitness curves of control- and turbulent-raised specimens. Discussionh trewavasae would have to negotiate the turbulent environments in which it would have difficulty feed- ing and maintaining mass (this study), as well as having to compete with more aggressive and strongly territo- rial male L. fuelleborni3,9. The hybrids used in this experiment, while viable and fertile (an inbred F2 generation Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x 6 www.nature.com/scientificreports/ Figure 5. Homologous landmarks used for geometric morphometric analysis. For clarity, the landmarks are displayed on an adult L. fuelleborni from Chidunga Rocks. The landmark positions are described in greater detail in the Methods. Figure 5. Homologous landmarks used for geometric morphometric analysis. For clarity, the landmarks are displayed on an adult L. fuelleborni from Chidunga Rocks. The landmark positions are described in greater detail in the Methods. currently exists in our laboratory aquaria; Pauers, unpubl. data), are not likely to exist in Lake Malaŵi. While they could likely thrive in a shallow, turbulent environment, their paternal species, L. trewavasae, could not. Similarly, although it might be possible for their maternal species, L. fuelleborni, to visit a deeper depth, perhaps at an island or submerged reef3,9, the hybrid young could not effectively compete with either parental species in these deep, calm habitats. Interestingly, our results suggest that both selection on morphology and hybridization in the Labeotropheus would have been concomitant with water level fluctuations in Lake Malaŵi. Throughout its history, Lake Malaŵi has undergone extreme lake level fluctuations, ranging from its current deep, full-basin level, to nearly dry about 100,000 years ago20,21. A decrease in lake level would have severely limited the rock-strewn habitats favored by the Labeotropheus, especially the deeper rocky shelves preferred by L. trewavasae20,21. As such, the available rocky habitat would have been shallow and likely turbulent, thus favoring a morphology that could successfully feed under these conditions; i.e., a deeper body and an upturned neurocranium. Further, any deep-dwelling, slender Labeotropheus would have likely been forced to hybridize with the deeper-bodied Labeotropheus sensu21; inter- estingly, the predominant shallow, turbulent conditions surrounding these rocky habitats would have been ideal for Labeotropheus hybrids, given the morphology we found them to adopt under these conditions. Once lake levels rose, and the deep rocky habitats reappeared, selection against slender Labeotropheus morphotypes would relax, and individuals with this body shape could once again exploit these habitats. Discussionh Conversely, selection against hybridization would strengthen, due to assortative mating based on male nuptial coloration22, and would help to promote the subsequent radiation of Labeotropheus21. p Directional selection has been a potent force in the evolution of the cichlids of Lake Malaŵi, and has been highly influential in the evolution of the oral jaws of L. fuelleborni23; as such, it is not surprising to find that it is also active in shaping the morphology of the neurocranium, snout, and body, as demonstrated in our current experiments. Feeding in turbulence imposes finite constraints on both the morphology and swimming perfor- mance of fishes reviewed in24, thus establishing a selective environment favoring what are often extreme morphol- ogies24,25. The experiments described herein placed fish that already have one of the most extreme morphologies of any cichlid from Lake Malaŵi8 in an experimental condition in which the morphology had to be further elaborated in order for these fishes to grow. The stocky, robust, L. fuelleborni, ostensibly specialized for feeding in turbulence, readily adapted to the experimental turbulence. On the other hand, the slender L. trewavasae, long suggested to be specialized for feeding in calm, structurally-complex habitats, was unable to effectively feed and grow in the experimental turbulent condition. It seems likely, then, that directional selection is responsible for L. fuelleborni’s successful exploitation of the shallow, turbulent nearshore regions of Lake Malaŵi. www.nature.com/scientificreports/ These pumps (Marineland MaxiJet Pro 1200; Spectrum Brands, Blacksburg, VA, USA) were each capable of producing a current of 4920 Lh−1, but were powered by a wave generator (Ocean Pulse Quadra; Transworld Aquatic Ent,; Inglewood, CA, USA) that created three pulses of water every minute (i.e., one pulse every 20 s). Each wave travelled at a rate of 0.12 ms−1, produc- ing the turbulent conditions in which these fishes were forced to feed.hi gi The young fish were raised in each condition for 45 days, then removed and euthanized with an overdose of MS-222 (tricaine methanesulfonate; 250 mgL−1). The fish were then stored in 70% EtOH until they could be photographed for morphometric analysis. p g p p y All of the procedures described above were approved by the UW Colleges Animal Care and Use Committee (ACUC), protocol # 2017-MP-03R, and were also approved by the Senior Animal Staff of the Milwaukee County Zoo (MCZ). All procedures were performed in accordance with the relevant guidelines and regulations set forth by the UW Colleges ACUC and the MCZ Senior Animal Staff, and followed all legal requirements of the United States of America. Geometric morphometric analysis. Preserved fishes were pinned with fins erect onto a felt-covered Styrofoam sheet and photographed from a distance of 10 cm with a digital camera attached to a copystand. These photographs were then imported to ImageJ and 19 homologous landmarks were superimposed onto the images. These landmarks, following26, were: (1) anterior tip of snout, (2) dorsal tip of premaxillary pedicel, (3) origin of dorsal fin, (4) insertion of dorsal fin, (5) dorsal insertion of caudal fin, (6) caudal border of hypural plate aligned with lower lateral line, (7) ventral insertion of caudal fin, (8) insertion of anal fin, (9) origin of anal fin, (10) base of pelvic fin spine, (11) a point on posterior margin of opercular membrane meeting ventral margin of head, (12) posterior end of dentary symphysis, (13) anteriormost margin of upper jaw soft tissue, (14) posterior end of jaws, (15) anterior margin of midline through the eye, (16) posterior margin of midline through the eye, (17) dorsal end of preopercle, (18) dorsalmost end of opercule, and (19) origin of pectoral fin (Fig. 5). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Species Treatment Replicate n L. fuelleborni Control 1 17 2 6 Turbulent 1 17 2 5 Hybrid Control 1 26 2 5 Turbulent 1 17 2 8 L. trewavasae Control 1 22 2 29 Turbulent 1 20 2 30 Table 4. Sample sizes of individuals raised 45 days in each treatment and replicate. Species Treatment Replicate n L. fuelleborni Control 1 17 2 6 Turbulent 1 17 2 5 Hybrid Control 1 26 2 5 Turbulent 1 17 2 8 L. trewavasae Control 1 22 2 29 Turbulent 1 20 2 30 Table 4. Sample sizes of individuals raised 45 days in each treatment and replicate. Table 4. Sample sizes of individuals raised 45 days in each treatment and replicate. accumulated on the bottom of each mesocosm were regularly removed via siphoning, and approximately 10% of the volume of each mesocosm was removed and replaced each week. accumulated on the bottom of each mesocosm were regularly removed via siphoning, and approximately 10% of the volume of each mesocosm was removed and replaced each week. A stack of paving blocks 53.33 cm L × 30.5 cm W × 40.6 cm H was used as a feeding station. Feeding blocks were created by smearing a gel-based vegetarian food (Mazuri Herbivore Aquatic Gel Diet – No Corn; PMI Nutrition, Inc., St. Louis, MO, USA) onto the upper surface (15.25 cm × 15.25 cm; 232.56 cm2) of a paving block, which was then air-dried overnight and stored in a refrigerator at about 8 °C. These feeding blocks were placed atop the feeding station and replaced when all the food had been eaten. The feeding blocks were an important part of this experiment, as they allowed the young Labeotropheus to feed naturally (i.e., by scraping food from a surface), and forced them to forage in the experimental turbulence (see below).h g p The control mesocosm was set up exactly as described above, and had a background current of approximately 0.017 ms−1. The turbulent mesocosm had two additional submersible water pumps mounted directly across from the feeding tower, with their outputs aimed at the feeding block. Methods E i Experimental animals. Wild caught adult specimens of Labeotropheus fuelleborni and L. trewavasae were obtained from a reputable Malaŵi-based exporter of cichlid fishes (Stuart M. Grant, Ltd.); both species were captured at the Chidunga Rocks in southwestern Lake Malaŵi. Twenty individuals of each species were obtained, ten of each sex. These adults were segregated by species and sex into four separate 160 L aquaria and were housed at the Aquarium and Reptile Center of the Milwaukee County Zoo. These fishes were fed to satiation daily with a mixture of spirulina flake food (e.g., Formula Two; Ocean Nutrition, Newark, CA, USA), cichlid pellets (e.g., Hikari Cichlid Excel; Kyorin Co., Ltd., Hyogo, Japan), and a variety of frozen foods (e.g., bloodworms, brine shrimp; various manufacturers). Water temperature was maintained at 28 °C, and water quality was maintained using standard box filters and twice-weekly 50% water changes.i gi y g In order to produce the experimental fishes, haphazardly-selected groups of adults (1–3 males with 2–5 females) were moved into separate 160 L aquaria, which were maintained as described above. Brooding females were isolated until their fry were released at about 21 days post-fertilization. Hybrids were produced by isolating male L. trewavasae with female L. fuelleborni. The sample sizes of individuals ultimately harvested from each replicate are shown in Table 4. Experimental rearing conditions. When the fry were between 25 and 35 days post fertilization, they were then moved to the experimental mesocosms. Two such mesocosms were built using large, 1.83 m diameter aquaculture tubs. The tubs were filled to a depth of 56 cm, giving a wetted volume of 1470 L, and were heated to a temperature of 28 °C using programmable titanium heaters. Filtration was provided by single submersible filter (Fluval U4; Rolf C. Hagen Corp., Mansfield, MA, USA) that produced a current of 260 Lh−1. The wastes that Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x 7 Data Availabilityh Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. y The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Referencesh g q 29. Schluter, D. & Nychka, D. Exploring fitness surfaces. Am. Nat. 143, 597–616 (1994). www.nature.com/scientificreports/ p p p g pi g The (x, y) coordinates of each landmark for each specimen were then used in a Generalized Procrustes Analysis27 in the program Coordgen8 to reduce the effects of size and orientation of each specimen. Next, a multiple regression of shape on geometric centroid size was performed using the Standard6 software to remove the effects of allometry from the data. Allometry-free data were then subjected to a thin-plate spline procedure to generate geometric descriptors of shape variation called relative warps (RW); this procedure was performed using the program TPSRelW. All deformation grids were generated with TPSRelW. Additionally, the angles among landmarks were calculated using the TradMorphGen tools within Coordgen8. All morphometric software is free for public use and can be downloaded from: http://www.life.bio.sunysb.edu/morph/. Additionally, we used Systat 10.0 to calculate the ANOVAs of the RW scores, and to create the bivariate plots of the RW scores. Selection analyses. In order to estimate the possible action of selection on morphology, we first estimated the daily growth rate for each individual over the course of the 45-day experiment by subtracting the mean stand- ard length (SL) of a cohort of 10 individuals of the same species collected and euthanized at day 0 of the experi- ment from its SL at day 45. Since RW1 explains more variation than any of the other RW factors, and because it describes morphological variation among both species and treatments, we used that as our proxy of body shape. Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x 8 www.nature.com/scientificreports/ We then standardized each individual’s RW1 score by subtracting the mean RW1 score of its experimental group from its own RW1 score, and dividing by the standard deviation of the group’s RW1 scores28. These data were then used to estimate fitness with a cubic spline in the program glmsWIN 1.029, which can be downloaded from: https://www.zoology.ubc.ca/∼schluter/wordpress/software/. We then standardized each individual’s RW1 score by subtracting the mean RW1 score of its experimental group from its own RW1 score, and dividing by the standard deviation of the group’s RW1 scores28. These data were then used to estimate fitness with a cubic spline in the program glmsWIN 1.029, which can be downloaded from: https://www.zoology.ubc.ca/∼schluter/wordpress/software/. Referencesh References 1. Fryer, G. The trophic interrelationships and ecology of some littoral communities of Lake Nyasa with especial reference to the fishes and a discussion of the evolution of a group of rock-frequenting Cichlidae Proc Zool Soc Lond 132 153-281 (1959) References 1. Fryer, G. The trophic interrelationships and ecology of some littoral communities of Lake Nyasa with especial reference to the fishes and a discussion of the evolution of a group of rock-frequenting Cichlidae. Proc. Zool. Soc. Lond. 132, 153-281 (1959).h g p q g 2. Fryer, G., & Iles, T. D. The Cichlid Fishes of the Great Lakes of Africa (Oliver and Boyd, 1972).h yh f f f y 3. Ribbink, A. J., Marsh, A. C., Marsh, B. A. & Sharp, B. J. The zoogeography, ecology and taxonomy of the genus Labeotropheus Ahl 1927, of Lake Malawi (Pisces: Cichlidae). Zool. J. Linn. Soc. 79, 223–43 (1983a).hi 4. Reinthal, P. N. The feeding habits of a group of herbivorous rock-dwelling cichlid fishes (Cichlidae, Perciformes) from Lake Malawi Africa. Env. Biol. Fish. 27, 215–233 (1990). 5. Ribbink, A. J. Distribution and ecology of the cichlids of the African Great Lakes in (ed. Keenleyside, M. H. A.) Cichlid Fishes Behaviour, Ecology and Evolution 36–59 (Chapman & Hall, 1991). Behaviour, Ecology and Evolution 36 59 (Chapman & Hall, 1991). 6. Ahl, E. Einige neue Fische der Familie Cichlidae aus dem Nyassa-see. Sitz. Berl. Gesell. Natur. Freund. Berlin 1926, 51–62 (1927). m Nyassa-see. Sitz. Berl. Gesell. Natur. Freund. Berlin 1926, 51–62 ( g y 7. Fryer, G. A new species of Labeotropheus from Lake Nyasa, with a redescription of Labeotropheus fuelleborni Ahl and some notes on the genus Labeotropheus (Pisces: Cichlidae. Rev. Zool. Bot. Afr. 54, 280–289 (1956).h 8. Concannon, M. R. & Albertson, R. C. The genetic and developmental ba cichlids. J. Exp. Zool. B (Mol. Dev. Evol.) 324, 662–670 (2015). 8. Concannon, M. R. & Albertson, R. C. The genetic and developmental basis of an exaggerated craniofacial trait in East African cichlids. J. Exp. Zool. B (Mol. Dev. Evol.) 324, 662–670 (2015).i 8. Concannon, M. R. & Albertson, R. C. The genetic and develo h cichlids. J. Exp. Zool. B (Mol. Dev. Evol.) 324, 662–670 (2015). 9. Ribbink, A. J., Marsh, B. A., Marsh, A. C., Ribbink, A. C. & Sharp, B. J. A preliminary survey of the cichlid fishes of rocky habitats in Lake Malawi. S. Afr. J. Zool. Referencesh 18, 149–310 (1983b).h f 10. Albertson, R. C., Streelman, J. T., Kocher, T. D. & Yelick, P. C. Integration and evolution of the cichlid mandible: The molecular basis of alternate feeding strategies. Proc. Nat. Acad. Sci. (USA) 102, 16287–16292 (2005).i g g ( ) ( ) 1. McElroy, D. M. & Kornfield, I. Novel jaw morphology in hybrids between Pseudotropheus zebra and Labeotropheus fuelleborn (Teleostei: Cichlidae) from Lake Malawi, Africa. Copeia 1993, 933–945 (1993). p 12. Albertson, R. C. & Kocher, T. D. Assessing morphological differences in an adaptive trait: a landmark-based morphometric approach. J. Exp. Zool. 289, 385–403 (2001).l p 13. Rupp, M. F. & Hulsey, C. D. Influence of substrate orientation on feeding kinematics and performance of algae grazing Lake Malawi cichlid fishes. J. Exp. Biol. 217, 3057–3066 (2014).h i p 14. Konings, A. F. 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Pauers, M. J. & McMillan, S. A. Geometric morphometrics reveals surprising diversity in the Lake Malawi cichlid genus L b t ph H d bi l i 748 145 160 (2015) 6. Pauers, M. J. & McMillan, S. A. Geometric morphometrics reveals surprising diversity in the Lake Malawi cichlid genu Labeotropheus. Hydrobiologia 748, 145–160 (2015). p y g 27. Rohlf, F. J. & Slice, D. Extensions of the Procrustes method for the optimal superimposition of landmarks. Syst. Biol. 39, 4 (1990). (1990). 8. Schluter, D. Estimating the form of natural selection on a quantitative trait. Evolution 42, 849–861 (1988). Schluter, D. Estimating the form of natural selection on a quantitati 8. Schluter, D. Estimating the form of natural selection on a quantitative trait. Evolution 42, 849–861 (1988). 9. Schluter, D. & Nychka, D. Exploring fitness surfaces. Am. Nat. 143, 597–616 (1994). 28. Schluter, D. Estimating the form of natural selection on a quantitative trait. Evolution 42, 849–861 (1988) 29. Schluter, D. & Nychka, D. Exploring fitness surfaces. Am. Nat. 143, 597–616 (1994). Acknowledgements David P. Nkhwazi (Stuart M. Grant, Ltd.) and Laif DeMaison (Old World Exotic Tropical Fish) were instrumental in obtaining the wild-caught Labeotropheus. The paving blocks were donated by Champion Brick (New Berlin, WI, USA). We thank the staff of the Aquarium and Reptile Center of the Milwaukee County Zoo, including Billie Harrison, Jeff Johnson, Chad Pappas, Craig Pavlik, and Melissa Spreda, for their skilled and attentive help throughout this project. Financial support for this project was provided by the American Cichlid Association Guy D. Jordan fund, the UW Colleges Summer Research Grant program, the UW Colleges Department of Biological Sciences, and the Orth Ichthyology Research Fund of the Milwaukee Public Museum. The Behavior, Ecology, and Evolution Discussion Group of the Milwaukee Public Museum offered many helpful comments and critiques of an early presentation of this project. We also thank former UW Colleges Vice Chancellor Joe Foy for his steadfast support of MJP’s research activities. RAH and KP participated in this project as part of the UW-Waukesha Undergraduate Honors Program and the UW-Waukesha Undergraduate Research Program. Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x 9 www.nature.com/scientificreports/ Author Contributions M.J.P. originally conceived the project, obtained funding, carried out the experimental treatments, analyzed the data, and wrote the final manuscript. K.R.F., R.A.H., and K.P. photographed the fish, superimposed the landmarks, managed and prepared the data for analysis, and assisted M.J.P. with data analysis and proofreading the final manuscript. R.A.H. and K.P. prepared early drafts of the manuscript. K.R.F. assisted with logistical aspects of the project, especially as the project began. Additional Informationh Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific REPOrtS \| (2018) 8:15842 \| DOI:10.1038/s41598-018-34135-x 10
https://openalex.org/W2264306296	https://kclpure.kcl.ac.uk/ws/files/46748505/DasMunshi_ECHASM_off_print_protocol_paper.pdf	English	null	Ethnicity and cardiovascular health inequalities in people with severe mental illnesses: protocol for the E-CHASM study	Social psychiatry and psychiatric epidemiology	2,016	cc-by	9,458	Citation for published version (APA): Das-Munshi, J., Ashworth, M., Gaughran, F. P., Hull, S., Morgan, C., Nazroo, J., Roberts, A., Rose, D. S., Schofield, P., Stewart, R. J., Thornicroft, G. J., & Prince, M. J. (2016). Ethnicity and cardiovascular health inequalities in people with severe mental illnesses: protocol for the E-CHASM study. Social Psychiatry and Psychiatric Epidemiology. https://doi.org/10.1007/s00127-016-1185-8 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. 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Download date: 24. Oct. 2024 Keywords Severe mental illness Ethnicity Keywords Severe mental illness Ethnicity Cardiovascular disease Schizophrenia Bipolar affective disorder 4 Centre for Primary Care and Public Health, Blizard Institute, Queen Mary University of London, Yvonne Carter Building, 58 Turner Street, London E1 2AB, UK 5 University of Manchester, Manchester, England J. Das-Munshi1 • M. Ashworth2 • F. Gaughran3 • S. Hull4 • C. Morgan1 • J. Nazroo5 • A. Roberts6 • D. Rose1 • P. Schoﬁeld2 • R. Stewart7 • G. Thornicroft1 • M. J. Prince1 J. Das-Munshi1 • M. Ashworth2 • F. Gaughran3 • S. Hull4 • C. Morgan1 • J. Nazroo5 • A. Roberts6 • D. Rose1 • P. Schoﬁeld2 • R. Stewart7 • G. Thornicroft1 • M. J. Prince1 J. Das-Munshi1 • M. Ashworth2 • F. Gaughran3 • S. Hull4 • C. Morgan1 • J. Nazroo5 • A. Roberts6 • D. Rose1 • P. Schoﬁeld2 • R. Stewart7 • G. Thornicroft1 • M. J. Prince1 Received: 17 November 2015 / Accepted: 18 January 2016 Received: 17 November 2015 / Accepted: 18 January 2016 The Author(s) 2016. This article is published with open access at Springerlink.com prevalence and disparities in accessing healthcare for ethnic minority people living with SMI exist. A nested qualitative study will be used to assess barriers to accessing healthcare, both from the perspectives of service users and providers. Results In primary care, 993,116 individuals, aged 18?, provided data from 186/189 (98 %) practices in four inner- cityboroughs(localgovernment areas)inLondon.Prevalence of SMI according to primary care records, ranged from 1.3–1.7 %, across boroughs. The primary care sample inclu- ded Bangladeshi [n = 94,643 (10 %)], Indian [n = 6086 (6 %)], Pakistani [n = 35,596 (4 %)], black Caribbean [n = 45,013 (5 %)], black African [n = 75,454 (8 %)] and Irish people [n = 13,745 (1 %)]. In the secondary care data- base, 12,432 individuals with SMI over 2007–2013 con- tributed information; prevalent diagnoses were schizophrenia [n = 6805 (55 %)], schizoaffective disorders [n = 1438 (12 %)] and bipolar affective disorder [n = 4112 (33 %)]. Largest ethnic minority groups in this sample were black Caribbean [1432 (12 %)] and black African (1393 (11 %)). Conclusions There is a dearth of research examining cardiovascular disease in minority ethnic groups with severe mental illnesses. The E-CHASM study will address Social Psychiatry and Psychiatric Epidemiology The International Journal for Research in Social and Genetic Epidemiology and Mental Health Services ISSN 0933-7954 Soc Psychiatry Psychiatr Epidemiol DOI 10.1007/s00127-016-1185-8 Ethnicity and cardiovascular health inequalities in people with severe mental illnesses: protocol for the E-CHASM study J. Das-Munshi, M. Ashworth, F. Gaughran, S. Hull, C. Morgan, J. Nazroo, A. Roberts, D. Rose, P. Schofield, R. Stewart, G. Thornicroft, et al. Ethnicity and cardiovascular health inequalities in people with severe mental illnesses: protocol for the E-CHASM study 1 23 Social Psychiatry and Psychiatric Epidemiology The International Journal for Research in Social and Genetic Epidemiology and Mental Health Services ISSN 0933-7954 Soc Psychiatry Psychiatr Epidemiol DOI 10.1007/s00127-016-1185-8 Ethnicity and cardiovascular health inequalities in people with severe mental illnesses: protocol for the E-CHASM study J. Das-Munshi, M. Ashworth, F. Gaughran, S. Hull, C. Morgan, J. Nazroo, A. Roberts, D. Rose, P. Schofield, R. Stewart, G. Thornicroft, et al. J. Das-Munshi, M. Ashworth, F. Gaughran, S. Hull, C. Morgan, J. Nazroo, A. Roberts, D. Rose, P. Schofield, R. Stewart, G. Thornicrof Social Psychiatry and Psychiatric Epidemiology The International Journal for Research in Social and Genetic Epidemiology and Mental Health Services ISSN 0933-7954 Soc Psychiatry Psychiatr Epidemiol DOI 10.1007/s00127-016-1185-8 et al. 1 23 Your article is published under the Creative Commons Attribution license which allows users to read, copy, distribute and make derivative works, as long as the author of the original work is cited. You may self- archive this article on your own website, an institutional repository or funder’s repository and make it publicly available immediately. 1 23 Soc Psychiatry Psychiatr Epidemiol DOI 10.1007/s00127-016-1185-8 STUDY PROTOCOLS AND SAMPLES Abstract Purpose People with severe mental illnesses (SMI) experience a 17- to 20-year reduction in life expectancy. One-third of deaths are due to cardiovascular disease. This study will establish the relationship of SMI with cardio- vascular disease in ethnic minority groups (Indian, Pak- istani, Bangladeshi, black Caribbean, black African and Irish), in the UK. Methods E-CHASM is a mixed methods study utilising data from 1.25 million electronic patient records. Secondary analysis of routine patient records will establish if differ- ences in cause-speciﬁc mortality, cardiovascular disease & J. Das-Munshi jayati.das-munshi@kcl.ac.uk 1 Department of Health Service and Population Research, Centre for Epidemiology and Public Health, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, De Crespigny Park, PO 33, London SE5 8AF, UK Conclusions There is a dearth of research examining cardiovascular disease in minority ethnic groups with severe mental illnesses. The E-CHASM study will address this knowledge gap. 2 Division of Health and Social Care Research, Department of Primary Care and Public Health Sciences, King’s College London, 3rd Floor, Addison House, Guy’s Campus, London SE1 1UL, UK 3 South London and Maudsley Trust and King’s College London, London, UK Keywords Severe mental illness Ethnicity Cardiovascular disease Schizophrenia Bipolar affective disorder Received: 17 November 2015 / Accepted: 18 January 2016 The Author(s) 2016. This article is published with open access at Springerlink.com Objectives 1. To understand the reasons for premature mortality in ethnic minority people living with severe mental illnesses; in particular to determine variations by ethnicity in the following: (a) The effect of severe mental illness on cardiovascular risk factors; Health inequalities may be even more pronounced among ethnic minority populations with severe mental illnesses [13, 14]. The reasons for this are unclear. There is much evidence to suggest that those from ethnic minority groups may experience disadvantage within the mental healthcare system. Black people are more likely to be compulsorily detained [15], be less satisﬁed with pre- scribing [16] and more likely to be prescribed high-potency antipsychotics at high doses [17–19]. In addition, physical health monitoring may not be to the same standard as for white patients [20]. The prevalence of cardiovascular dis- ease is known to be elevated in people living with severe mental illnesses and is also known to be of a greater con- cern for some ethnic minority groups [21]. The exact nat- ure of the interaction between being of an ethnic minority background and living with severe mental illness, for the risk of cardiovascular disease—is less clear. Previous research examining cardiovascular disease health inequal- ities by ethnicity in severe mental illness populations has been limited by an over-reliance on small convenience samples recruited from clinics, with limited representa- tiveness and without the inclusion of adequate numbers of people from ethnic minority groups to enable assessment of prevalence of cardiovascular disease [14]. (b) Cause-speciﬁc mortality patterns among people with severe mental illnesses; (c) Quality of care received, relevant to premature mortality; 2. To develop and validate a measure for individual- level socioeconomic position for application in a large secondary care electronic mental health records data resource, which will be used to improve the assessment of the association of self-ascribed eth- nicity with health-related outcomes. 3. To assess barriers to equitable physical healthcare amongst ethnic minority people living with severe mental illnesses, from the perspectives of service users, their carers and clinicians. Background 6 Natural Language Processing Group, Department of Computer Science, University of Shefﬁeld, Shefﬁeld, England People living with severe mental illnesses such as schizophrenia have a reduced life expectancy relative to the general population which is up to 20 years earlier in men and 17 years in women, in high income countries [1, 2]. A 7 Institute of Psychiatry, Psychology and Neuroscience, King’s College London, Room M1.06, De Crespigny Park, London SE5 8AF, UK 12 3 Soc Psychiatry Psychiatr Epidemiol large proportion of deaths are from chronic diseases, including coronary heart disease and stroke [3]. Increased mortality may be related to people with severe mental ill- nesses receiving poorer quality physical healthcare [4–6]. There is also a higher prevalence of metabolic risk factors such as obesity [7], hyperlipidaemia [8], diabetes [9] together with higher smoking rates [10], in these popula- tions. Anti-psychotic medications, especially at higher doses, are associated with death from stroke and coronary heart disease [11]. People with severe mental illnesses experience barriers to seeking timely help for co-morbid medical problems [12]. Finally, there may be shared factors underlying premature mortality and severe mental illness, such as social disadvantage [12]. quantitative ﬁndings, adopting a mixed methods design [22]. Using this approach, it may be possible to understand trends revealed in quantitative data analysis, particularly from the perspectives of service users, carers and clini- cians/service providers, which will help to elucidate mechanisms underlying quantitative ﬁndings. Hypotheses (for quantitative data analyses) Compared to white British people with severe mental ill- nesses, ethnic minority service users with severe mental illnesses will: 1. Have an elevated prevalence of cardiovascular disease risk factors (hypertension, diabetes, obesity, raised serum cholesterol, and smoking) and be less likely to have had these adequately managed, as determined by national standards for clinical management. The E-CHASM study (ethnicity and cardiovascular health inequalities in severe mental illness) described in this protocol will draw upon electronic health records from a large secondary care mental health Trust in England, as well as data from primary care, to enable analyses exam- ining the mechanisms for premature mortality due to car- diovascular disease in ethnic minority people with severe mental illnesses. The catchment areas for the study (south east/east London) represent an ethnically and socioeco- nomically diverse part of London, typical of many inner- cities where ethnic minority communities reside and where the burden of chronic health conditions is greatest. 2. Be less likely to have cardiovascular disease risk factors adequately screened or managed, when pre- scribed neuroleptic medication; 3. Be more likely to be prescribed multiple antipsychotics or antipsychotics at higher doses or outside recom- mended dose ranges. 4. Experience differing causes of mortality (i.e., cause- speciﬁc mortality fractions), in particular excess risk of mortality due to coronary artery disease. E-CHASM will additionally utilise an embedded qual- itative study, with integration of qualitative ﬁndings with E-CHASM will additionally utilise an embedded qual- itative study, with integration of qualitative ﬁndings with 123 123 123 Soc Psychiatry Psychiatr Epidemiol Design To address key objectives, this study will utilise anon- ymised patient data from a variety of sources, some of which will be linked together (e.g., secondary care patient data linked to Ofﬁce for National Statistics (ONS) mor- tality data). This programme of research will follow a quantitative study design leading on to a nested qualitative study. In the ﬁnal phase, results from the quantitative and qualitative studies will be integrated. Psychotropic medications Information on prescriptions of antipsychotic medications according to formulation (oral/depot) and dose, will be extracted and classiﬁed [36] prior to analyses. Qualitative study aims extracted through data entered into structured ﬁelds in primary care electronic patient records (Read Codes [31]). Healthcare records contain information on consultation rates, clinical measurement values, prescribing and health- screening [28–30]. The aim of the qualitative phase of this study is to identify barriers to delivering equitable physical healthcare (from clinician perspectives) or in accessing healthcare (service user and/or carer perspectives). Overview of data from primary care Overview of data from primary care Setting The London boroughs represented in the study (Lambeth, Tower Hamlets, Newham, City and Hackney) are notable for being home to the largest ethnic minority communities in the UK, including Bangladeshi, black Caribbean and black African communities; up to 51 % in these areas comprise people of an ethnic minority back- ground [23]. These areas are also characterised by high population density and poverty [23, 24]. Tower Hamlets, Newham and Hackney have the highest levels of depriva- tion in England [25]. The location of the study is charac- teristic of many other urban locations where ethnic minority communities reside within the UK [24]. Qualitative study questions Information on patient age, gender and ethnicity will be collected for analyses. Data will be linked to indices for area-level deprivation, such as the index of multiple deprivation [32], at small geographic level. 1. What are the barriers to having physical health moni- tored and treated in black and minority ethnic people living with severe mental illness? Severe mental illnesses 2. How are physical health problems related to cardio- vascular disease managed by black and minority ethnic participants living with severe mental illnesses? 2. How are physical health problems related to cardio- vascular disease managed by black and minority ethnic participants living with severe mental illnesses? General practitioners are ﬁnancially incentivised to main- tain a register of people with severe mental illnesses. Individuals with a diagnosis of schizophrenia, bipolar affective disorder or non-organic psychosis, comprise people on this register [31]. The use of computer-based electronic records to identify patients with severe mental illnesses in primary care has previously been validated, with a sensitivity of 91 % and positive predictive value of 91 % for non-organic psychosis, assessed against a syn- drome checklist derived from the Present State Examina- tion and International Classiﬁcation of Disease-9 (ICD-9) [33], applied to clinical case notes [34]. Recent work has shown that these diagnostic groupings remain stable over time [35]. Quantitative research methodology Diagnostic read codes will be used to ascertain presence of main cardiovascular health indicators. Diabetes mellitus, hypertension, weight (body mass index), smoking status and presence of hyperlipidaemia will be ascertained by presence of diagnostic codes, blood test results and other measurements (e.g., glycated haemoglobin (HbA1c), lipid proﬁle readings, etc.) Cause-speciﬁc mortality have received care from a large secondary mental health service provider organisation (South London and Maudsley NHS Foundation Trust)37. South London and Maudsley is one of the largest mental health Trusts in Europe serving a base population of approximately 1.2 million people [37] living in south east London and using electronic health records across all its services since 2006 (with some ser- vices adopting these earlier). This data source contains all patient contacts with Trust services including out-patient appointments and in-patient admissions. The information from the electronic health record is accessed via the Clinical Record Interactive Search (CRIS) software sys- tem, which permits free text and structured ﬁelds to be searched for relevant information regarding patient char- acteristics, interventions and outcomes. All data since 2007 will be used in the E-CHASM study. All electronic patient records contained within this dataset have a unique NHS patient identiﬁer which can be linked to death certiﬁcate information nationally. Lists of deceased patients are downloaded on a monthly basis from the NHS care records service. To ascertain cause of death, information from linked death certiﬁcates will be extracted and categorised according to ICD-10 [39]. Development of a measure for individual-level socioeconomic position Much important information is captured within the free- text information ﬁelds of electronic patient records [40, 41], particularly in mental healthcare. Using computa- tional techniques such as natural language processing may help to unlock this information from within these text ﬁelds [41]. Within the SLaM-BRC Case Register, this has already been applied to derive information on cognition [42], smoking [43], pharmacotherapy [38] and symptoms [44]. The software [General Architecture for Text Engineering (GATE)] is an open source package used for natural language processing [45]. For example, in a study designed to assess smoking use, natural lan- guage processing supplemented information within structured ﬁelds, leading to an increase in proportions identiﬁed as smokers, from 11.6 % (when reliant on structured ﬁelds alone) to 64 % (when supplemented by natural language processing of free text) [43]. This approach is robust and repeatable [43]. Algorithms using GATE will be developed to derive indicators for socioeconomic position, through ﬁrst ascertaining a pri- ori keywords indicative of socioeconomic position, fol- lowed by an iterative process of: (1) developing a bespoke gazetteer of terms related to socioeconomic position (together with synonyms), and rules and models for extracting socioeconomic position; (2) evaluating the real world use and applicability of these terms and rules in routine clinical records. At least two indicators of socioeconomic position will be derived: education and occupational social class. Education is a valuable mea- sure of socioeconomic position as it reﬂects early life socioeconomic position and is strongly related to parental characteristics [46]. Occupational social class is an important measure of socioeconomic position as it taps into individual social standing, conditions relating to work-based stress, and is also predictive of income and material resources [46]. Both have clear associations with mental and physical health [46, 47]. Psychotropic medications Detail on antipsychotic medications will be derived from the health records and classiﬁed into formulation (oral/ depot) and dose [36]. Information on medications pre- scribed will be extracted from structured ﬁelds. In order to minimise missing data on prescribed medications, natural language processing with a bespoke algorithm will also be used to extract relevant information on prescribing from free text [38]. Demographic indicators Information on age, gender and self-ascribed ethnicity will be used. Area-level deprivation indicators matched to the last census will be used in initial analyses; however, work over the course of this study will lead to the derivation of a variable for individual-level socioeconomic position, as detailed below. Overview of data from secondary care Measures from primary care The quality and outcomes framework (QOF), a pay-for-performance scheme [26] introduced into primary care in the UK in 2004 [27], ensures that data quality is good for key indicators of health [28–30]. Information from primary care records will be South London and Maudsley NHS Foundation Trust Biomedical Research Centre (SLaM BRC) Case Register South London and Maudsley NHS Foundation Trust Biomedical Research Centre (SLaM BRC) Case Register This is an anonymised data resource drawn from the electronic health records of over 250,000 service users who 12 3 Soc Psychiatry Psychiatr Epidemiol Mental disorder Within South London and Maudsley Trust (SLaM), clinical teams are required to assign mental disorder diagnoses for all service users [37]. Diagnoses are entered into structured ﬁelds on the electronic record. Due to auditing of diagno- sis, completion rates for diagnostic ﬁelds are high [37]. Using information from these ﬁelds, supplemented by natural language processing of free text and clinical note ﬁelds [38], the following ICD-10 diagnostic groups will be included in analyses: schizophreniform disorders (F20– F29), mania and bipolar affective disorder (F30, F31). 123 12 Soc Psychiatry Psychiatr Epidemiol Analyses ‘Poor quality’ of care will be deemed present when Table 1 Smallest effect sizes (odds ratios) detectable for the largest and smallest ethnic minority groups at 80 % power (with two sided 5 % signiﬁcance levels), for exposures with a prevalence of 10, 20 and 50 % in the reference (white British) group Primary care data source N Prevalence of outcome in white British group White British 238,211 10 % 20 % 50 % Smallest detectable odds ratio Bangladeshi 93,143 1.03 1.02 1.01 Irish 13,459 1.08 1.05 1.03 Secondary care data source N Prevalence of outcome in white British group white British 28,618 10 % 20 % 50 % Smallest detectable odds ratio ‘Other’ white 4477 1.14 1.09 1.05 Indian 711 1.35 1.23 1.11 Sample size calculation The following table details detectable effect sizes at 80 % power. There is greater power to detect associations in the primary care sample for each of the ethnic groups, as it is a larger sample (Table 1). 2. To determine ethnic variations in the effect of SMI on cardiovascular risk factors. Data from primary care will be used. The association of severe mental illness with cardiovascular risk factors (such as type 2 diabetes mellitus, hypertension, obesity, tobacco use and 10-year cardiovascular mortality risk, such as QRISK [48]) will be assessed using multivariable logistic regression, adjusting or stratifying for age and gender as appropriate. Odds ratios stratiﬁed by ethnicity for the association of severe mental illness with cardiovascular disease risk will be obtained, in order to assess for ethnic variations in the effect of severe mental illness on cardio- vascular risk factors. Formal tests of statistical interaction will be used to assess for heterogeneity in the association of severe mental illness with cardiovascular disease risk fac- tors across strata. The role of putative mediators, such as health-related behaviours, e.g., smoking, antipsychotic medication prescriptions and weight or body mass index, will be assessed using formal procedures to test for medi- ation [49]. Table 1 Smallest effect sizes (odds ratios) detectable for the largest and smallest ethnic minority groups at 80 % power (with two sided 5 % signiﬁcance levels), for exposures with a prevalence of 10, 20 and 50 % in the reference (white British) group Analyses ‘Poor quality’ of care will be deemed present when therapeutic interventions fall outside of nationally recog- nised guidelines, in the UK. This might include a failure to adequately screen and manage physical health comorbidi- ties (as detected in primary care data sources), or in the prescribing of antipsychotic medications in excess of rec- ommended dose. Multivariable logistic regression will assess the association of ethnicity (with ‘white British’ as the reference) with each of the care indicators, adjusting or stratifying by age and gender, as appropriate. 1. To determine ethnic variations in cause-speciﬁc mor- tality patterns among people with severe mental illnesses. 1. To determine ethnic variations in cause-speciﬁc mor- tality patterns among people with severe mental illnesses. Data from secondary care will be used. ‘Cohorts’ of indi- viduals by severe mental illness diagnoses (e.g., F20 Schizophreniform disorders F30/F31 Bipolar Affective Disorder and Manic episodes) will be followed from 2007 until the latest date at which linked census information on mortality and cause of death are available. Mortality rates by severe mental illness diagnosis will be obtained, indi- rectly age- and gender-standardised to the population of England and Wales. Indirectly standardised SMRs by eth- nic group will also be obtained. 4. Validation of the measure for individual-level socioe- conomic position, derived using structured ﬁeld infor- mation and natural language processing. The construct and concurrent validity of the GATE- derived socioeconomic position indicators of education and occupational social class will be assessed using structural equation modelling against a nested cohort of 558 individ- uals with psychosis [50]. These individuals presented to South London and Maudsley Trust services with a ﬁrst epi- sode of psychosis between 1st May 2010 and 30th April 2012. Individuals within this cohort were aged 18–64. Information on education and occupation were extracted from patient records by research workers, using the Medical Research Council (MRC) socio-demographic schedule [51]. 2. To determine ethnic variations in the effect of SMI on cardiovascular risk factors. 2. To determine ethnic variations in the effect of SMI on cardiovascular risk factors. Topic guide for qualitative study Topic guides will be semi-structured. Domains to be cov- ered in the topic guide will include: experiences of accessing/using mental and physical healthcare, the use of alternative therapies and medical models, experiences of physical symptoms, use of biomedical treatments and dis- ease monitoring, perceptions of stigma/discrimination from health service providers and individuals. Other domains will include perceived barriers to care, including language, recent migration and knowledge of local services. The topic guide will be further developed following feedback from service user representatives. Individual qualitative interviews will then be conducted to further explore the personal experience and relevance of themes identiﬁed from the focus groups. It is envisaged that up to twenty individual interviews will be conducted for this purpose. Purposive sampling will be used to identify participants for both the focus groups and the individual interviews. The criteria for this will be determined after examination of the quantitative data (which may for example give an indication of which ethnic minority groups with severe mental illnesses experience physical health inequalities). This process is described in more detail next. Analysis of qualitative data and integration with quantitative ﬁndings Thematic content analysis will be used to identify salient themes, until no further themes emerge. Analyses will be iterative as emergent themes will be used to generate hypotheses which may be tested in the quantitative dataset. Coding of qualitative data will be through appropriate software (N-Vivo) [53]. Emergent themes and coding frameworks will be cross-checked with researchers with expertise in qualitative research. Find- ings from the qualitative phase will be triangulated with those from the quantitative phase. A triangulation pro- tocol will be used to identify ‘meta-themes’ relevant to ﬁndings from both quantitative and qualitative phases of the study [54] and in particular, areas of convergence or divergence. Methodology To address the objectives for the qualitative work, focus groups and interviews will be conducted with patients and 3. To determine ethnic variations in quality of care received, relevant to premature mortality. 3. To determine ethnic variations in quality of care received, relevant to premature mortality. 12 3 3 Soc Psychiatry Psychiatr Epidemiol clinicians. Focus groups stimulate discussion and involve group processes that can help people to explore and clarify views and provide insight into cultural values and norms. Up to eight focus groups, each comprising 6–8 participants will be facilitated by two interviewers, with one group comprising clinicians and at least one group containing individuals who identify their ethnicity as ‘white Bri- tish’. In practice, the actual number of focus groups to be conducted will be determined by the range of relevant characteristics that emerge from the quantitative work and how these might best be reﬂected and balanced within the focus groups [52]. The purpose of the groups will be to understand the perspectives of service users and their carers, in particular experiences of living with cardiovascular disease co-morbid with severe mental illnesses and to identify barriers as well as facilitators to accessing healthcare. ﬁndings from the quantitative analyses, which will high- light where inequalities with cardiovascular health in people living with severe mental illness are most pro- nounced. Stratiﬁcation [52] will be used to ensure a diversity of people representative of the population are included in the study, dependent on the types of question generated by quantitative ﬁndings and may for example include people living with severe mental illnesses who have also been diagnosed with type 2 diabetes mellitus. Sampling frame for qualitative research For people living with severe mental illness, the ‘Consent for Contact’ programme [47], a register of South London and Maudsley Trust service users who are willing to be contacted about research projects on the basis of informa- tion in their record, will be used to purposively sample participants who have a severe mental illness co-morbid with a known physical health condition. To date, of a total of 9564 service users approached to take part in the ‘Consent for Contact’ programme, 72 % have consented to being contacted for mental health research. There are approximately 1340 individuals with severe mental ill- nesses, who will form the main pool of participants to be approached to take part in focus groups and interviews for the qualitative part of the study. As this register is linked to the electronic patient record data source detailed above, purposive sampling will be based on characteristics con- sidered important for the composition of the groups (e.g., ethnicity and/or presence of a physical health problem). The composition of focus groups will be informed by Ethical standards All data will be anonymised and managed according to UK National Health Service (NHS) information governance (IG) requirements. Ethical approval to examine data from the South London and Maudsley Trust Biomedical Research Centre Case Registry (SLaM BRC case registry) as an anonymised dataset for secondary analysis has been obtained from 12 123 123 Soc Psychiatry Psychiatr Epidemiol Table 2 Characteristics of primary care database, sample restricted to 18? Lambeth Tower Hamlets Newham City and Hackney Participants N (%) 295,516 (30) 214,600 (21.6) 282,512 (28.4) 200,488 (20.2) Practices N (%) 47 (25) 37 (20) 64 (34) 41 (22) Proportion resident in most deprived areasa N (%) 254,593 (86.2) 169,036 (90.0) 263,681 (98.8) 176,463 (97.7) Proportion ethnic minorities N (%) 152,307 (62.1) 133,727 (68.2) 220,549 (84.0) 118,637 (66.9) Prevalence severe mental illness N 4718 3477 3706 3484 % (95 % CI) 1.60 (1.55, 1.64) 1.62 (1.57, 1.67) 1.31 (1.27, 1.35) 1.74 (1.68, 1.80) Prevalence type 2 diabetes N 13,372 13,479 20,309 10,754 % (95 % CI) 4.54 (4.47, 4.62) 6.30 (6.20, 6.40) 7.20 (7.11, 7.30) 5.38 (5.28, 5.48) Prevalence hypertension N 32,454 22,238 38,632 24,993 % (95 % CI) 10.98 (10.87, 11.10) 10.36 (10.23, 10.49) 13.67 (13.55, 13.80) 12.47 (12.32, 12.61) Prevalence ischaemic heart disease N 5103 4792 6759 4226 % (95 % CI) 1.73 (1.68, 1.77) 2.23 (2.17, 2.30) 2.39 (2.34, 2.45) 2.11 (2.05, 2.17) Current or ex-smoker N 132,741 48,266 47,749 46,412 % (95 % CI) 46.62 (46.44, 46.80) 50.40 (50.08, 50.72) 40.31 (40.02, 40.59) 50.33 (50.00, 50.65) a Bottom two quintiles for index of multiple deprivation 2000 at lower super output level; prevalence estimates based on number of patients on quality and outcomes framework (QoF) registers for each disease, crude estimates Table 2 Characteristics of primary care database, sample restricted to 18? a Bottom two quintiles for index of multiple deprivation 2000 at lower super output level; prevalence estimates based on number of patients on quality and outcomes framework (QoF) registers for each disease, crude estimates ﬁnalised, following completion of initial analyses of quantitative data. Oxfordshire REC C in 2008 and renewed in 2013 (refer- ence number 08/H0606/71?5). Methods to de-identify data have been published and are robust [55]. Separate approvals to conduct the analyses proposed within this protocol have been granted by the CRIS Oversight Committee. Oxfordshire REC C in 2008 and renewed in 2013 (refer- ence number 08/H0606/71?5). Ethical standards Methods to de-identify data have been published and are robust [55]. Separate approvals to conduct the analyses proposed within this protocol have been granted by the CRIS Oversight Committee. Primary care The South London Primary Care Research Governance Team reviewed the process of anonymised data analysis for patient data from Lambeth and approved the usage of aggregated anonymised patient data for research purposes. Lambeth Clinical Commissioning Group, Information Governance Steering Group (Lambeth CCG IGCG) has to approve each research project based on individual appli- cations using a standardised proforma, the ‘Privacy Impact Statement’. The primary care database comprises approximately 1.06 million patient records, including 358,614 anonymised electronic patient records registered to 47 (of 48) general practices in Lambeth and 697,600 anonymised records of patients registered to 142 (of 144) general practices in East London (Tower Hamlets, Newham, City and Hackney) (in total 98.3 % of practices). Data on age and gender are near complete, since these are recorded routinely at patient registration. Self-ascribed ethnicity according to Ofﬁce for National Statistics (ONS) census categories is available for 80–90 % of patients, following local schemes to improve the recording of ethnicity [28–30, 56]. For data from east London (Newham, Tower Hamlet, City and Hackney), each of the practices opted into the study by signing forms permitting the Clinical Effective- ness Group (CEG) to use anonymised aggregated data for audits and research projects supported by the CEG. Infor- mation Technology (IT) information governance commit- tees provided approval for each of the three localities covered by the CEG. People registered to general practitioners/family doctors within the primary care database are more likely to reside in areas which are deprived and a high proportion of res- idents in each of the boroughs report their ethnicity as being of minority status (Table 2). Prevalence of severe mental illnesses ranges from 1.3–1.7 %, by borough Qualitative study Separate ethical approval will be sought for the qualitative part of the study once the topic guide and likely composition of focus groups have been 12 3 Soc Psychiatry Psychiatr Epidemiol Fig. 1 Prevalence of severe mental illnesses across the study sites, by borough Fig. 1 Prevalence of severe mental illnesses across the study sites, by borough diagnoses for severe mental illnesses by ethnicity, for this database. (Table 2). Figure 1 highlights the geographical distribution of severe mental illness across the study sites. Primary care Compared to people registered to general practitioners and not known to have a severe mental illness, the severe mental illness sample in primary care were older, with a higher proportion of men, a larger proportion of people reporting their ethnicity as white British or black Caribbean and were more likely to reside in deprived areas (Table 3). Discussion People living with severe mental illnesses experience a dramatic reduction in life expectancy [1], a large pro- portion is accounted for through cardiovascular disease [3]. Although complex [57], a parallel body of work has highlighted the particular problem of cardiovascular disease for many ethnic minority groups [21, 58, 59]. It is, therefore, surprising that there is a dearth of evidence, relating to cardiovascular disease in ethnic minority groups living with severe mental illnesses [14]. This is a concern, as this represents preventable causes of death. The present study, E-CHASM, will address this gap in knowledge. Secondary care The secondary care database currently comprises approxi- mately 260,000 anonymised patient records [37], and has increased consistently by around 20,000 per year. Age and gender are complete in this database. Ethnicity is self- ascribed according to standardised criteria, consistent with the last census. Information on self-ascribed ethnicity was 93–97 % complete in 2007–2013. Table 4 displays ICD-10 12 123 123 Soc Psychiatry Psychiatr Epidemiol Table 3 Characteristics of people living with severe mental illnesses in primary care database, sample restricted to adults aged 18? Not on SMI registera N = 997,731 Severe mental illness (SMI register)a N = 15,385 Total N % N % N Age (mean, SD) 40 (15.5) 47 (15.3) 993,116 Sex Men 494,304 51 8488 55 502,792 Women 483,426 49 6897 45 490,323 Ethnicity White British 238,211 27 4403 31 242,614 Irish 13,459 2 286 2 13,745 ‘Other’ white 171,493 20 1442 10 172,935 Indian 60,298 7 566 4 60,864 Pakistani 35,215 4 381 3 35,596 Bangladeshi 93,143 11 1500 10 94,643 Black Caribbean 43,367 5 1646 11 45,013 Black African 74,037 9 1417 10 75,454 ‘Other’/Chinese 110,094 13 2027 14 112,121 Mixed ethnicity 27,877 3 717 5 28,594 Area-level deprivation (quintiles) 1 Most deprived 587,820 64 10,437 71 589,257 2 261,966 29 3550 24 265,516 3 51,207 6 521 4 51,728 4 10,396 1.1 96 0.7 10,492 5 Least deprived 4626 0.5 21 0.1 4647 p \ 0.001 for all sociodemographic variables; comparing SMI to non-SMI group p \ 0.001 for all sociodemographic variables; comparing SMI to non-SMI group p \ 0.001 for all sociodemographic variables; comparing SMI to non-SMI group Table 4 Breakdown of severe mental illness diagnosis by ethnicity in secondary care database, 2007–2013 Schizophrenia Schizoaffective disorder Bipolar affective disorder N = 6885 N = 1438 N = 4112 Ethnicity, n (%) White British 2271 (33) 519 (36) 2033 (49) ‘Other’ white 473 (7) 93 (6) 376 (9) Irish 141 (2) 37(3) 132 (3) Indian 131 (2) 21 (1) 64 (2) Pakistani 55 (0.1) 10 (0.7) 24 (0.6) Bangladeshi 29 (0.4) 8 (0.6) 18 (0.4) Black Caribbean 1004 (15) 173 (12) 255 (6) Black African 926 (13) 194 (13) 273 (7) ‘Other’/Chinese 1472 (21) 284 (20) 606 (15) Mixed ethnicity 97 (1) 22 (1.5) 48 (1) Not stated/missing 286 (4) 71 (4.9) 257 (6) illness are, however, broadly consistent with those published by Public Health England [59]. Table 4 Breakdown of severe mental illness diagnosis by ethnicity in secondary care database, 2007–2013 Table 3 Characteristics of people living with severe mental illnesses in primary care database, sample restricted to adults aged 18? Dissemination Analyses will be disseminated in peer-reviewed manu- scripts and through conference proceedings. If requested, analyses will also be prepared as reports or presentations for interested stakeholders. Findings relating directly to clinical care will be fed back to clinical care networks, with a view to informing guideline development. Secondary care Integration of ﬁndings across qualitative and quantitative data sources [54] will help to understand mechanisms underlying quantitative ﬁndings as well as identify barriers to care from the perspectives of service users, carer and service providers. A future application of this data source could be to assess discrepancies in care provided across primary and secondary care. This would be based on data linkages between primary and secondary care, which could be explored in future work. Limitations relate to analysing routine electronic patient records, where missing data may be associated with bias and loss of precision [62]. There may also be concerns around the quality of the data entered on databases and variables to adjust for known confounders may not be available [63]. It may be possible to apply specialist techniques to address this [62–64]. For the cross-sectional phases of the study, it will not be possible to conclude temporality of associations. Conclusions There is currently an absence of evidence relating to life expectancy differences in ethnic minority people living with severe mental illnesses such as schizophrenia. In particular, little is known about the experience of cardio- vascular disease in these populations and whether there are additional barriers or inequities in service provision. E-CHASM will seek to address these gaps in knowledge through a combination of quantitative analysis of electronic health records and qualitative interviews. Acknowledgments We are grateful to Ms Sherifat Oduola for providing advice on the ‘consent-for-contact’ programme as well as information relating to the cohort of people with ﬁrst episode psy- chosis. We are grateful to Professor Michael Dewey for advice. We are also grateful to Dr Chin-Kuo Chang and Mr Matthew Broadbent and Mr Hitesh Shetty for advice on usage of BRC-CRIS data. Strength and limitations Strengths of this study include the power to conduct sta- tistical analyses in groups of individuals who form a minority in the population and thus address the current scarcity of research in this ﬁeld. Other strengths include the usage of natural language processing to derive a measure for individual-level socioeconomic position. If successful, this will provide a methodological advantage, as almost all previous work using electronic health records has tended to Secondary care Cardiovascular disease indicators presented here are broadly consistent with prevalence estimates published for London [60]. The results presented suggest that the prevalence of severe mental illness in the study catchment area is greater than previously published estimates for prevalence of psychotic disorders in Britain, which have been noted to range from 0.4 to 0.8 %, with considerable variability between geographi- cal regions [60]. Local areal estimates for severe mental The feasibility of E-CHASM rests on its usage of rou- tine electronic patient records to establish differences in 12 Soc Psychiatry Psychiatr Epidemiol prevalence and treatment access. In particular, analyses of records from patients registered to general practices in an ethnically and socioeconomically diverse region in a major inner-city conurbation, alongside analysis of records from a large mental health Trust serving these populations, will enhance the study. The enriched representation of popu- lations normally under-represented or absent in similar work [14] will allow the assessment of ethnic minority physical health inequalities in severe mental illness popu- lations. Methodological techniques such as natural lan- guage processing to data-mine free text within secondary care records [41, 45] will enable development of measures for socioeconomic position through robust and repeat- able methods which will also enable the automation of checking of a large volume of records which would otherwise be impossible [41]. As far as we are aware, the derivation of an individual-level measure for socioeco- nomic position from routine electronic health records has not been previously attempted. Finally a novel application of using electronic patient records in research is in the application of the ‘consent for contact’ programme at South London and Maudsley Trust. Thus, following anal- ysis of quantitative data, it will be possible to purposively sample potential participants who will be invited to take part in focus groups and individual interviews for the qualitative phase of this study, based on important attri- butes (e.g., type of diagnosis, ethnicity, presence of phys- ical comorbidity). Thus, the ﬁndings from the quantitative phase will directly inform qualitative data collection. This form of integration, known as ‘connecting’, will draw from the strengths of deductive methods in the quantitative phase to inform study design for the qualitative phase [22]. Integration of ﬁndings across qualitative and quantitative data sources [54] will help to understand mechanisms underlying quantitative ﬁndings as well as identify barriers to care from the perspectives of service users, carer and service providers. Secondary care A future application of this data source could be to assess discrepancies in care provided across primary and secondary care. This would be based on data linkages between primary and secondary care, which could be explored in future work. rely on area-level measures for deprivation. The mixed methods design of the study will enhance possibilities of understanding trends in quantitative analyses as well as highlighting barriers to equitable care from the perspec- tives’ of service users and service providers. In all of the quantitative data sources, ethnicity is self-ascribed. This is an addition over previous work which has tended to rely on country of birth [61]. prevalence and treatment access. In particular, analyses of records from patients registered to general practices in an ethnically and socioeconomically diverse region in a major inner-city conurbation, alongside analysis of records from a large mental health Trust serving these populations, will enhance the study. The enriched representation of popu- lations normally under-represented or absent in similar work [14] will allow the assessment of ethnic minority physical health inequalities in severe mental illness popu- lations. Methodological techniques such as natural lan- guage processing to data-mine free text within secondary care records [41, 45] will enable development of measures for socioeconomic position through robust and repeat- able methods which will also enable the automation of checking of a large volume of records which would otherwise be impossible [41]. As far as we are aware, the derivation of an individual-level measure for socioeco- nomic position from routine electronic health records has not been previously attempted. Finally a novel application of using electronic patient records in research is in the application of the ‘consent for contact’ programme at South London and Maudsley Trust. Thus, following anal- ysis of quantitative data, it will be possible to purposively sample potential participants who will be invited to take part in focus groups and individual interviews for the qualitative phase of this study, based on important attri- butes (e.g., type of diagnosis, ethnicity, presence of phys- ical comorbidity). Thus, the ﬁndings from the quantitative phase will directly inform qualitative data collection. This form of integration, known as ‘connecting’, will draw from the strengths of deductive methods in the quantitative phase to inform study design for the qualitative phase [22]. Compliance with ethical standards Conﬂicts of interest FG has received honoraria for advisory work and lectures from Roche, Lundbeck, and Sunovion. FG is part of a research team indirectly supported by Janssen and has a family member with professional links to Lilly and GSK. No other conﬂicts of interest declared. 123 Soc Psychiatry Psychiatr Epidemiol Declaration of funding JD is funded by the Health Foundation working with the Academy of Medical Sciences. CM is supported by a European Research Council Consolidator Award (Ref: ERC-CoG- 2014-Proposal 648837, REACH). RS is funded by the NIHR Spe- cialist Biomedical Research Centre for Mental Health at the South London and Maudsley NHS Foundation Trust and Institute of Psy- chiatry, King’s College London. GT and FG are supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care South London at King’s College London Foundation Trust. GT acknowledges ﬁnancial support from the Department of Health via the National Institute for Health Research (NIHR) Biomedical Research Centre and Dementia Unit awarded to South London and Maudsley NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. GT is supported by the European Union Seventh Framework Programme (FP7/2007–2013) Emerald project. AR is funded by the European Union Horizon 2020 pro- gramme OpenMinTeD and KConnect projects, by the National Institute for Health Research (NIHR) Biomedical Research Centre and Dementia Biomedical Research Unit at South London and Maudsley NHS Foundation Trust and King’s College London, and by QBurst. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health or any of the funding bodies. 9. 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https://openalex.org/W3042254226	https://pubs.rsc.org/en/content/articlepdf/2020/ra/d0ra01233e	English	null	A mitochondrion-targeted dual-site fluorescent probe for the discriminative detection of SO<sub>3</sub><sup>2−</sup> and HSO<sub>3</sub><sup>−</sup> in living HepG-2 cells	RSC advances	2,020	cc-by	6,236	College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, South Puzhu Road, Nanjing 211816, China. E-mail: wengeyang11@163. com; Fax: +86-25-58139393; Tel: +86-25-58139393 † Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra01233e PAPER Open Access Article. Published on 14 July 2020. Downloaded on 10/24/2024 5:07:30 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 14 July 2020. Downloaded on 10/24/2024 5:0 This article is licensed under a Creative Commons Attribution 3.0 U Sulfur dioxide, known as an environmental pollutant, produced during industrial productions is also a common food additive that is permitted worldwide. In living organisms, sulfur dioxide forms hydrates of sulﬁte (SO2$H2O), bisulﬁte (HSO3 ) and sulﬁte (SO3 2) under physiological pH conditions; these three exist in a dynamic balance and play a role in maintaining redox balance, further participating in a wide range of physiological and pathological processes. On the basis of the diﬀerences in nucleophilicity between SO3 2 and HSO3 , for the ﬁrst time, we built a mitochondrion-targeted dual-site ﬂuorescent probe (Mito-CDTH-CHO) based on benzopyran for the highly speciﬁc detection of SO3 2 and HSO3 with two diverse emission channels. Mito-CDTH-CHO can discriminatively respond to the levels of HSO3 and SO3 2. Besides, its advantages of low cytotoxicity, superior biocompatibility and excellent mitochondrial enrichment ability contribute to the detection and observation of the distribution of sulfur dioxide derivatives in living organisms as well as allowing further studies on the physiological functions of sulfur dioxide. Received 10th February 2020 Accepted 28th June 2020 Received 10th February 2020 Accepted 28th June 2020 Received 10th February 2020 Accepted 28th June 2020 DOI: 10.1039/d0ra01233e rsc.li/rsc-advances DOI: 10.1039/d0ra01233e rsc.li/rsc-advances healthy donors. HSO3 /SO3 2 also relax aortic rings in a dose- dependent manner at high concentrations ranging from 0.5 to 12 mM.10 However, whether HSO3 and SO3 2 are independent or synergistic in action remains largely unknown. Therefore, the accurate and independent determination of the levels of SO3 2 and HSO3 is fairly necessary and valuable for further investi- gating the physiological functions of sulfur dioxide in living organisms. 1. Introduction 1. Sulfur dioxide (SO2), the most common and simple irritating gas, is one of the main pollutants in the atmosphere.1 In recent years, increasing physiological functions of sulfur dioxide have been discovered in mammals.2–4 How does SO2 work in the internal environment of living organisms? It has been reported in numerous studies that SO2 is not independent in action or directly aﬀects, but dissociated to SO3 2 and HSO3 (SO2 derivatives) in neutral uid or plasma (HSO3 /SO3 2, 1 : 3 M/ M),5,6 which mainly account for its toxicity. There is a dynamic conversion equilibrium between sulfur dioxide and sulte, which also exists in bisulte and sulte.7 Over the past decades, uorescence imaging technology has drawn considerable attention beneting from its outstanding performances, such as eminent non-invasiveness, excellent signal-to-noise ratio, high sensitivity, extraordinary reliability, cheap availability and easy operation.14 Since Qian and Zhang et al. rst reported a uorescent probe based on the Michael addition reaction for HSO3 in 2013,15 a large number of uo- rescent probes have been developed to detect HSO3 /SO3 2 in recent years.16–20 Although they work on a simple mechanism and are facile to synthesize, they cannot exactly distinguish between HSO3 and SO3 2, thus producing the same uores- cence signal. Herein, a dual-site uorescent probe for SO2 derivatives (HSO3 and SO3 2) based on benzopyran, Mito- CDTH-CHO, was designed and synthesized. The probe is capable of discriminatively responding to the levels of HSO3 and SO3 2 with distinct uorescence signals. Furthermore, Mito-CDTH-CHO exhibited superior selectivity, lower cytotox- icity, good sensitivity, and readily available for uorescence imaging in vitro and in vivo. To the best of our knowledge, the probes that possess obvious uorescence sensing for HSO3 and SO3 2 are still rare. RSC Advances Cite this: RSC Adv., 2020, 10, 26349 Received 10th February 2020 Accepted 28th June 2020 DOI: 10.1039/d0ra01233e rsc.li/rsc-advances PAPER ed on 14 July 2020. Downloaded on 10/24/2024 5:07:30 AM. ensed under a Creative Commons Attribution 3.0 Unported Licence. A mitochondrion-targeted dual-site ﬂuorescent probe for the discriminative detection of SO3 2 and HSO3 in living HepG-2 cells† Zhenmei Deng, Fangzhao Li, Guomin Zhao, Wenge Yang * and Yonghong Hu * 2.2. Synthesis of probe Mito-CDTH-CHO The pH values of the solutions were directly obtained by preparing a series of specic pH buﬀers, including acetate buﬀer, phosphate buﬀer, and sodium hydroxide/potassium chloride/boric acid buﬀer. The probe was synthesized according to the reported literature via a facile two-step reaction.21,22 The synthesis routes of the probe are depicted in Scheme S1,† and it was characterized via high- resolution mass spectrometry, 1H NMR and 13C NMR (see ESI†). Synthesis of Mito-CDTH. Freshly distilled cyclohexanone was added dropwise to a solution of concentrated H2SO4 cooled down to 0 C in advance. To a concentrated H2SO4 solution of 2-(4- diethylamino-2-hydroxybenzoyl), benzoic acid was added drop- wise in freshly distilled cyclohexanone at 0 C. Further, heating up to 90 C, the mixture was vigorously stirred for 2 h, poured into ice, the perchloric acid (70%) was added, the supernatant was ltered oﬀ, and the residue was washed with cold water for three times. The residue was dried under vacuum and further puried via silica gel column chromatography (CH2Cl2 : MeOH ¼ 20 : 1, v/ v) to aﬀord Mito-CDTH as a bright red solid (372 mg, 68%). ESI- MS calcd for C24H26NO3 [M + H]+ 376.1921, found 376.1913. 2.6. Cell cytotoxicity assay The cytotoxicity was measured using a CCK-8 kit. Hela cells were cultured in Dulbecco's modied Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics at 37 C under 5% CO2 for 24 h. Hela cells were cultured with a fresh medium containing various concentrations of Mito- CDTH-CHO (0–40 mM) for another 12 h. Next, Hela cells were washed three times with PBS and incubated with diluted CCK-8 reagent for 1 h, and then the cell viability was determined by a microplate reader. The procedure was repeated three times for each concentration. Synthesis of Mito-CDTH-CHO. To an acetic acid solution (30 ml) of Mito-CDTH (376 mg, 1 mmol), terephthalaldehyde (268 mg, 2 mmol) was added. The reaction solution was stirred at 110 C for 3 h, and the solvent was evaporated under a reduced pressure. The crude product was extracted with CH2Cl2 (100 ml) and water (300 ml), washed with a saturated ammonium chloride solution and dried over anhydrous sodium sulfate. Aer evaporating using a rotary evaporator, the purple target compound (300 mg, 61%) was obtained via silica gel column chromatography (CH2Cl2 : MeOH ¼ 50 : 1, v/v). 2.5. Spectral analysis 1 mM of the probe stock solution was prepared by dissolving 1 mg Mito-CDTH-CHO in 2 ml anhydrous ethanol, and diluted with PBS solution (10 mM, pH ¼ 7.4, 6.0 or 8.0, containing 2% EtOH) for nal test solutions. 2.7. Cell culture and imaging HepG-2 cells were cultured in a DMEM medium (containing 1% penicillin/streptomycin and 10% FBS) under an air condition at 37 C under 5% CO2. HepG-2 cells at the logarithmic growth phase were implanted into 25 mm glass-bottomed dishes and incubated overnight. Aer the attachment of cells, the cells were treated with diﬀerent pH values (pH ¼ 6.0, 7.4 and 8.0) of the DMEM medium for 3 h. The pH of the DMEM medium was adjusted by adding a specic concentration of hydrochloric acid or sodium hydroxide.23 HepG-2 cells were washed with PBS three times and incubated with Mito-CDTH-CHO (20 mM) in an untreated DMEM medium. Confocal uorescence images were recorded using a Zeiss LSM 800 confocal laser scanning microscope. The green channel was collected at 460–520 nm at an excitation of 390, and the blue uorescence channel was covered over the range of 420–470 nm at an excitation of 370 nm. 1H NMR (300 MHz, DMSO-d6) d 10.03 (s, 1H), 7.95 (d, J ¼ 7.8 Hz, 3H), 7.86–7.63 (m, 4H), 7.47 (s, 1H), 7.34 (d, J ¼ 7.6 Hz, 1H), 6.56 (d, J ¼ 2.3 Hz, 1H), 6.52–6.35 (m, 2H), 3.36 (q, J ¼ 7.0 Hz, 4H), 2.80 (d, J ¼ 15.6 Hz, 1H), 2.67 (s, 1H), 1.91 (d, J ¼ 12.9 Hz, 1H), 1.62 (d, J ¼ 9.5 Hz, 3H), 1.11 (t, J ¼ 6.9 Hz, 6H). 13C NMR (75 MHz, DMSO-d6) d 191.99, 149.12, 142.35, 134.78, 134.40, 132.23, 129.65, 129.53, 129.04, 127.80, 124.44, 123.99, 123.39, 109.19, 96.69, 43.43, 26.41, 22.36, 21.54, 12.05. ESI-MS calcd for C32H30NO4 [M + H]+ 492.2133, found 492.2160. 1. Introduction Among them, HSO3 /SO3 2 at high concentrations are catalyzed to generate a variety of sulfur oxy radicals, which are known to give rise to negligible damage to the body.8 Numerous studies have conrmed that abnormally high sulte levels are closely related to respiratory diseases,9 cardiovascular diseases10 as well as neurological diseases, such as migraine, stroke, brain cancer,11 lung cancer12 and liver cancer.13 Besides, clinical studies suggest that the concentration of sulfur dioxide gas ranges from 1 to 2000 mM in living organisms, and the total concentration of serum sulte ranges from 0 to 10 mM in RSC Adv., 2020, 10, 26349–26357 \| 26349 This journal is © The Royal Society of Chemistry 2020 View Article Online View Article Online Paper View Article Online RSC Advances RSC Advances Paper dilution of a commercial hypochlorite solution in puried water and measured using a spectrophotometer (3292 nm ¼ 350 M1 cm1). TBHP was prepared by the dilution of a commercial tert-butyl hydroperoxide stock solution. Perox- ynitrite stock (ONOO) was prepared by a previously reported procedure23 and measured using a spectrophotometer (3302 nm ¼ 1670 M1 cm1) 1 M of stock solutions (Cys, GSH, HS, SO4 2, S2O3 2, S2, SCN, H2O2, ClO, TBHP, Hcy, NO2 , Sx2, Cl, Br, I, CO3 2, HCO3 , PO4 2, HPO4 , AcO, SO3 2, and HSO3 ) were prepared by the dissolution of 10 mmol solid in puried water, and diluted to the desired concentrations when needed. 2.1. Materials and instruments All the reagents were supplied by commercial suppliers and were directly used without further purication. Absorption spectra were recorded on a UNICO UV-4802 spectrophotometer. Fluorescence spectra were obtained on a uorescence spectro- photometer (Lengguang tech CO., Ltd. F97XP, China). 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE III 400 Nanobay at 500 MHz for 1H NMR and 300 MHz for 13C NMR (TMS as an internal standard). High-resolution mass spectra (HRMS) were recorded on a MicrOTOF Bruker. The pH values were measured with an acidity meter (alkalis, pH 400, China). 2.3. Preparation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), active sulfur species (RSS) and anions 2.3. Preparation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), active sulfur species (RSS) and anions H2O2 was prepared by the direct dilution of a commercial hydrogen peroxide stock solution. NaClO was obtained by the This journal is © The Royal Society of Chemistry 2020 26350 \| RSC Adv., 2020, 10, 26349–26357 RSC Advances View Article Online Paper Scheme 1 Rational design and sensing mechanism of the probe for HSO3 and SO3 2. Scheme 1 Rational design and sensing mechanism of the probe for HSO3 and SO3 2. 3. Results and discussions proposed that (Scheme 1), on the one hand, the oxygen positive ion on the benzopyran ring acts as a strong electron- withdrawing group, which reduces the electron cloud density of the C]C double bond and enable the C]C double bond strong electrophilicity. On the other hand, the C]O double bond conjugated to the benzene ring also possesses weaker This journal is © The Royal Society of Chemistry 2020 3.1. Design and synthesis of Mito-CDTH-CHO According to the previous reports on the response mechanism of detecting sulfur dioxide type uorescent probes,24–30 we Fig. 1 Fluorescence and UV responses of Mito-CDTH-CHO toward SO2 derivatives. (a) The UV-Vis absorption spectra change of Mito-CDTH- CHO (20 mM) with Na2SO3 (20 mM) in PBS buﬀer (pH 7.4, containing 2% EtOH). (b) UV-Vis absorption spectra change of Mito-CDTH-CHO (20 mM) with NaHSO3 (20 mM) in PBS buﬀer (pH 6.0, containing 2% EtOH). (c) Fluorescence spectra changes of Mito-CDTH-CHO (20 mM) with 50 mM SO2 derivatives in PBS buﬀer (containing 2% EtOH). Black: Mito-CDTH-CHO; blue: NaHSO3, lex ¼ 370 nm; green: Na2SO3, lex ¼ 390 nm. (d) Fluorescence spectra changes of Mito-CDTH-CHO (20 mM) in the presence of various concentrations Na2SO3 (0–600 mM), lex ¼ 390 nm. Open Access Article. Published o This article is licens Fig. 1 Fluorescence and UV responses of Mito-CDTH-CHO toward SO2 derivatives. (a) The UV-Vis absorption spectra change of Mito-CDTH- CHO (20 mM) with Na2SO3 (20 mM) in PBS buﬀer (pH 7.4, containing 2% EtOH). (b) UV-Vis absorption spectra change of Mito-CDTH-CHO (20 mM) with NaHSO3 (20 mM) in PBS buﬀer (pH 6.0, containing 2% EtOH). (c) Fluorescence spectra changes of Mito-CDTH-CHO (20 mM) with 50 mM SO2 derivatives in PBS buﬀer (containing 2% EtOH). Black: Mito-CDTH-CHO; blue: NaHSO3, lex ¼ 370 nm; green: Na2SO3, lex ¼ 390 nm. (d) Fluorescence spectra changes of Mito-CDTH-CHO (20 mM) in the presence of various concentrations Na2SO3 (0–600 mM), lex ¼ 390 nm. This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 26349–26357 \| 26351 View Article Online View Article Online RSC Advances Fig. 2 Fluorescence responses of Mito-CDTH-CHO toward SO2 derivatives. (a) The linear curve of Mito-CDTH-CHO (20 mM) ﬂuorescence intensity at 492 nm with Na2SO3 concentrations range from 10–100 mM. (b) Fluorescence spectra changes of Mito-CDTH-CHO (20 mM) after the addition of various concentrations NaHSO3 (0–1000 mM). PBS buﬀer, pH 6.0, containing 2% EtOH, lex ¼ 370 nm. (c) The linear curve of Mito- CDTH-CHO (20 mM) ﬂuorescence intensity at 456 nm with NaHSO3 concentrations range from 40–200 mM in PBS buﬀer. (d) The ﬂuorescence intensity values of Mito-CDTH-CHO (20 mM) after interacting with 500 mM SO2 derivatives, reactive nitrogen species, reactive oxygen species, active sulfur species and anions. 3.1. Design and synthesis of Mito-CDTH-CHO (1) Mito-CDTH-CHO, (2) Cys, (3) GSH, (4) HS, (5) SO4 2, (6) S2O3 2, (7) S2, (8) SCN, (9) H2O2, (10) NaClO, (11) TBHP, (12) Hcy, (13) NO2 , (14) Sx2, (15) Cl, (16) Br, (17) I, (18) CO3 2, (19) HCO3 , (20) PO4 2, (21) HPO4 , (22) AcO, (23) SO3 2, (24) HSO3 . RSC Advances Paper Open Access Article. Published on 14 July 2020. Downloaded on 10/24/2024 5:07:30 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. on 14 July 2020. Downloaded on 10/24/2024 5:07:30 AM. sed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 14 July 2020. Downloaded on 10/24/2024 5:07:30 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 The 1H NMR titration of Mito-CDTH-CHO with Na2SO3. Fig. 2 Fluorescence responses of Mito-CDTH-CHO toward SO2 derivatives. (a) The linear curve of Mito-CDTH-CHO (20 mM) ﬂuorescence intensity at 492 nm with Na2SO3 concentrations range from 10–100 mM. (b) Fluorescence spectra changes of Mito-CDTH-CHO (20 mM) after the addition of various concentrations NaHSO3 (0–1000 mM). PBS buﬀer, pH 6.0, containing 2% EtOH, lex ¼ 370 nm. (c) The linear curve of Mito- CDTH-CHO (20 mM) ﬂuorescence intensity at 456 nm with NaHSO3 concentrations range from 40–200 mM in PBS buﬀer. (d) The ﬂuorescence intensity values of Mito-CDTH-CHO (20 mM) after interacting with 500 mM SO2 derivatives, reactive nitrogen species, reactive oxygen species, active sulfur species and anions. (1) Mito-CDTH-CHO, (2) Cys, (3) GSH, (4) HS, (5) SO4 2, (6) S2O3 2, (7) S2, (8) SCN, (9) H2O2, (10) NaClO, (11) TBHP, (12) Hcy, (13) NO2 , (14) Sx2, (15) Cl, (16) Br, (17) I, (18) CO3 2, (19) HCO3 , (20) PO4 2, (21) HPO4 , (22) AcO, (23) SO3 2, (24) HSO3 . 26352 \| RSC Adv., 2020, 10, 26349–26357 This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 14 July 2020. Downloaded on This article is licensed under a Creative Commons A Fig. 2 Fluorescence responses of Mito-CDTH-CHO toward SO2 derivatives. (a) The linear curve of Mito-CDTH-CHO (20 mM) ﬂuorescence intensity at 492 nm with Na2SO3 concentrations range from 10–100 mM. (b) Fluorescence spectra changes of Mito-CDTH-CHO (20 mM) after the addition of various concentrations NaHSO3 (0–1000 mM). PBS buﬀer, pH 6.0, containing 2% EtOH, lex ¼ 370 nm. 3.1. Design and synthesis of Mito-CDTH-CHO (c) The linear curve of Mito- CDTH-CHO (20 mM) ﬂuorescence intensity at 456 nm with NaHSO3 concentrations range from 40–200 mM in PBS buﬀer. (d) The ﬂuorescence intensity values of Mito-CDTH-CHO (20 mM) after interacting with 500 mM SO2 derivatives, reactive nitrogen species, reactive oxygen species, active sulfur species and anions. (1) Mito-CDTH-CHO, (2) Cys, (3) GSH, (4) HS, (5) SO4 2, (6) S2O3 2, (7) S2, (8) SCN, (9) H2O2, (10) NaClO, (11) TBHP, (12) Hcy, (13) NO2 , (14) Sx2, (15) Cl, (16) Br, (17) I, (18) CO3 2, (19) HCO3 , (20) PO4 2, (21) HPO4 , (22) AcO, (23) SO3 2, (24) HSO3 . Fig. 3 The 1H NMR titration of Mito-CDTH-CHO with Na2SO3. 26352 \| RSC Adv., 2020, 10, 26349–26357 This journal is © The Royal Society of Chemistry 2020 This journal is © The Royal Society of Chemistry 2020 RSC Advances View Article Online Paper Paper Fig. 4 The 1H NMR titration of Mito-CDTH-CHO with NaHSO3. ative Commons Attribution 3.0 Unported Licence. Fig. 4 The 1H NMR titration of Mito-CDTH-CHO with NaHSO3. electrophilicity, which makes the strong nucleophilic HSO3 attack the C]O and C]C double bonds. When the probe Mito- CDTH-CHO conjugate structure is broken, strong blue uores- cence is emitted. The weaker nucleophilic SO3 2 could only attack the C]C double bond, hence exhibited red-shied green uorescence. range of 10–100 mM. Moreover, the detection limit was calcu- lated to be 100 nM. For HSO3 , Fig. 2b and c revealed that the uorescence intensity at 456 nm constantly increased aer the addition of 0–1000 mM NaHSO3 in the phosphate buﬀer solu- tion (pH ¼ 6.0, 37 C, lex ¼ 370 nm), in a wide linear range (40– 200 mM). The detection limit was 80 nM (Fig. 2c). Aerward, we evaluated the selectivity and pH stability of the probe Mito-CDTH-CHO towards sulfur dioxide derivatives. As described in Fig. 2d, SO3 2/HSO3 led to a signicant uorescence enhancement at 492 nm and 456 nm, This journal is © The Royal Society of Chemistry 2020 3.2. Vitro sensing of Mito-CDTH-CHO for SO3 2/HSO3 From what had been dis- cussed above, Mito-CDTH-CHO presented excellent selectivity toward SO3 2 and HSO3 in separated emission regions (492 and 456 nm). respectively. While other interfering substances, including reactive nitrogen species (NO2 and NO3 ), reactive oxygen species (ClO, H2O2, TBHP), active sulfur species (SO4 2, HS, S2O3 2, S2, SCN, Sx2, Cys, Hcy, GSH) and anions (I, Br, Cl, CO3 2, HCO3 , PO4 3, HPO4 2, ACO), did not produce remarkable uorescence response. From what had been dis- cussed above, Mito-CDTH-CHO presented excellent selectivity toward SO3 2 and HSO3 in separated emission regions (492 and 456 nm). In general, compared to other uorescence probes sensing SO2, the most evident superiority of Mito-CDTH-CHO is selec- tivity for SO3 2 and HSO3 . Most uorescent probes for detecting SO2 are not selective toward SO3 2 and HSO3 due to their very similar chemical properties, showing that the same response toward SO3 2 (HSO3 ) when detecting HSO3 (SO3 2). In addition, superior water solubility, suitable detection limit, and accurate mitochondrial targeting performance also indi- cate that Mito-CDTH-CHO is a fairly qualied uorescent probe for the accurate detection of sulfur dioxide derivatives (see Table S1†). Open Access Article. Published on 14 July 2020. Downloaded on 10/24/2024 5:07:30 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 14 July 2020. Downloaded on 10/24/2024 5:07:30 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The pH interference of the probe for SO2 derivatives was discussed. In the absence of SO3 2 and HSO3 , the probe had little uorescence and was unaﬀected by the variation of pH values (Fig. S5†). When Na2SO3 or NaHSO3 was added, the uorescence intensity changed with the mutual conversion balance between SO3 2 and HSO3 in the range of pH 4 to 10 (Fig. S6†). In the range of acidic pH (4–6), HSO3 ion dominates, thus Mito-CDTH-CHO exhibited stronger uorescence at 456 nm than in neutral and weak basic pH ranges (Fig. S7†). In basic pH ranges (7–10), SO3 2 accounts for the main part, so the 3.2. Vitro sensing of Mito-CDTH-CHO for SO3 2/HSO3 First of all, we tested the UV-Vis absorption of Mito-CDTH-CHO in the absence and presence of SO2 derivatives (SO3 2 and HSO3 ) in PBS buﬀer. There were mainly two absorption peaks at 300–700 nm, centered at 330 nm and 553 nm, respectively. Mito-CDTH-CHO exhibited similar UV spectra changes aer it reacted with SO3 2 (Fig. 1a) and HSO3 (Fig. 1b). The absorption intensity at 553 nm sharply decreased, which means for the breaking of the conjugate system, and a new absorption at 365 nm was elevated. Fig. 5 Cell viability of Mito-CDTH-CHO in a standard CCK-8 kit in living HepG-2 cells for 24 h. The experiment was repeated three times (S.D.). To explain the uorescence response distinction, the dual- site uorescence response of probe toward SO3 2 and HSO3 was investigated. As shown in Fig. 1c, the probe Mito-CDTH- CHO (20 mM) had almost no uorescence in the absence of SO3 2/HSO3 at 400–600 nm. However, aer the reaction with 50 of mM Na2SO3, the strong green uorescence was emitted (lex ¼ 390 nm, lem ¼ 492 nm). When at the same concentration in the case of NaHSO3, the solution exhibited luminous blue emission at a shorter wavelength (lex ¼ 370 nm, lem ¼ 456 nm). These results provide a preliminary proof that the probe Mito- CDTH-CHO conjugate structure is destroyed by SO3 2 and HSO3 . As shown in Fig. 1d and 2a, for SO3 2, with the addition of the Na2SO3 (0–600 mM), the emission intensity at 492 nm increased signicantly (pH ¼ 7.4, 37 C, lex ¼ 390 nm), and an excellent linear relationship (R2 ¼ 0.992) was obtained in the Fig. 5 Cell viability of Mito-CDTH-CHO in a standard CCK-8 kit in living HepG-2 cells for 24 h. The experiment was repeated three times (S.D.). RSC Adv., 2020, 10, 26349–26357 \| 26353 This journal is © The Royal Society of Chemistry 2020 View Article Online View Article Online Paper RSC Advances uorescence intensity increased with the pH value increase (Fig. S6†). respectively. While other interfering substances, including reactive nitrogen species (NO2 and NO3 ), reactive oxygen species (ClO, H2O2, TBHP), active sulfur species (SO4 2, HS, S2O3 2, S2, SCN, Sx2, Cys, Hcy, GSH) and anions (I, Br, Cl, CO3 2, HCO3 , PO4 3, HPO4 2, ACO), did not produce remarkable uorescence response. 3.3. The proposed mechanism of Mito-CDTH-CHO for SO2 derivatives detection In order to illuminate the reaction mechanism between Mito- CDTH-CHO and SO2 derivatives, the NMR titration experiments in DMSO-d6/D2O (8 : 2) were performed. As shown in Fig. 3, a proton at 6.34 ppm represents the double bond conjugated to Fig. 6 Confocal microscopy colocalization images of Mito-CDTH-CHO and Mito-Tracker Green in HepG-2 cells. (a and e) the bright ﬁeld of HepG-2 cells. (b and f) ﬂuorescence image of Mito-Tracker Green (lex ¼ 488 nm, lem ¼ 500–540 nm). (c and g) ﬂuorescence image of Mito- CDTH-CHO (lex ¼ 370 nm, lem ¼ 400–460 nm). (d and h) merged image of (b and c), (f and g), respectively. (i) Intensity scatter plot of blue and green channels and (j) normalized intensity proﬁle of the linear region of part a across the HepG-2 cells. Fig. 6 Confocal microscopy colocalization images of Mito-CDTH-CHO and Mito-Tracker Green in HepG-2 cells. (a and e) the bright ﬁeld of HepG-2 cells. (b and f) ﬂuorescence image of Mito-Tracker Green (lex ¼ 488 nm, lem ¼ 500–540 nm). (c and g) ﬂuorescence image of Mito- CDTH-CHO (lex ¼ 370 nm, lem ¼ 400–460 nm). (d and h) merged image of (b and c), (f and g), respectively. (i) Intensity scatter plot of blue and green channels and (j) normalized intensity proﬁle of the linear region of part a across the HepG-2 cells. 26354 \| RSC Adv., 2020, 10, 26349–26357 This journal is © The Royal Society of Chemistry 2020 26354 \| RSC Adv., 2020, 10, 26349–26357 RSC Advances View Article Online RSC Advances View Article Online Paper Paper tracker) and Mito-CDTH-CHO were co-incubated in HepG-2 cells. The uorescence imaging from Mito-CDTH-CHO in the blue channel (Fig. 6c and g) overlapped well with the Mito- tracker in the green channel (Fig. 6b and f), resulting in the Pearson's correlation coeﬃcient of 0.98. Furthermore, the region of interest (ROI) is illustrated in Fig. 6j, and the normalized uorescence intensity of Mito-CDTH-CHO changed in coordination with the normalized uorescence intensity of Mito-Tracker Green. These results suggested that Mito-CDTH- CHO possesses the excellent ability to target mitochondrial of subcellular organelle in HepG-2 cells. benzopyrone of the probe Mito-CDTH-CHO. As the concentra- tion of Na2SO3 increased from 1 to 10 eq., the proton peak disappeared, while the singlet at 4.89 ppm appeared. The response mechanism of the probe Mito-CDTH-CHO to NaHSO3 was also conrmed (Fig. 3.4. Cellular imaging of Mito-CDTH-CHO In view of the excellent performances of the probe Mito-CDTH- CHO in vitro, the capability of the discriminative detection of SO3 2 and HSO3 was investigated. Prior to bioimaging exper- iments, the cytotoxic assay was carried out by a CCK-8 method in HepG-2 cells, the results indicated that Mito-CDTH-CHO had low cytotoxicity (Fig. 5). According to the literature,31–33 cationic small molecules could enter into mitochondria and interact with anionic species via the electrostatic interaction. The design of Mito-CDTH-CHO is based on our considerations that the benzopyran cation (containing oxygen positive ions) can act as a mitochondrion- targeting moiety. The positive charge and hydrophobic prop- erties of the benzopyran cation are supposed to mediate the localization of Mito-CDTH-CHO inside the mitochondrial membrane. Thus, we speculate that Mito-CDTH-CHO will mainly distribute in the mitochondria. In order to verify our hypothesis, the mitochondrial colocalization experiment was carried out. The commercial mitochondrion tracker (Mito- HepG-2 cells were pre-treated with a probe (20 mM) in the DMEM medium and then incubated with Na2SO3 (200 mM) for 30 min. As shown in Fig. 8, due to the equilibrium conversion between SO3 2 and HSO3 in a neutral uid, HepG-2 cells exhibited distinct uorescence (a1) in the green channel and weak uorescence (a2) in the blue channel. In contrast, aer incubating the HepG-2 cells with NaHSO3 (200 mM) for 30 min, clear uorescence in the blue channel (b1) and weak uorescence (b2) in the green channel were observed. Inspired by the above Fig. 7 Confocal microscopy colocalization images of Mito-CDTH-CHO and Mito-Tracker RED in HepG-2 cells. (a) Bright ﬁeld of HepG-2 cells. (b) Fluorescence image of Mito-Tracker RED (lex ¼ 548 nm, lem ¼ 560–620 nm). (c) Fluorescence image of Mito-CDTH-CHO (lex ¼ 390 nm, lem ¼ 460–520 nm). (d) The merges images of (a and b). (e) Intensity scatter plot of red and green channels. (f) Normalized intensity proﬁle of the linear region of part a across the HepG-2 cells. Scale bar ¼ 20 mm. Fig. 7 Confocal microscopy colocalization images of Mito-CDTH-CHO and Mito-Tracker RED in HepG-2 cells. (a) Bright ﬁeld of HepG-2 cells. (b) Fluorescence image of Mito-Tracker RED (lex ¼ 548 nm, lem ¼ 560–620 nm). (c) Fluorescence image of Mito-CDTH-CHO (lex ¼ 390 nm, lem ¼ 460–520 nm). (d) The merges images of (a and b). (e) Intensity scatter plot of red and green channels. 3.3. The proposed mechanism of Mito-CDTH-CHO for SO2 derivatives detection 4), except the singlet at 4.89 ppm, an additional hydroxyl proton peak at 5.12 ppm emerged, which is attributed to the diﬀerence in nucleophilicity between SO3 2 and HSO3 . These results further veried that the dual-site sensing of Mito-CDTH-CHO toward Na2SO3 and NaHSO3 via diﬀerent double bond nucleophilic addition reactions. Open Access Article. Published on 14 July 2020. Downloaded on 10/24/2024 5:07:30 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. For the sake of better experimental results, we conducted the control experiments with a lyso-tracker. As illustrated in Fig. 7, the green uorescence of Mito-CDTH-CHO is not overlapped at all with the red uorescence of LysoTracker RED with the Pearson's correlation coeﬃcients (Rr) of 0.3308 and an overlap coeﬃcient (R) of 0.6277 (Fig. 7e). The green uorescence of Mito-CDTH-CHO and red uorescence of LysoTracker RED changes in the intensity proles of ROIs are not synchronized at all (Fig. 7f). The result further indicates that Mito-CDTH-CHO mainly localizes in the mitochondria of living cells. This journal is © The Royal Society of Chemistry 2020 Acknowledgements This work was supported by the National Key Laboratory of Materials Chemistry Independent Project Fund ZK201904 (2019). Jiangsu Province Agricultural Science and Technology Independent Innovation Fund Project CX (19) 3101. Special funds for the transformation of scientic and technological achievements SBA 2019030143. 3.4. Cellular imaging of Mito-CDTH-CHO (f) Normalized intensity proﬁle of the linear region of part a across the HepG-2 cells. Scale bar ¼ 20 mm. This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 26349–26357 \| 26355 This journal is © The Royal Society of Chemistry 2020 View Article Online RSC Advances Fig. 8 The confocal ﬂuorescence images of probe and SO2 derivatives in HepG-2 cells. (a1–a4) HepG-2 cells were pre-treated with Mito-CDTH- CHO (20 mM), then incubated with Na2SO3 (200 mM) for 30 min. (b1–b4) HepG-2 cells were incubated with NaHSO3 (200 mM) for 30 min. (c1–c4) After incubated with Na2SO3 (200 mM) for 30 min. (d1–d4) After incubated with NaHSO3 (200 mM) for 30 min. All cells were pre-treated with diﬀerent pH values (pH ¼ 6.0, 7.4 and 8.0) DMEM medium for 3 h. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 14 July 2020. Downloaded on 10/24/2024 5:07:30 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 8 The confocal ﬂuorescence images of probe and SO2 derivatives in HepG-2 cells. (a1–a4) HepG-2 cells were pre-treated with Mito-CDTH- CHO (20 mM), then incubated with Na2SO3 (200 mM) for 30 min. (b1–b4) HepG-2 cells were incubated with NaHSO3 (200 mM) for 30 min. (c1–c4) After incubated with Na2SO3 (200 mM) for 30 min. (d1–d4) After incubated with NaHSO3 (200 mM) for 30 min. All cells were pre-treated with diﬀerent pH values (pH ¼ 6.0, 7.4 and 8.0) DMEM medium for 3 h. of the physiological and pathological processes of SO2 derivatives. experimental results, HepG-2 cells were cultured with NaHSO3 (200 mM) for 30 min, it is worth noting that the uorescence in the green channel disappeared (d1), whereas the uorescence in the blue channel enhanced (d2). Similarly, in the HepG-2 cells incubated with Na2SO3 in the DMEM medium for 30 min, there was uorescence enhancement in the green channel (c1) and almost no uorescence in the blue channel (c2). Therefore, probe Mito-CDTH-CHO can detect the intracellular SO3 2 and HSO3 levels with diﬀerent uorescence signals. 4. Conclusions In short, to the best of our knowledge, for the rst time, a dual- site uorescence probe for HSO3 and SO3 2 with two diﬀerent emission signals was designed and synthesized. Mito-CDTH- CHO can distinguishingly sense the levels of HSO3 and SO3 2 with diﬀerent uorescence signals under separate pH condi- tions in living biological systems and possesses low cytotoxicity, excellent biocompatibility and outstanding mitochondrial tar- geting. In the meantime, the sensing mechanism of the double bond nucleophilic addition was successfully validated using the NMR titration experiments. 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https://openalex.org/W4292314165	https://estudogeral.uc.pt/bitstream/10316/101202/1/wevj-13-00155-v2.pdf	English	null	Heterogeneous Impact of Electrification of Road Transport on Premature Deaths from Outdoor Air Pollution: A Macroeconomic Evidence from 29 European Countries	World electric vehicle journal	2,022	cc-by	17,240	Emad Kazemzadeh 1 , Matheus Koengkan 2,* , José Alberto Fuinhas 3 , Mônica Teixeira 4 and Alexandre Mejdalani 4 1 Department of Economics, Faculty of Economics and Administrative Sciences, Ferdowsi University of Mashhad, Mashhad 9177948974, Iran 2 Governance, Competitiveness and Public Policies (GOVCOPP), Department of Economics, Management, Industrial Engineering and Tourism (DEGEIT), University of Aveiro, 3810-193 Aveiro, Portugal 3 Centre for Business and Economics Research (CeBER), Faculty of Economics, University of Coimbra, 3004-512 Coimbra, Portugal 4 Department of Economics, Federal Fluminense University, Niteroi 24020-150, Brazil 1 Department of Economics, Faculty of Economics and Administrative Sciences, Ferdowsi University of Mashhad, Mashhad 9177948974, Iran 2 Governance, Competitiveness and Public Policies (GOVCOPP), Department of Economics, Management, Industrial Engineering and Tourism (DEGEIT), University of Aveiro, 3810-193 Aveiro, Portugal 3 Centre for Business and Economics Research (CeBER), Faculty of Economics, University of Coimbra, 3004-512 Coimbra, Portugal 4 Department of Economics, Federal Fluminense University, Niteroi 24020-150, Brazil y 2 Governance, Competitiveness and Public Policies (GOVCOPP), Department of Economics, Industrial Engineering and Tourism (DEGEIT), University of Aveiro, 3810-193 Aveiro, Portu 3 C f B i d E i R h (C BER) F l f E i U i i f g g ( ), y , 3 Centre for Business and Economics Research (CeBER), Faculty of Economics, Unive 3004-512 Coimbra, Portugal g 4 Department of Economics, Federal Fluminense University, Niteroi 24020-150, Brazil 4 Department of Economics, Federal Fluminense University, Niteroi 24020-150, Brazil * Correspondence: matheuskoengkan@ua.pt Abstract: One of the biggest problems associated with vehicles that use internal combustion engines is that they cause elevated levels of pollution in the places they travel through, especially if they cause congestion. However, it is not only the level, but also probably the concentration of gases emitted by internal combustion engines in the places where they move around that is particularly lethal. Can the road transport sector’s electriﬁcation mitigate premature deaths from outdoor air pollution? Our main hypothesis is that replacing internal combustion engine vehicles with electrical ones contributes to mitigating people’s exposure to high concentrations of air pollution. To answer the research question, a panel of 29 European countries, from 2010 to 2020, using the method of moments quantile regression and ordinary least squares, was examined. Results support the concept that economic growth, renewable energy consumption, and electric vehicles in all quantiles have a negative impact on premature mortality due to air pollution. These impacts are higher on premature mortality in lower quantiles, but gradually decrease with increasing quantile levels. Citation: Kazemzadeh, E.; Koengkan, M.; Fuinhas, J.A.; Teixeira, M.; Mejdalani, A. Heterogeneous Impact of Electriﬁcation of Road Transport on Premature Deaths from Outdoor Air Pollution: A Macroeconomic Evidence from 29 European Countries. World Electr. Veh. J. 2022, 13, 155. https://doi.org/10.3390/ wevj13080155 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: electriﬁcation of road transport; European countries; method of moments quantile regression; outdoor air pollution; premature deaths Emad Kazemzadeh 1 , Matheus Koengkan 2,* , José Alberto Fuinhas 3 , Mônica Teixeira 4 and Alexandre Mejdalani 4 The results also reveal that methane emissions, in all quantiles except 10th, have a negative effect on premature mortality. Nitrous oxide emissions positively impact premature mortality in all quantiles except the 10th, and this impact increases at high quantiles. Fine particulate matter positively impacts premature mortality in all quantiles, with the same at all levels. The ordinary least squares, used as a robustness check, conﬁrm that economic growth, renewable energy consumption, and methane emissions have reduced impacts on premature mortality due to outdoor air pollution. However, nitrous oxide emissions and ﬁne particulate matter increase premature mortality. These results reinforce the importance of policymakers implementing policies for road electriﬁcation. Heterogeneous Impact of Electriﬁcation of Road Transport on Premature Deaths from Outdoor Air Pollution: A Macroeconomic Evidence from 29 European Countries Emad Kazemzadeh 1 , Matheus Koengkan 2,* , José Alberto Fuinhas 3 , Mônica Teixeira 4 and Alexandre Mejdalani 4 Share of deaths from outdoor air pollution Figure 1. Share of premature deaths from outdoor air pollution in the EU-27, between 1990–2019. This figure was created by the authors with data from Our World in Data [3]. Figure 1. Share of premature deaths from outdoor air pollution in the EU-27, between 1990–2019. This ﬁgure was created by the authors with data from Our World in Data [3]. The figure above shows that premature deaths from outdoor air pollution in the EU- 27 were reduced by −45% between 1990 to 2019. This decline is attributed to programmes to mitigate air pollution in EU countries (e.g., electrification of road transport and energy transition) [5]. According to Eurostat [6], the exposure to air pollution by particulate mat- t (PM2 5) d d b 14% i th EU 27 ( h i Fi 2 b l ) The ﬁgure above shows that premature deaths from outdoor air pollution in the EU-27 were reduced by −45% between 1990 to 2019. This decline is attributed to programmes to mitigate air pollution in EU countries (e.g., electriﬁcation of road transport and energy transition) [5]. According to Eurostat [6], the exposure to air pollution by particulate matter (PM2.5) dropped by −14% in the EU-27 (as shown in Figure 2 below). ter (PM2.5) dropped by −14% in the EU-27 (as shown in Figure 2 below). The figure above shows that the exposure to air pollution by PM2.5 was 14.5% in 2000, and this value dropped to 12.60% in 2019. Consistent with the European Environ- mental Agency [5], this reduction in exposure to air pollution mitigated the premature deaths attributed to air pollution in 33% of the EU-27. Therefore, if air quality continues to improve and the number of premature deaths per year continues to fall at a comparable t th th ll ti t t ill b hi d b 2032 The ﬁgure above shows that the exposure to air pollution by PM2.5 was 14.5% in 2000, and this value dropped to 12.60% in 2019. Consistent with the European Environmental Agency [5], this reduction in exposure to air pollution mitigated the premature deaths attributed to air pollution in 33% of the EU-27. Therefore, if air quality continues to improve and the number of premature deaths per year continues to fall at a comparable rate, then the zero-pollution target will be achieved by 2032. rate, then the zero-pollution target will be achieved by 2032. 1. Introduction Copyright: © 2022 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/). The combustion processes from motor vehicles, solid fuel burning, and industry are responsible for the increase in outdoor air pollution [1]. Outdoor air pollution is composed of six pollutants (e.g., carbon monoxide, lead, nitrogen dioxide, ozone, particulate matter of different size fractions, and sulphur dioxide) [2]. The intensiﬁcation of outdoor air pollution is one of the signiﬁcant causes of global health problems [3]. Consistent with the World Health Organisation (WHO) [4], the gases from outdoor air pollution mentioned before are a risk factor for several of the world’s leading causes of death (e.g., stroke, lung cancer, https://www.mdpi.com/journal/wevj World Electr. Veh. J. 2022, 13, 155. https://doi.org/10.3390/wevj13080155 World Electr. Veh. J. 2022, 13, 155 2 of 21 heart disease, and respiratory diseases). This problem tends to be worsened in high-income countries, and in transition countries from low to middle incomes [2]. cancer, heart disease, and respiratory diseases). This problem tends to be worsened in high-income countries, and in transition countries from low to middle incomes [2]. In 1990, the number of premature deaths attributed to outdoor air pollution was 2.14 In 1990, the number of premature deaths attributed to outdoor air pollution was 2.14 million, and in 2017 this value reached 3.41 million deaths around the world. In some countries, outdoor air pollution accounts for more than 8% of deaths. For example, in Egypt, outdoor air pollution accounted for 12% of deaths in 2017, 10% in China and Turkey, and 8% in India [3]. In the European Union (EU), which is the object of study in this empirical investigation, the share of premature deaths attributed to outdoor air pollution was 6.72% in 1990, and this value reached 3.71% in 2019 (as shown in Figure 1 below). In 1990, the number of premature deaths attributed to outdoor air pollution was 2.14 million, and in 2017 this value reached 3.41 million deaths around the world. In some countries, outdoor air pollution accounts for more than 8% of deaths. For example, in Egypt, outdoor air pollution accounted for 12% of deaths in 2017, 10% in China and Tur- key, and 8% in India [3]. 1. Introduction In the European Union (EU), which is the object of study in this empirical investigation, the share of premature deaths attributed to outdoor air pollution was 6.72% in 1990, and this value reached 3.71% in 2019 (as shown in Figure 1 below). 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 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 2015 2016 2017 2018 2019 Share of deaths (%) Share of premature deaths from outdoor air pollution in the EU-27 between 1990-2019 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 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 2015 2016 2017 2018 2019 Share of deaths (%) Share of premature deaths from outdoor air pollution in the EU-27 between 1990-2019 Share of premature deaths from outdoor air pollution in the EU-27 between 1990-2019 Exposure to air pollution by particulate matter (PM2.5) Figure 2. Exposure to air pollution by particulate matter (PM2.5) in the EU-27 between 2000–2019. This figure was created by the authors with data from Eurostat [7]. Figure 2. Exposure to air pollution by particulate matter (PM2.5) in the EU-27 between 2000–2019. This ﬁgure was created by the authors with data from Eurostat [7]. This concern in reducing the exposure to air pollution is associated with the signifi- cant part of poorer people in the EU who lives next to busy roads or industrial areas and, thus, face higher levels of exposure to air pollution. In some cities, wealthier people live in central, polluted areas, while in other European cities, poorer communities inhabit the main areas [5]. Therefore, the higher population exposure to PM2.5 in particular regions translates into higher premature deaths attributed to air pollution, generating more eco- nomic costs. Europa [8] said that the economic costs of premature deaths from air pollu- tion amount to well over €20 billion a year in the EU. As mentioned above by the European Environmental Agency [5], the reduction of air pollution in the EU is related to several initiatives, such as the electrification of road transport, and energy transition. Indeed, the evidence that the electrification of the transport sector can mitigate air pollution in EU countries was discovered by some au- thors (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]). For example, Kazemzadeh et al. [9] studied the impact of battery-electric vehicles (BEVs) and plug-in hybrid electric vehicles (PHEVs) on PM2 5 emissions in 29 European Countries Therefore the capacity of BEVs As mentioned above by the European Environmental Agency [5], the reduction of air pollution in the EU is related to several initiatives, such as the electriﬁcation of road transport, and energy transition. Indeed, the evidence that the electriﬁcation of the transport sector can mitigate air pollution in EU countries was discovered by some authors (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]). For example, Kazemzadeh et al. [9] studied the impact of battery-electric vehicles (BEVs) and plug-in hybrid electric vehicles (PHEVs) on PM2.5 emissions in 29 European Countries. Therefore, the capacity of BEVs and PHEVs to reduce PM2.5 emissions is correlated with the increase in energy efﬁciency in electric vehicles that consume less electricity from non-renewable energy sources. This reduction in electricity consumption reﬂects a decrease in air pollution emissions. Fuinhas et al. Share of deaths from outdoor air pollution [9], where the increase of electric vehicles in the EU-27 ﬂeet will mitigate the consumption of fossil fuels and, consequently, the GHGs. Moreover, the same authors suggest that the rise in energy efﬁciency is also responsible for reducing emissions in the region. As Kazemzadeh et al. [9] mentioned, electric vehicles are gradually penetrating the market in the EU. Electric vehicles represented a market share of only 3.5% in 2019. On the other hand, the BEVs accounted for 2.19%, while the PHEVs represented a 1.21% market share. In the EU, more than half of these registrations were made in Germany, Norway, the Netherlands, France, and the United Kingdom (e.g., Kazemzadeh et al. [9]; and Fuinhas et al. [10]. Indeed, according to the European Alternative Fuels Observatory (EAFO) [11] and Koengkan et al. [12], the number of BEVs in the ﬂeet in the EU that includes, for example, passenger cars, light commercial vehicles, buses, and trucks, was 4440 units in 2008, but reached 2190. 328 units in 2021, while the number of PHEVs in the ﬂeet, that includes passenger cars, light commercial vehicles, buses, and trucks, was 163 units in 2012, but reached 965,460 units in 2021 (see Figure 3 below). 0.00 5.00 10.00 15.00 20.00 25.00 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Exposue to particule matter (%) Exposure to air pollution by particulate matter (PM2.5) in the EU-27, between 2000-2019 Exposure to air pollution by particulate matter (PM2.5) 0.00 5.00 10.00 15.00 20.00 25.00 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Exposue to particule matter (%) Exposure to air pollution by particulate matter (PM2.5) in the EU-27, between 2000-2019 Exposure to air pollution by particulate matter (PM2.5) Share of deaths from outdoor air pollution p g y This concern in reducing the exposure to air pollution is associated with the signiﬁcant part of poorer people in the EU who lives next to busy roads or industrial areas and, thus, face higher levels of exposure to air pollution. In some cities, wealthier people live in central, polluted areas, while in other European cities, poorer communities inhabit the main areas [5]. Therefore, the higher population exposure to PM2.5 in particular regions translates into higher premature deaths attributed to air pollution, generating more economic costs. Europa [8] said that the economic costs of premature deaths from air pollution amount to well over €20 billion a year in the EU. 3 of 21 3 of 22 3 of 21 3 of 22 World Electr. Veh. J. 2022, 13, 155 World Electr. Veh. J. 2022, 13, x FO Figure 2. Exposure to air pollution by particulate matter (PM2.5) in the EU-27 between 2000–2019. This figure was created by the authors with data from Eurostat [7]. This concern in reducing the exposure to air pollution is associated with the signifi- cant part of poorer people in the EU who lives next to busy roads or industrial areas and, thus, face higher levels of exposure to air pollution. In some cities, wealthier people live in central, polluted areas, while in other European cities, poorer communities inhabit the main areas [5]. Therefore, the higher population exposure to PM2.5 in particular regions translates into higher premature deaths attributed to air pollution, generating more eco- nomic costs. Europa [8] said that the economic costs of premature deaths from air pollu- tion amount to well over €20 billion a year in the EU. As mentioned above by the European Environmental Agency [5], the reduction of air pollution in the EU is related to several initiatives, such as the electrification of road transport, and energy transition. Indeed, the evidence that the electrification of the transport sector can mitigate air pollution in EU countries was discovered by some au- thors (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]). For example, Kazemzadeh et al. [9] studied the impact of battery-electric vehicles (BEVs) and plug-in hybrid electric vehicles (PHEVs) on PM2.5 emissions in 29 European Countries. Therefore, the capacity of BEVs and PHEVs to reduce PM2.5 emissions is correlated with the increase in energy efficiency in electric vehicles that consume less electricity from non-renewable energy sources. Share of deaths from outdoor air pollution This reduction in electricity consumption reflects a decrease in air pollution emissions. Fuinhas et al. [10] also studied the impact of electric vehicles on greenhouse gas emissions (GHGs) in the EU-27 countries. The authors complement the explanation of Kazemzadeh et al. [9], where the increase of electric vehicles in the EU-27 fleet will mitigate the consumption of fossil fuels and, consequently, the GHGs. Moreover, the same authors suggest that the rise in energy efficiency is also responsible for reducing emissions in the region. As Kazemzadeh et al. [9] mentioned, electric vehicles are gradually penetrating the market in the EU. Electric vehicles represented a market share of only 3.5% in 2019. On the other hand, the BEVs accounted for 2.19%, while the PHEVs represented a 1.21% mar- ket share. In the EU, more than half of these registrations were made in Germany, Nor- way, the Netherlands, France, and the United Kingdom (e.g., Kazemzadeh et al. [9]; and 0.00 5.00 10.00 15.00 20.00 25.00 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Exposue to particule matter (%) Exposure to air pollution by particulate matter (PM2.5) in the EU-27, between 2000-2019 Exposure to air pollution by particulate matter (PM2.5) Figure 2. Exposure to air pollution by particulate matter (PM2.5) in the EU-27 between 2000–2019. This ﬁgure was created by the authors with data from Eurostat [7]. As mentioned above by the European Environmental Agency [5], the reduction of air pollution in the EU is related to several initiatives, such as the electriﬁcation of road transport, and energy transition. Indeed, the evidence that the electriﬁcation of the transport sector can mitigate air pollution in EU countries was discovered by some authors (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]). For example, Kazemzadeh et al. [9] studied the impact of battery-electric vehicles (BEVs) and plug-in hybrid electric vehicles (PHEVs) on PM2.5 emissions in 29 European Countries. Therefore, the capacity of BEVs and PHEVs to reduce PM2.5 emissions is correlated with the increase in energy efﬁciency in electric vehicles that consume less electricity from non-renewable energy sources. This reduction in electricity consumption reﬂects a decrease in air pollution emissions. Fuinhas et al. [10] also studied the impact of electric vehicles on greenhouse gas emissions (GHGs) in the EU-27 countries. The authors complement the explanation of Kazemzadeh et al. Exposure to air pollution by particulate matter (PM2.5) The number of BEVs and PHEVs in the ﬂeet in the EU between 2008 to 2021. This ﬁgure was created by the authors with data from the European Alternative Fuels Observatory (EAFO) [11]. Figure 3. The number of BEVs and PHEVs in the fleet in the EU between 2008 to 2021. This figure was created by the authors with data from the European Alternative Fuels Observatory (EAFO) [11]. Figure 3. The number of BEVs and PHEVs in the ﬂeet in the EU between 2008 to 2021. This ﬁgure was created by the authors with data from the European Alternative Fuels Observatory (EAFO) [11]. In other words, if the process of electrification of the transport sector mitigates the air pollution in the EU countries, as found by Kazemzadeh et al. [9] and Fuinhas et al. [10], then this same process also could mitigate the premature deaths from outdoor air pollu- tion as a consequence of the reduction in air pollution. In the literature the impact of electrification of the transport sector on premature In other words, if the process of electriﬁcation of the transport sector mitigates the air pollution in the EU countries, as found by Kazemzadeh et al. [9] and Fuinhas et al. [10], then this same process also could mitigate the premature deaths from outdoor air pollution as a consequence of the reduction in air pollution. In the literature, the impact of electrification of the transport sector on premature deaths from outdoor air pollution or similar studies has not been approached. Most stud- ies linked to the electrification of the transport sector have focused on aspects related to the consumption of energy and air pollution, leaving aside the health aspects associated with air pollution. Therefore, there is a gap in the literature associated with the possible externalities caused by the electrification of the transport sector on health aspects, since the process of electrification mitigates the consumption of fossil fuels and air pollution (e.g., PM2.5 and CO2 emissions) and, consequently, also the problems health associated to air pollution. What are electric ehicles replacing? Electric ehicles are mostly replacing internal In the literature, the impact of electrification of the transport sector on premature deaths from outdoor air pollution or similar studies has not been approached. Exposure to air pollution by particulate matter (PM2.5) Most studies linked to the electrification of the transport sector have focused on aspects related to the consumption of energy and air pollution, leaving aside the health aspects associated with air pollution. Therefore, there is a gap in the literature associated with the possible externalities caused by the electrification of the transport sector on health aspects, since the process of electrification mitigates the consumption of fossil fuels and air pollution (e.g., PM2.5 and CO2 emissions) and, consequently, also the problems health associated to air pollution. What are electric vehicles replacing? Electric vehicles are mostly replacing internal combustion vehicles. One of the biggest problems associated with vehicles that use inter- nal combustion engines is that they cause high levels of pollution in the places they travel around, especially if they cause congestion. However, it is not just the level, but probably also the concentration of gases emitted by internal combustion engines in the place where they move around, that is particularly lethal. Due to a gap in the literature regarding this topic of study, this investigation raises the following central question: Can the road transport sector’s electrification mitigate premature deaths from outdoor air pollution? Our main hypothesis is that replacing internal combustion engine vehicles with electrical ones contributes to mitigating people’s exposure to high concentrations of air pollution. Consequently, the increased number of electric vehicles in the fleet is expected to d d h f d ll i Th l i hi b l ifi i What are electric vehicles replacing? Electric vehicles are mostly replacing internal combustion vehicles. One of the biggest problems associated with vehicles that use internal combustion engines is that they cause high levels of pollution in the places they travel around, especially if they cause congestion. However, it is not just the level, but probably also the concentration of gases emitted by internal combustion engines in the place where they move around, that is particularly lethal. Due to a gap in the literature regarding this topic of study, this investigation raises the following central question: Can the road transport sector’s electriﬁcation mitigate premature deaths from outdoor air pollution? Our main hypothesis is that replacing internal combustion engine vehicles with electrical ones contributes to mitigating people’s exposure to high concentrations of air pollution. reduce premature deaths from outdoor pollution. Exposure to air pollution by particulate matter (PM2.5) [10] also studied the impact of electric vehicles on greenhouse gas emissions (GHGs) in the EU-27 countries. The authors complement the explanation of Kazemzadeh et al. [9], where the increase of electric vehicles in the EU-27 ﬂeet will mitigate the consumption of fossil fuels and, consequently, the GHGs. Moreover, the same authors suggest that the rise in energy efﬁciency is also responsible for reducing emissions in the region. (PHEVs) on PM2.5 emissions in 29 European Countries. Therefore, the capacity of BEVs and PHEVs to reduce PM2.5 emissions is correlated with the increase in energy efficiency in electric vehicles that consume less electricity from non-renewable energy sources. This reduction in electricity consumption reflects a decrease in air pollution emissions. Fuinhas et al. [10] also studied the impact of electric vehicles on greenhouse gas emissions (GHGs) in the EU-27 countries. The authors complement the explanation of Kazemzadeh et al. [9], where the increase of electric vehicles in the EU-27 fleet will mitigate the consumption of fossil fuels and, consequently, the GHGs. Moreover, the same authors suggest that the rise in energy efficiency is also responsible for reducing emissions in the region. As Kazemzadeh et al. [9] mentioned, electric vehicles are gradually penetrating the market in the EU. Electric vehicles represented a market share of only 3.5% in 2019. On th th h d th BEV t d f 2 19% hil th PHEV t d 1 21% As Kazemzadeh et al. [9] mentioned, electric vehicles are gradually penetrating the market in the EU. Electric vehicles represented a market share of only 3.5% in 2019. On the other hand, the BEVs accounted for 2.19%, while the PHEVs represented a 1.21% market share. In the EU, more than half of these registrations were made in Germany, Norway, the Netherlands, France, and the United Kingdom (e.g., Kazemzadeh et al. [9]; and Fuinhas et al. [10]. Indeed, according to the European Alternative Fuels Observatory (EAFO) [11] and Koengkan et al. [12], the number of BEVs in the ﬂeet in the EU that includes, for example, passenger cars, light commercial vehicles, buses, and trucks, was 4440 units in 2008, but reached 2190. 328 units in 2021, while the number of PHEVs in the ﬂeet, that includes passenger cars, light commercial vehicles, buses, and trucks, was 163 units in 2012, but reached 965,460 units in 2021 (see Figure 3 below). World Electr. Veh. J. Exposure to air pollution by particulate matter (PM2.5) The relationship between electriﬁcation and premature deaths was expected to be nonlinear. Indeed, it is expectable that the percentage of deaths from outdoor air pollution will react to their causes as it increases. Therefore, an empirical analysis will be performed to answer this central question. The econometric model includes, as control variables, the essential variables identiﬁed in the literature as having explanatory power on deaths from outdoor pollution. This econometric analysis will be based on the macroeconomic panel data of 29 European countries (e.g., the EU-27 European countries plus the United Kingdom and Norway) between 2010 and 2020. The method of moments quantile regression (MM-QR) and ordinary least squares (OLS) with ﬁxed effects will be used for this task Figure 3. The number of BEVs and PHEVs in the fleet in the EU between 2008 to 2021. This figure was created by the authors with data from the European Alternative Fuels Observatory (EAFO) [11]. 0 500,000 1,000,000 1,500,000 2,000,000 2,500,000 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Number of BEVs and PHEVs on the fleet Number of BEVs and PHEVs on the fleet in the EU between 2008-2021 BEV PHEV Figure 3. The number of BEVs and PHEVs in the ﬂeet in the EU between 2008 to 2021. This ﬁgure was created by the authors with data from the European Alternative Fuels Observatory (EAFO) [11]. Figure 3. The number of BEVs and PHEVs in the fleet in the EU between 2008 to 2021. This figure was created by the authors with data from the European Alternative Fuels Observatory (EAFO) [11]. 0 500,000 1,000,000 1,500,000 2,000,000 2,500,000 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Number of BEVs and PHEVs on the fleet Number of BEVs and PHEVs on the fleet in the EU between 2008-2021 BEV PHEV Figure 3. The number of BEVs and PHEVs in the ﬂeet in the EU between 2008 to 2021. This ﬁg was created by the authors with data from the European Alternative Fuels Observatory (EAFO) [ Number of BEVs and PHEVs on the fleet in the EU between 2008-2021 Figure 3. The number of BEVs and PHEVs in the fleet in the EU between 2008 to 2021. This figure was created by the authors with data from the European Alternative Fuels Observatory (EAFO) [11]. Figure 3. Exposure to air pollution by particulate matter (PM2.5) 2022, 13, 155 4 of 21 , s ( g ) Figure 3. The number of BEVs and PHEVs in the fleet in the EU between 2008 to 2021. This figure was created by the authors with data from the European Alternative Fuels Observatory (EAFO) [11]. In other words, if the process of electrification of the transport sector mitigates the air pollution in the EU countries, as found by Kazemzadeh et al. [9] and Fuinhas et al. [10], then this same process also could mitigate the premature deaths from outdoor air pollu- tion as a consequence of the reduction in air pollution. In the literature, the impact of electrification of the transport sector on premature deaths from outdoor air pollution or similar studies has not been approached. Most stud- ies linked to the electrification of the transport sector have focused on aspects related to the consumption of energy and air pollution, leaving aside the health aspects associated with air pollution. Therefore, there is a gap in the literature associated with the possible externalities caused by the electrification of the transport sector on health aspects, since the process of electrification mitigates the consumption of fossil fuels and air pollution (e.g., PM2.5 and CO2 emissions) and, consequently, also the problems health associated to air pollution. What are electric vehicles replacing? Electric vehicles are mostly replacing internal combustion vehicles. One of the biggest problems associated with vehicles that use inter- nal combustion engines is that they cause high levels of pollution in the places they travel around, especially if they cause congestion. However, it is not just the level, but probably also the concentration of gases emitted by internal combustion engines in the place where they move around, that is particularly lethal. Due to a gap in the literature regarding this topic of study, this investigation raises the following central question: Can the road transport sector’s electrification mitigate premature deaths from outdoor air pollution? Our main hypothesis is that replacing internal combustion engine vehicles with electrical ones contributes to mitigating people’s exposure to high concentrations of air pollution. Consequently, the increased number of electric vehicles in the fleet is expected to reduce premature deaths from outdoor pollution. Exposure to air pollution by particulate matter (PM2.5) The relationship between electrification 0 500,000 1,000,000 1,500,000 2,000,000 2,500,000 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Number of BEVs and PHEVs on the fleet Number of BEVs and PHEVs on the fleet in the EU between 2008-2021 BEV PHEV Figure 3. The number of BEVs and PHEVs in the ﬂeet in the EU between 2008 to 2021. This ﬁgure was created by the authors with data from the European Alternative Fuels Observatory (EAFO) [11]. In other words, if the process of electriﬁcation of the transport sector mitigates the air pollution in the EU countries, as found by Kazemzadeh et al. [9] and Fuinhas et al. [10], then this same process also could mitigate the premature deaths from outdoor air pollution as a consequence of the reduction in air pollution. In the literature, the impact of electrification of the transport sector on premature deaths from outdoor air pollution or similar studies has not been approached. Most studies linked to the electrification of the transport sector have focused on aspects related to the consumption of energy and air pollution, leaving aside the health aspects associated with air pollution. Therefore, there is a gap in the literature associated with the possible externalities caused by the electrification of the transport sector on health aspects, since the process of electrification mitigates the consumption of fossil fuels and air pollution (e.g., PM2.5 and CO2 emissions) and, consequently, also the problems health associated to air pollution. What are electric vehicles replacing? Electric vehicles are mostly replacing internal combustion vehicles. One of the biggest problems associated with vehicles that use internal combustion engines is that they cause high levels of pollution in the places they travel around, especially if they cause congestion. However, it is not just the level, but probably also the concentration of gases emitted by internal combustion engines in the place where they move around, that is particularly lethal. Due to a gap in the literature regarding this topic of study, this investigation raises the following central question: Can the road transport sector’s electriﬁcation mitigate premature deaths from outdoor air pollution? Our main hypothesis is that replacing internal combustion engine vehicles with electrical ones contributes to mitigating people’s exposure to high concentrations of air pollution. Consequently, the increased number of electric vehicles in the ﬂeet is expected to reduce premature deaths from outdoor pollution. Exposure to air pollution by particulate matter (PM2.5) The relationship between electrification Consequently, the increased number of electric vehicles in the ﬂeet is expected to reduce premature deaths from outdoor pollution. The relationship between electriﬁcation and premature deaths was expected to be nonlinear. Indeed, it is expectable that the percentage of deaths from outdoor air pollution will react to their causes as it increases. Therefore, an empirical analysis will be performed to answer this central question. The econometric model includes, as control variables, the essential variables identiﬁed in the literature as having explanatory power on deaths from outdoor pollution. This econometric analysis will be based on the macroeconomic panel data of 29 European countries (e.g., the EU-27 European countries plus the United Kingdom and Norway) between 2010 and 2020. The method of moments quantile regression (MM-QR) and ordinary least squares (OLS) with ﬁxed effects will be used for this task. World Electr. Veh. J. 2022, 13, 155 5 of 21 Additionally, as stated above, the impact of electrification of the transport sector on premature deaths from outdoor air pollution is not addressed by the literature. For this reason, this investigation opted to use the literature regarding the impact of electric vehicles on air pollution to help answer the central question and explain the results that will be found (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]; Plötz et al. [13]; Zhao et al. [14]; Andersson and Börjesson, [15]; Vilchez and Jochem [16]; Ahmadi and Kjeang [17]; Miotti et al. [18]; and Bauer et al. [19]). Most of these studies came from the engineering areas. In the literature, few studies from social sciences have approached the impact of electric vehicles on air pollution. Moreover, another motivation to realise this investigation is related to the capacity of the electrification of road transport to mitigate the PM2.5 and CO2 emissions as discovered by Kazemzadeh et al. [9] and Fuinhas et al. [10]. Therefore, if the electrification of road transport mitigates the PM2.5 and CO2 emissions, then this process also could mitigate the deaths caused by PM2.5 as well as CO2 emissions. Thus, this investigation will extend and complement the studies realised by Kazemzadeh et al. [9] and Fuinhas et al. [10]. This investigation will introduce a new analysis regarding the impact of the electriﬁca- tion of the transport sector on premature deaths from outdoor air pollution in the EU-27 countries, plus the United Kingdom and Norway. Exposure to air pollution by particulate matter (PM2.5) This topic of research has never been approached before by the literature. Therefore, this study can open new opportunities for studying the link between the electriﬁcation of the transport sector and the health aspects of air pollution. Furthermore, this investigation is innovative because it uses econometric and macroeconomic approaches to identify the possible effect of the electriﬁcation of the transport sector on premature deaths from air pollution. It is the ﬁrst time these methodol- ogy approaches have been employed. Lastly, the results and explanations of this study will support policymakers and governments in developing consistent policies and initiatives that promote the process of electriﬁcation of the road transport sector in European countries and developing countries. The rest of this article is set out as follows. Section 2 provides an overview of the literature. Section 3 describes the methodology approach and data used in the study. Section 4 presents the empirical results. Section 5 discusses the main ﬁndings, Section 6 presents the conclusions and policy implications. Finally, Section 7 reveals the limitations and future research. 2. Literature Review [23]; Gai et al. [24]; Pan et al. [25]; Liang et al. [26]; and Requia et al. [27]). [ ]; [ ]; [ ]; g [ ]; q [ ]) Requia et al. [27] studied the effects of electric vehicles on air quality, greenhouse gas emissions and human health. The authors reviewed a total of 4734 articles. The results showed that the electric vehicle type, energy source, driving conditions, charging pattern, and govern- ment policies have an impact on reducing pollution emissions. Choma et al. [23] investigated the United States and found that electric vehicles increased air quality. Rizza et al. [21], in a study for Turin (Italy), stated that the 5% share of electric vehicles reduces NO2, PM10, and PM2.5 pollution by 52%, 35%, and 49%, respectively, and that the results also showed that significant social benefits are obtained from reducing the pollution concentration. Pan et al. [25] examined the effects of electric vehicles on air pollution and health in the Greater Houston area. The authors found that increased O3 and PM2.5 concentrations would result in 122 premature deaths compared to 2013. Gai et al. [24] identified the health and environmental benefits of using electric vehicles in Toronto, Canada. The authors found that even with the worst-case scenario of 100% of electric vehicles being powered by natural gas, an electric car could still have significant health benefits. Furthermore, if all-electric cars are charged with renewable sources, 330 premature deaths per year can be prevented. Horton et al. [22] explained the impact of public transportation on public health and air pollution in China. The authors found that widespread use of heavy-duty electric vehicles reduced nitric oxide and PM2.5 and, thus, reduced 562 premature deaths from pollution. In contrast, light electric vehicles reduce air pollution and mortality less than heavy-duty electric vehicles. In another study on the electriﬁcation beneﬁts of China’s transportation ﬂeet, Liang et al. [26] found that the electriﬁcation of 27% of personal and commercial vehicles could easily reduce PM2.5, nitrogen dioxide, and ozone concentrations by 2030. Kazemzadeh et al. [9] analysed the effects of battery electric vehicles and plug-in hybrid vehicles in 29 European countries on environmental quality from 2010 to 2019. The authors found that electric cars reduced PM2.5 emissions, and these effects were more signiﬁcant in high quantiles. In another investigation using the MM-QR model for 29 European countries, Fuinhas et al. 2. Literature Review As mentioned in the previous section, none of the existing literature approaches the impact of electric vehicles or the electriﬁcation of road transport on premature deaths from outdoor air pollution. However, most studies on electric vehicles addressed issues associated with air pollution in the literature, as shown in Figure 4 below. World Electr. Veh. J. 2022, 13, 155 6 of 23 very close to each other, forming almost a circle, which shows the proximity of the themes addressed by the articles. Figure 4. Wordnet of publications related to electric vehicles. This figure was created using the VOSviever software. The gap i the lite atu e co ce i g the i pact of elect ic ehicles o the elect ifica Figure 4. Wordnet of publications related to electric vehicles. This ﬁgure was created using the VOSviever software. Figure 4. Wordnet of publications related to electric vehicles. This figure was created using the VOSviever software. Figure 4. Wordnet of publications related to electric vehicles. This ﬁgure was created using the VOSviever software. World Electr. Veh. J. 2022, 13, 155 6 of 21 In Figure 4 above, the VOSviewer software separates the words into categories (clus- ters), dividing the items by colours. Each circle represents a word, and the size means the word’s relevance in the search—the greater the number of co-occurrences, the greater the proximity between words [20]. Indeed, in the map created, it can be seen that the software created six categories (e.g., greenhouse gases, atmospheric pollution, atmospheric emission, air pollution effects, greenhouse effects, and air pollution). These six categories are very close to each other, forming almost a circle, which shows the proximity of the themes addressed by the articles. The gap in the literature concerning the impact of electric vehicles or the electrification of road transport on premature deaths from outdoor air pollution led this investigation to more closely use the literature related to this topic (e.g., the effect of electric vehicles or the electrifica- tion of road transport on air pollution). This investigation selected the most important articles that approach the impact of electric cars or the electrification of road transport on air pollution (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]; Plötz et al. [13]; Zhao et al. [14]; Andersson and Börjesson [15]; Vilchez and Jochem [16]; Miotti et al. [18]; Rizza et al. [21]; Horton et al. [22]; Choma et al. 3.1. Data A group of 29 European countries will be analysed for the period from 2010 to 2020. That is, macroeconomic data from EU-27 countries (e.g., Austria, Belgium, Bulgaria, Croatia, Cyprus, Czechia, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, the Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, and Sweden), plus two others (the United Kingdom and Norway), will be used. The United Kingdom and Norway were added to this investigation because these two countries have high EV participation in their fleet. Furthermore, the period from 2010 to 2020 will be used due to the availability of macroeconomic data for this group of countries. The variables used in this empirical investigation are shown in Table 1 below. 8 of 2 8 of 2 8 of 2 8 of 2 8 of 2 Table 1. Variables and sources. Abbreviation Variables Source QR Code Dependent variable DOAP Deaths from outdoor air pollution (%) Our World in Data [3] Table 1. Variables and sources. Abbreviation Variables Source QR Code Dependent variable DOAP Deaths from outdoor air pollution (%) Our World in Data [3] Independent variables EVs The total number of electric vehicles registered in the fleet. This variable is the sum of BEVs, PHEVs, electric buses (EBUSS), electric light commercial vehicles (ELCVs), and electric heavy-duty (EHD) vehicles. Moreover, BEVs and PHEVs are passenger vehicles. European Alternative Fuels Observatory (EAFO) [11] GDP Gross domestic product (GDP) per capita (con- stant = 2010 $). World Bank Data (WBD) [28] RENE Renewable energy consumption per capita WBD [28] PM2.5 Particulate matter (micrograms per cubic metre) until 2019. Eurostat [29] Independent variables EVs The total number of electric vehicles registered in the ﬂeet. This variable is the sum of BEVs, PHEVs, electric buses (EBUSS), electric light commercial vehicles (ELCVs), and electric heavy-duty (EHD) vehicles. Moreover, BEVs and PHEVs are passenger vehicles. European Alternative Fuels Observatory (EAFO) [11] Table 1. Variables and sources. Abbreviation Variables Source QR Code Dependent variable DOAP Deaths from outdoor air pollution (%) Our World in Data [3] Independent variables EVs The total number of electric vehicles registered in the fleet. This variable is the sum of BEVs, PHEVs, electric buses (EBUSS), electric light commercial vehicles (ELCVs), and electric heavy-duty (EHD) vehicles. Moreover, BEVs and PHEVs are passenger vehicles. European Alternative Fuels Observatory (EAFO) [11] GDP Gross domestic product (GDP) per capita (con- stant = 2010 $). 2. Literature Review [10] stated that electric vehicles reduce greenhouse gas emissions in the middle quantiles (25th, 50th, and 75th). In a survey to control carbon pollution using PHEVs, Zhao et al. [14] found that PHEVs reduced carbon emissions. Plötz et al. [13] examined the effects of PHEVs and BEVs on CO2 emission reduction in Germany and the United States. The authors found that PHEVs charged from renewable electricity could help reduce GHGs, and that PHEVs reduced CO2 emissions more than EVs. Andersson and Börjesson [15] stated that plug-in hybrid vehicles might improve climate quality in the future more than electric vehicles. Vilchez and Jochem [16] investigated the effect of Powertrain technologies on GHGs by 2030 in several countries (China, Germany, Japan, France, India, and the United States) using a system dynamics model. The results showed that electric vehicles might reduce GHGs in the transportation system. However, it is possible that GHGs from the construction, scrapping, and power generation processes in these vehicles will increase. Finally, Miotti et al. [18] investigated the effects of fuel World Electr. Veh. J. 2022, 13, 155 7 of 21 7 of 21 cell vehicles (FCV) on GHGs. The authors found that if hydrogen were supplied from renewable electricity, FCVs could reduce GHGs by up to 50% compared to ICEVs. However, if hydrogen is supplied from natural gas, FCVs do not mitigate GHGs. Most of the studies that have been carried out in this ﬁeld so far have investigated the relationship between electric vehicles and environmental quality (e.g., CO2 emissions, PM2.5, PM10, NO2, and O3) in different countries and regions. Yet, increased air pollution leads to higher rates of premature mortality by pollution. Nevertheless, to our knowledge, no studies have examined this scientiﬁc gap. For this purpose, we investigate the effects of road transport electriﬁcation on premature deaths due to outdoor air pollution. In this study, the MM-QR model is used for analysis, making it possible to investigate the effects of electric vehicles at different quantiles on premature death due to outdoor air pollution. The next part of this research introduces the data/variables and methodology. 3. Data and Method Approach As mentioned previously, this section shows the data/variables and methodological approach used. Hence, the ﬁrst Section 3.1, will demonstrate the data/variables, while Section 3.2 will outline the methodological approach. 3.1. Data World Bank Data (WBD) [28] RENE Renewable energy consumption per capita WBD [28] PM2.5 Particulate matter (micrograms per cubic metre) until 2019. Eurostat [29] GDP Gross domestic product (GDP) per capita (constant = 2010 $). World Bank Data (WBD) [28] Table 1. Variables and sources. Abbreviation Variables Source QR Code Dependent variable DOAP Deaths from outdoor air pollution (%) Our World in Data [3] Independent variables EVs The total number of electric vehicles registered in the fleet. This variable is the sum of BEVs, PHEVs, electric buses (EBUSS), electric light commercial vehicles (ELCVs), and electric heavy-duty (EHD) vehicles. Moreover, BEVs and PHEVs are passenger vehicles. European Alternative Fuels Observatory (EAFO) [11] GDP Gross domestic product (GDP) per capita (con- stant = 2010 $). World Bank Data (WBD) [28] RENE Renewable energy consumption per capita WBD [28] PM2.5 Particulate matter (micrograms per cubic metre) until 2019. Eurostat [29] RENE Renewable energy consumption per capita WBD [28] Table 1. Variables and sources. Abbreviation Variables Source QR Code Dependent variable DOAP Deaths from outdoor air pollution (%) Our World in Data [3] Independent variables EVs The total number of electric vehicles registered in the fleet. This variable is the sum of BEVs, PHEVs, electric buses (EBUSS), electric light commercial vehicles (ELCVs), and electric heavy-duty (EHD) vehicles. Moreover, BEVs and PHEVs are passenger vehicles. European Alternative Fuels Observatory (EAFO) [11] GDP Gross domestic product (GDP) per capita (con- stant = 2010 $). World Bank Data (WBD) [28] RENE Renewable energy consumption per capita WBD [28] PM2.5 Particulate matter (micrograms per cubic metre) until 2019. Eurostat [29] PM2.5 Particulate matter (micrograms per cubic metre) until 2019. Eurostat [29] Table 1. Variables and sources. Abbreviation Variables Source QR Code Dependent variable DOAP Deaths from outdoor air pollution (%) Our World in Data [3] Independent variables EVs The total number of electric vehicles registered in the fleet. This variable is the sum of BEVs, PHEVs, electric buses (EBUSS), electric light commercial vehicles (ELCVs), and electric heavy-duty (EHD) vehicles. Moreover, BEVs and PHEVs are passenger vehicles. European Alternative Fuels Observatory (EAFO) [11] GDP Gross domestic product (GDP) per capita (con- stant = 2010 $). World Bank Data (WBD) [28] RENE Renewable energy consumption per capita WBD [28] PM2.5 Particulate matter (micrograms per cubic metre) until 2019. Eurostat [29] Table 1. Variables and sources. Table 1. Variables and source Table 1. Variables and source Table 1. 3.1. Data Variables and source Table 1. Variables and source Table 1. Variables and source World Electr. Veh. J. 2022, 13, 155 RENE RENE 8 of 21 Table 1. Cont. Abbreviation Variables Source QR Code CH4 Methane emissions from the transportation sector in a tonne. Eurostat [29] PM2.5 Particulate matter (micrograms per cubic metre) until 2019. Eurostat [29] CH4 Methane emissions from the transportation sec- tor in a tonne. Eurostat [29] N2O Nitrous oxide emissions from the transportation sector in a tonne. Eurostat [29] Notes: This was created by the authors N2O Nitrous oxide emissions from the transportation sector in a tonne. Eurostat [29] PM2.5 Particulate matter (micrograms per cubic metre) until 2019. Eurostat [29] CH4 Methane emissions from the transportation sec- tor in a tonne. Eurostat [29] N2O Nitrous oxide emissions from the transportation sector in a tonne. Eurostat [29] Notes: This was created by the authors. Notes: This was created by the authors. Table 1. Cont. ticulate matter ticulate matter The variable DOAP will be the dependent or explained variable of the econometri approach, while EVs, GDP, RENE, PM2.5, CH4, and N2O are the independent or explana tory variables. Furthermore, the variables GDP, RENE, PM2.5, CH4, and N2O are also th control variables in the econometric model (as shown in Figure 5 below). The variable DOAP will be the dependent or explained variable of the econometri approach, while EVs, GDP, RENE, PM2.5, CH4, and N2O are the independent or explana tory variables. Furthermore, the variables GDP, RENE, PM2.5, CH4, and N2O are also th control variables in the econometric model (as shown in Figure 5 below). The variable DOAP will be the dependent or explained variable of the econometric approach, while EVs, GDP, RENE, PM2.5, CH4, and N2O are the independent or explana- tory variables. Furthermore, the variables GDP, RENE, PM2.5, CH4, and N2O are also the control variables in the econometric model (as shown in Figure 5 below). 9 of 23 Figure 5 Venn diagram with variables of the model The authors created this figure Figure 5. Venn diagram with variables of the model. The authors created this ﬁgure. Fi 5 V di i h i bl f h d l Th h d hi fi Figure 5. Venn diagram with variables of the model. The authors created this ﬁgure. g g g Koengkan et al. 3.2. Method Approach 3.2. Method Approach This empirical investigation will follow the methodological strategy used by several authors (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]; and Koengkan et al. [12]). Figure 6 This empirical investigation will follow the methodological strategy used by several authors (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]; and Koengkan et al. [12]). Figure 6 below shows the methodology strategy this investigation will follow. below shows the methodology strategy this investigation will follow. After presenting the methodology strategy that this investigation will follow, it is also necessary to present the preliminary tests that will be computed before the MM-QR and OLS with ﬁxed effects models. 3.1. Data [12] used these control variables before, investigating the impact of renewable energy policies on outdoor and indoor air pollution deaths in Latin American and Caribbean countries. Indeed, the variable EVs was not addressed by the literature. This investigation is innovative because it is the first time this variable has been used to explain the variable premature deaths from outdoor air pollution. The following subsec- ti ill h th th d l i l h th t thi i ti ti ill Koengkan et al. [12] used these control variables before, investigating the impact of renewable energy policies on outdoor and indoor air pollution deaths in Latin American and Caribbean countries. Indeed, the variable EVs was not addressed by the literature. This investigation is innovative because it is the ﬁrst time this variable has been used to explain the variable premature deaths from outdoor air pollution. The following subsection will show the methodological approach that this investigation will use. 3.2.1. Preliminary Tests The preliminary tests must be computed before estimating the MM-QR and OLS with ﬁxed effects models. Thus, in this empirical investigation, the following tests will be used, such as (a) the Shapiro–Francia test [30] to verify the presence of normality in the panel data; (b) the Shapiro–Wilk test [31] to verify the presence of normality in the panel data; (c) the variance inﬂation factor (VIF) test [32] to identify the presence of multicollinearity between the variables in the econometric models; (d) a cross-sectional dependence (CSD) test [33] to identify the presence of cross-sectional dependence in the variables; (e) a homogeneity World Electr. Veh. J. 2022, 13, 155 9 of 21 ature. d t 9 of 21 ature. d t slope test [34] to assess the presence of slope homogeneity in the model; (f) a panel unit root test (CIPS) [35] to assess the presence of unit roots in the variables of the model; (g) the Westerlund cointegration test [36] to identify the presence of cointegration in the stationary variables; and (h) the Hausman test [37] to identify the presence of random effects or ﬁxed effects. Moreover, all these preliminary tests were used before by Kazemzadeh et al. [9], Fuinhas et al. [10], and Koengkan et al. [12]. tion will show the methodological approach that this investigation will use. 3.2. Method Approach This empirical investigation will follow the methodological strategy used by several authors (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]; and Koengkan et al. [12]). Figure 6 below shows the methodology strategy this investigation will follow. Figure 6. Methodology strategy. The authors created this figure. Figure 6. Methodology strategy. The authors created this ﬁgure. Figure 6. Methodology strategy. The authors created this figure. Figure 6. Methodology strategy. The authors created this ﬁgure. 3.2.2. Method of Moments Quantile Regression (MM-QR) The theory related to quantile regression was developed by Koenker and Bassett [38], who extended knowledge of ordinary quantiles in the location model to a more general class of linear models in which conditional quantiles have a linear form. Median regression is the most generic form and aims to estimate the median of the dependent variable, conditioned to the value of the independent variables. The remaining conditional quantiles are estimated by minimizing an asymmetrically weighted sum of absolute errors. Furthermore, the quantile regression method characterizes the entire conditional distribution of a dependent variable given a set of regressors. The impact of the electriﬁcation of the transport sector on premature deaths from outdoor air pollution in the EU-27 countries, plus the United Kingdom and Norway, were examined in this study, employing the method of moments quantile regression (MM-QR) with panel ﬁxed effects, as proposed by Machado and Silva [39]. The MM-QR method is different from the traditional method introduced by Koenker and Bassett [38], as it is based on conditional means. However, the authors point out that the MM-QR method recognizes the same conditional quantiles and is robust. Furthermore, unlike the panel quantile regressions discussed in the works by Canay [40], Lamarche [41], and Koenker [42], Machado and Silva [39] used the MM-QR method with ﬁxed effects. Therefore, with this method, it is possible to capture the unobserved distribution heterogeneity between countries within a panel. The MM-QR model proposed by Machado and Silva [39] uses the conditional scale function to estimate regression quantiles. In addition, differences can be noted through the parameters estimated for each quantile. Furthermore, the MM-QR technique has some advantages compared to other methods. It provides information about how the explanatory World Electr. Veh. J. 2022, 13, 155 10 of 21 10 of 21 variables can inﬂuence the entire conditional distribution and, thus, enables the use of valid methods only in the estimation of conditional means, such as when it is difﬁcult to perceive heterogeneity and endogeneity problems in panel data models. Additionally, it identiﬁes individual effects in panel data models while providing information about how the conditional distribution is affected by regressors, while also allowing the estimation of regression quantiles not to cross, which is an important assumption but is not considered in empirical applications. In addition, Fuinhas et al. 3.2.2. Method of Moments Quantile Regression (MM-QR) [10] emphasize that the MM-QR method considers that the covariate affects only the variable of interest through the location channel and scale functions regarding a mere location shift. Thus, it is possible to examine the effects of conditional heterogeneous covariance on the determinants of deaths from outdoor air pollution at different distribution quantiles for the countries analyzed in this work. Based on the work of Machado and Silva [39], it is possible to perform a location- scale model in which Equation (1) follows the following form to estimate the conditional quantiles QDOAP(τ\|Xit) : DOAPit = αi + X′ itβ + i + Z′ it γ Uit (1) (1) where probability, Pr δi + Z′ it γ > 0 = 1 and the unknown parameters (αi , δi), i = 1, 2, 3, . . . , n describe individual i fixed effects, and Z is a known differentiable (with Pr = 1) transforma- tion of the elements of X. Thus, Xit is identically and independently distributed. Furthermore, Xit is independent across time (t). In addition, Uit is the error term which is independently and identically distributed over individuals (i) across time (t), and is statistically independent of Xit. Equation (1) can be expanded, as follows: QDOAP (τ\|Xit) = (αi + δi q (τ)) + X′ it β + Z′ it γq (τ) (2) (2) From Equation (2), αi (τ) = αi + δi q (τ) is the scalar coefﬁcient, which represents the quantile τ ﬁxed effect for an individual country or distributive effect on τ. From Equation (2), αi (τ) = αi + δi q (τ) is the scalar coefﬁcient, which represents the quantile τ ﬁxed effect for an individual country or distributive effect on τ. Finally, it is important to emphasize that according to Fuinhas et al. [10], the distribu- tive impact varies from the classic ﬁxed effect, as it is not location ﬁxed. Additionally, Machado and Silva [39] corroborate that the impact of the distribution shows the time- invariant traits that allow other variables to have different effects in the analyzed countries. .3. Ordinary Least Squares (OLS) with Fixed Effects 3.2.3. Ordinary Least Squares (OLS) with Fixed Effects The ﬁxed effects model can be considered as a generalization of a constant inter- cept/slope model for the panel, including a dummy variable for the effects of omitted variables, which remain constant over time. This way, individual effects can be freely correlated with the other regressors. In addition, Fuinhas et al. [10] highlight that the ordinary least squares with ﬁxed effects can estimate the slope and intercepts for a set of observations, and it is also possible to estimate the average response for the ﬁxed predictors. p g p p That said, the OLS with ﬁxed effects addressed in this study can be represented as follows in Equation (3): LDOAPit = β0 + β1LGDPit + β2LEVsit + β3LRENEit + β4LPM2.5it +β5LCH4it + β6LN2Oit + εit (3) (3) In this model, β0 is the intercept, and β is the value of ﬁxed covariates being ﬁtted to predict the dependent variable LDOAPit, εi is the error term, and each variable enters regression for country i at year t. Furthermore, according to Fuinhas et al. [10], the OLS model shows the relationship between the covariates, but it cannot be extended to non- central locations in the case of shapeshifts and is inﬂuenced by outliers. World Electr. Veh. J. 2022, 13, 155 d El t V h J 2022 13 155 11 of 21 11 of 21 Figure 7. QR code—all commands of Stata that were used in this in Figure 7. QR code—all commands of Stata that were used in this investigation. 4. Empirical Results Moreover, this investigation computed all preliminary tests and model estimations us- ing Stata 17.0. The following section will present the empirical results of this investigation. This section the results of the 4. Empirical Results the results of the preliminary tests, and the second subsection contains the MM-QR and OLS fixed effects models. This section consists of the following two subsections: the ﬁrst subsection contains the results of the preliminary tests, and the second subsection contains the results of the MM-QR and OLS ﬁxed effects models. Figure 7. QR code—all commands of Stata that were used in this in Figure 7. QR code—all commands of Stata that were used in this investigation. 3.2.4. Stata Commands After presenting the preliminary tests and the MM-QR and OLS with ﬁxed effects models, it is necessary to display the Stata commands used in this empirical investigation. Table 2 below shows the Stata commands of preliminary tests and model estimations. piro–Wilk test Swilk lation factor (VIF) test Vif Table 2. Stata commands. p ( ) genity slope test Table 2. Stata commands. Preliminary Tests Test Stata command Descriptive statistics of variables sum (a) Shapiro–Francia test Sfrancia (b) Shapiro–Wilk test Swilk (c) Variance inﬂation factor (VIF) test Vif (d) Cross-sectional dependence (CSD) test xtcd (e) Homogenity slope test xthst (f) Panel unit root test (CIPS) Multipurt (g) Westerlund cointegration test xtwest, with option constant (h) Hausman test Hausman, with option sigmamore MM-QR and OLS with ﬁxed effects models MM-QR xtqreg, quantile (0.10 0.25 0.5 0.75 0.90) OLS with ﬁxed effects xtreg, fe Notes: This was created by the authors. p ( ) ogenity slope test xthst nit root test (CIPS) Multipurt nd cointegration test xtwest, with option constant ausman test Hausman, with option sigmamor MM-QR and OLS with fixed effects models MM-QR xtqreg, quantile (0.10 0.25 0.5 0.75 0 h fixed effects xtreg, fe Notes: This was created by the authors. All Stata commands used in this empirical investigation and model r well as information on how to conduct each test, can be found in the QR Co Figure 7). Moreover this investigation computed all preliminary tests and mod All Stata commands used in this empirical investigation and model regressions, as well as information on how to conduct each test, can be found in the QR Code below (see Figure 7). using Stata 17.0. The following section will present the empirical results of tion. 4.1. Preliminary Te Preliminary 4.1. Preliminary Tests Preliminary tests are required before estimating the model. The prel mentioned in the methodology part are given in this section. The first step i statistical characteristics of the data/variables (as shown in Table 3 below). T ber of observations is 319; the mean, standard deviation, minimum, and m premature pollution due to outdoor pollution (LDOAP) are 2.98, 0.47, 1.77 Preliminary tests are required before estimating the model. The preliminary tests men- tioned in the methodology part are given in this section. The first step is to check the statistical characteristics of the data/variables (as shown in Table 3 below). The total number of observa- tions is 319; the mean, standard deviation, minimum, and maximum for premature pollution due to outdoor pollution (LDOAP) are 2.98, 0.47, 1.77, and 3.89, respectively. World Electr. Veh. J. 2022, 13, 155 12 of 21 Table 3. Descriptive statistics of variables. Variables Descriptive Statistics Obs. Mean Std.-Dev. Min. Max. LDOAP 319 2.983314 0.4757265 1.774952 3.89182 LGDP 319 10.58265 0.3718944 9.766579 11.64343 LRENE 319 1.822005 1.691715 −2.302585 5.44846 LEVs 319 6.768906 2.622617 0.6931472 12.93599 LCH4 319 6.517416 2.081914 2.320425 11.71836 LN2O 319 5.763331 1.282468 1.819699 8.332102 LPM2.5 319 2.579939 0.4364631 1.526056 3.720862 Notes: Here, Obs. is the number of observations in the model, Std.-Dev. is the standard deviation, and Min and Max are the minimum and maximum. The first preliminary test required in the MM-QR model is to check the data normality. The MM-QR model is used to investigate the effects in different quantiles, and this model is applied when the data distribution is abnormal. On the other hand, using this model does not make sense if the data is normal. For this purpose, two Shapiro–Wilk and Shapiro–Francia tests have been used to check normality. Table 4 below shows that all variables reject the null hypothesis that the data is normal. Therefore, the MM-QR model can be applied. Table 4. Normal distribution tests. Table 4. Normal distribution tests. Table 4. Normal distribution tests. Variables Shapiro–Wilk Test Shapiro–Francia Test Obs Statistic Statistic LDOAP 0.97456 * 0.97687 * 319 LGDP 0.96862 * 0.96983 * 319 LRENE 0.98483 * 0.98715 * 319 LEVs 0.98656 * 0.98894 319 LCH4 0.97264 * 0.97473 * 319 LN2O 0.98324 * 0.98451 * 319 LPM2.5 0.98005 * 0.98154 * 319 Notes: *, denote statistical signiﬁcance at the 1% and 5% levels, respectively. The next test is the variance inﬂation factor (VIF), examining multicollinearity. 4.1. Preliminary Te Preliminary 4.1. Preliminary Tests Table 5, below, shows the VIF test results. Table 5. VIF test. Table 5. VIF test. Independent Variables Dependent Variable (LDOAP) VIF-Test VIF Mean VIF LGDP 1.64 2.43 LRENE 3.69 LEVs 2.12 LCH4 1.52 LN2O 3.99 LPM2.5 1.61 Notes: This was created by the authors. The model does not have multicollinearity if the VIF value of the variables is less than 10 and the average VIF value is less than 6. Therefore, as shown in Table 5 above, the absence of multicollinearity is conﬁrmed. The next step is to examine the cross-sectional dependence and homogeneity slope test (HS test). The Pesaran cross-sectional dependence CD test [33] and the HS test [34] have been applied in this research. The null hypothesis in these two tests is that there is no World Electr. Veh. J. 2022, 13, 155 13 of 21 13 of 21 cross-sectional dependence and no existence of a homogeneity slope. Table 6, above, shows that the model variables reject the null hypothesis in the CD test, indicating cross-sectional dependencies. Likewise, the HS test results also reject the null hypothesis and indicate the model has a heterogeneity slope. The following preliminary test is to examine the panel unit root test. Table 7 below shows the results of the Pesaran CIPS test [35]. Table 6. Pesaran CD and homogeneity slope tests. Pesaran CD Test Variables CD Statistics p-Value LDOAP 60.68 * 0.000 LGDP 50.14 * 0.000 LRENE 52.84 * 0.000 LEVs 53.70 * 0.000 LCH4 6.06 * 0.000 LN2O 18.15 * 0.000 LPM2.5 29.22 * 0.000 Homogeneity Slope test Models Delta Adjusted Delta Model~LDOAP 2.840 * 3.096 * Notes: * denotes statistical signiﬁcance at the 1% level. Table 7. Panel unit root test. Variables Panel Unit Root Test (CIPS) (Zt-Bar) Without Trend With Trend Lags Adjusted t Adjusted t LDOAP 1 −6.606 * −4.738 * LGDP 1 −8.144 * −6.487 * LRENE 1 −7.413 * −6.090 * LEVs 1 −7.390 * −6.306 * LCH4 1 −6.726 * −5.440 * LN2O 1 −9.201 * −7.866 * LPM2.5 1 −9.345 * −8.538 * Notes: * denotes statistically signiﬁcant at the 1% level. Table 6. Pesaran CD and homogeneity slope tests. 4.2. MM-QR and OLS with Fixed Effects Results In this part of the study, MM-QR and OLS with ﬁxed effects models have been applied to estimate the effects of road transport electriﬁcation on mortality due to air pollution. Quantiles 10th, 25th, 50th, 75th, and 90th are considered for the MM-QR model, and OLS with ﬁxed effects has been used to check the robustness of the model. Table 10 shows the model’s estimation results. Table 10. Estimation results from the MM-QR regression model and OLS with ﬁxed effects. Table 10. Estimation results from the MM-QR regression model and OLS with ﬁxed effects. Independent Variables Main Method Robustness Check MM-QR OLS Dependent Variable (LDOAP) Quantiles Fixed Effects 10th 25th 50th 75th 90th LGDP −0.4060 * −0.3719 * −0.3533 * −0.3270 * −0.3161 * −0.2546 * LRENE −0.0609 −0.0574 * −0.0555 * −0.0528 * −0.0517 * −0.0471 * LEVs −0.0566 * −0.0494 * −0.0454 * −0.0398 * −0.0375 * −0.0792 *** LCH4 −0.0088 −0.0167 * −0.0211 −0.0272 * −0.0297 *** −0.0097 * LN2O 0.0578 0.0761 * 0.0861 * 0.1002 * 0.1060 * 0.1024 * LPM2.5 0.5371 * 0.5345 * 0.5331 * 0.5311 * 0.5302 * 0.5423 * constant 5.7822 * 5.5279 * 5.3895 * 5.1938 * 5.1128 * 4.3495 * Notes: , , denote statistical signiﬁcance at the 1%, 5%, and 10% levels, respectively. Table 10 shows that economic growth (LGDP), renewable energy consumption (LRENE), and electric vehicles (LEVs) in all quantiles (10th, 25th, 50th, 75th, and 90th) have significant negative effects on premature mortality due to air pollution (LDOAP). As can be seen, the effects of LGDP, LRENE, and LEVs are higher on LDOAP in lower quantiles. This effect gradually decreases with increasing quantile levels. These results show that economic growth, renewable energy consumption, and electric vehicles will reduce premature deaths from air pollution. The results also show that methane emissions (LCH4) in all quantiles, except the 10th, significantly negatively affects LDOAP. On the other hand, nitrous oxide emissions (LN2O) and particulate matter (LPM2.5) positively affect LDOAP. Indeed, LN2O has a signifi- cant positive effect on LDOAP in all except the 10th quantile, and this effect is greater at high quantiles. The LPM2.5 has positive and significant effects on premature mortality (LDOAP) in all quantiles, and this effect is almost the same at all levels. Furthermore, OLS with fixed effects is applied to evaluate the robustness of the model. 4.1. Preliminary Te Preliminary 4.1. Preliminary Tests Pesaran CD Test Variables CD Statistics p-Value LDOAP 60.68 * 0.000 LGDP 50.14 * 0.000 LRENE 52.84 * 0.000 LEVs 53.70 * 0.000 LCH4 6.06 * 0.000 LN2O 18.15 * 0.000 LPM2.5 29.22 * 0.000 Homogeneity Slope test Models Delta Adjusted Delta Model~LDOAP 2.840 * 3.096 * Notes: * denotes statistical signiﬁcance at the 1% level. Table 6. Pesaran CD and homogeneity slope tests. Table 7. Panel unit root test. Variables Panel Unit Root Test (CIPS) (Zt-Bar) Without Trend With Trend Lags Adjusted t Adjusted t LDOAP 1 −6.606 * −4.738 * LGDP 1 −8.144 * −6.487 * LRENE 1 −7.413 * −6.090 * LEVs 1 −7.390 * −6.306 * LCH4 1 −6.726 * −5.440 * LN2O 1 −9.201 * −7.866 * LPM2.5 1 −9.345 * −8.538 * Notes: * denotes statistically signiﬁcant at the 1% level. The null hypothesis in this test is the existence of a panel unit root. As shown in Table 7 above, the panel unit root test results are presented with one lag and with and without a trend. As can be seen, all variables—LDOAP, LGDP, LRENE, LEVs, LCH4, LN2O, and LPM2.5—reject the null hypothesis at the 1% signiﬁcant level for both the with and without a trend modes. Moreover, these results conﬁrm the stationary nature of the variables. The cointegration test is performed after the unit root test (as shown in Table 8 below). Table 8. Westerlund panel cointegration test of all variables of the model. Table 8. Westerlund panel cointegration test of all variables of the model. Variables LDOAP, LGDP, LRENE, LEVs, LCH4, LN2O, and LPM2.5 Statistic Value Z-Value Robust p-Value Gt −2.404 1.489 0.260 Ga −7.107 3.714 0.180 Pt −7.426 1.040 0.140 Pa −6.586 2.480 0.110 Notes: Gt and Ga test the cointegration for each country individually, and Pt and Pa test the panel cointegration. As shown in Table 8 above, the Westerlund panel cointegration test was applied to investigate panel cointegration. The results of this study reject the null hypothesis that World Electr. Veh. J. 2022, 13, 155 14 of 21 14 of 21 there is no cointegration. Therefore, it can be stated that there is a long-term relationship between the independent variables and premature mortality due to air pollution. The last preliminary test applied in this study is the Hausman test. This test is used to check for the presence of panels with random effects versus ﬁxed effects. 4.1. Preliminary Te Preliminary 4.1. Preliminary Tests The null hypothesis is the existence of a panel with random effects. Table 9 below shows the results from the Hausman test. Table 9. Hausman test. Models Chi2(5) Prob. Model—LDOAP 19.63 * 0.0032 Notes: *** denotes statistically signiﬁcant at the 1% level. Table 9. Hausman test. As shown in Table 9 above, the null hypothesis is rejected. For this purpose, a panel with ﬁxed effects is used to estimate the model. Therefore, the MM-QR and OLS with ﬁxed effects models were used after the preliminary tests. Hence, the nega tion of transport 5. Discussions tion of transport sector) on the dependent variable (LDOAP) could be associated with the capacity of the electrification of the transport sector to mitigate the consumption of energy from fossil fuels and, consequently, air pollution, such as CO2 emissions and PM2.5 in European countries. Reducing the consumption of non-renewable energy sources and air pollution via the electrification of the transport sector could help to reduce premature deaths from outdoor air pollution. Indeed, some authors in the medicine area, such as Horton et al. [22] and Choma et al. [23] have indicated that the mitigation of nitric oxide and fine particulate matter by heavy-duty electric vehicles results in a reduction in prem- ature deaths caused by outdoor air pollution. In the literature, evidence that the electrification of the transport sector can mitigate the consumption of energy and also air pollution (e.g., CO2 emissions, PM2.5, and GHGs) f d b th ( K d h t l [9] F i h t l [10] d K k This section focuses on discussing the results found in this empirical investigation. Hence, the negative impact of the independent variable (LEVs) (a proxy of the electriﬁcation of transport sector) on the dependent variable (LDOAP) could be associated with the capacity of the electriﬁcation of the transport sector to mitigate the consumption of energy from fossil fuels and, consequently, air pollution, such as CO2 emissions and PM2.5 in European countries. Reducing the consumption of non-renewable energy sources and air pollution via the electriﬁcation of the transport sector could help to reduce premature deaths from outdoor air pollution. Indeed, some authors in the medicine area, such as Horton et al. [22] and Choma et al. [23] have indicated that the mitigation of nitric oxide and ﬁne particulate matter by heavy-duty electric vehicles results in a reduction in premature deaths caused by outdoor air pollution. was found by some authors (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]; and Koengkan et al. [12]). Kazemzadeh et al. [9] said that the capacity of electric vehicles to reduce energy In the literature, evidence that the electrification of the transport sector can mitigate the consumption of energy and also air pollution (e.g., CO2 emissions, PM2.5, and GHGs) was found by some authors (e.g., Kazemzadeh et al. [9]; Fuinhas et al. [10]; and Koengkan et al. [12]). Kazemzadeh et al. 4.2. MM-QR and OLS with Fixed Effects Results For this purpose, the results of OLS are compared with the 50th quantile [2]. The OLS results confirm that economic growth, renewable energy consumption, and methane emissions significantly reduce mortality due to outdoor air pollution, while LN2O and LPM2.5 emissions increase LDOAP. p Moreover, Figure 8 below summarises the impact of independent variables on the dependent ones. This ﬁgure was based on results from Table 10 above. World Electr. Veh. J. 2022, 13, 155 15 of 21 ons for Figure 8. Summary of each variable’s effect. The authors created this figure. Figure 8. Summary of each variable’s effect. The authors created this ﬁgure. Figure 8. Summary of each variable’s effect. The authors created this figure. Figure 8. Summary of each variable’s effect. The authors created this ﬁgure. 5. Discussions This section focuses on discussing the results found in this empirical investigation. The following section will present the discussions and the possible explanations for the results. Hence, the nega tion of transport 5. Discussions [9] said that the capacity of electric vehicles to reduce energy consumption and air pollution could be related to increasing energy efficiency. The introduction of new materials, electric motors, and improved batteries led electric vehicles to consume less electricity. World Electr. Veh. J. 2022, 13, 155 16 of 21 16 of 21 Therefore, consistent with the same authors, the increase in the energy efficiency of electric vehicles will mitigate the final electricity consumption. Evidence that electric cars reduce energy consumption was found by Koengkan et al. [12], which studied the impact of BEVs on final energy consumption in 29 European countries between 2010–2020. The authors pointed out that this reduction is due to the BEVs becoming more energy-efficient than conventional cars. The same authors suggest that reducing final energy consumption by BEVs will also reduce the consumption of fossil fuels and, consequently, air pollution. According to Fuinhas et al. [10], the energy consumption from electric vehicles decreased from 264 Wh/km to 150 Wh/km. Therefore, this is an indication that electric cars have become more efficient. Moreover, the same authors add that electric vehicles’ energy consumption will be 0.10 (kWh/km) between 2016–2030. Therefore, the explanation given by Fuinhas et al. [10] confirms the elucidations of Kazemzadeh et al. [9] and Koengkan et al. [12]. Although this investigation has found that the process of the electriﬁcation of the transport sector can help to mitigate premature deaths from outdoor air pollution, the impact of electriﬁcation is negligible. It is possible that the low effect of the independent variable (LEVs) on the dependent one could be related to the high participation of fossil fuels in the energy matrix in the EU countries, which consequently limits the possible effect of electric vehicles on premature deaths from outdoor air pollution. This is because the electricity consumed by electric cars comes from power stations burning fossil fuels. The high participation of fossil fuels in the energy matrix in the European countries is conﬁrmed by Europa [8], which found that, in 2019, 39% of the electricity consumed came from power stations burning fossil fuels, 35% came from renewable energy sources, and 26% came from nuclear power plants. Another possible explanation for the low impact of the independent variable could be correlated to the low participation of electric vehicles in the ﬂeets of the studied countries. Hence, the nega tion of transport 5. Discussions Indeed, the clarification that was given above by Koengkan et al. [2] helps to explain the negative impact of the independent variable (LRENE) on the dependent variable (LDOAP). Therefore, this negative impact was also found by Koengkan et al. [2], Koengkan et al. [43], Koengkan et al. [44]; and Fuinhas et al. [45]. It is related to the increase of initiatives related to the decarbonisation of the economy (e.g., energy transition). For example, in the case of the EU-28, the share of energy from renewable sources in the final consumption of energy in 2004 was 8.5% and, in 2020, this value reached 22.4% [7]. In this period, the participation of renewable energy sources had a growth rate of more than Moreover, Koengkan et al. [2] state that this negative impact of GDP also could be associated with the process of the decarbonisation of the economy with the adoption of initiatives that encourage the process of energy transition from fossil fuels to renewable energy sources, the electriﬁcation of urban transport, and the development and commer- cialisation of technologies with high energy efﬁciency. All these will mitigate air pollution and, consequently, premature deaths. As mentioned in the introduction section, the EU countries have been adopting several initiatives to decarbonise their economies, such as encouraging the process of energy transition and electriﬁcation of urban transport. p p p gy g 160%. Therefore, although this investigation has found that renewable energy consump- tion can help to mitigate premature deaths from outdoor air pollution, its impact is negli- gible. Indeed, the low effect of this variable could be related to the timid participation of Indeed, the clariﬁcation that was given above by Koengkan et al. [2] helps to explain the negative impact of the independent variable (LRENE) on the dependent variable (LDOAP). Therefore, this negative impact was also found by Koengkan et al. [2], Koengkan et al. [43], Koengkan et al. [44]; and Fuinhas et al. [45]. It is related to the increase of initiatives related to the decarbonisation of the economy (e.g., energy transition). For example, in the case of the EU-28, the share of energy from renewable sources in the ﬁnal consumption of energy in 2004 was 8.5% and, in 2020, this value reached 22.4% [7]. In this period, the participation of renewable energy sources had a growth rate of more than 160%. Hence, the nega tion of transport 5. Discussions As the introduction also states, in Europe, electric vehicles represented a market share of only 3.5% in 2019. The BEVs accounted for 2.19%, while the PHEVs represented 1.21% [11]. Therefore, the low participation of electric vehicles in the ﬂeet causes a limited effect on combating the increase of gases that cause premature deaths from outdoor air pollution. While this investigation has presented possible explanations regarding the impact of trans- port electriﬁcation on premature deaths from air pollution, it is necessary to realise more studies on this topic. Furthermore, as mentioned in the introduction section, this is the ﬁrst investigation that uses macroeconomic data to approach this topic, which means that this investigation is in the initial stage of maturity. Consequently, this investigation elaborated the following Figure 9 to summarise the aforementioned possible explanations. g g p p Regarding the negative impact of the independent variable (LGDP) on the dependent variable (LDOAP), this was also found in similar studies (e.g., Koengkan et al. [2], and Koengkan et al. [42]). Koengkan et al. [42] studied the effect of renewable energy consump- tion on outdoor air pollution death rates in Latin America and the Caribbean region. The capacity of the independent variable (LGDP) to mitigate premature deaths from outdoor air pollution could be related to the increased investment in the health systems caused by economic development. According to the authors, economic growth can reduce death rates because it allows for the ﬁnancing of better health systems. Indeed, this effect of eco- nomic growth on improving health systems can lower the mortality rate from air pollution. In the EU, for example, the healthcare expenditure amounted to 9.9% of GDP in 2018, and in 2020 this value reached 11% of GDP. Among the EU member states, the most signiﬁcant shares were recorded in Germany (11.5% of GDP) and France (11.3%), followed by Sweden (10.9%). In contrast, Luxembourg is the lowest healthcare expenditure shares (5.3% of GDP), while Romania spends 5.6% of GDP [6]. 17 of 21 the EU such as 17 of 21 the EU such as World Electr. Veh. J. 2022, 13, 155 g g p gy p Figure 9. Summary of linkages. The authors created this figure. Figure 9. Summary of linkages. The authors created this ﬁgure. g g p gy p Figure 9. Summary of linkages. The authors created this figure. Figure 9. Summary of linkages. The authors created this ﬁgure. Hence, the nega tion of transport 5. Discussions Therefore, although this investigation has found that renewable energy consumption can help to mitigate premature deaths from outdoor air pollution, its impact is negligible. Indeed, the low effect of this variable could be related to the timid participation of green energy sources in the energy matrix in European countries, consequently limiting their World Electr. Veh. J. 2022, 13, 155 18 of 21 18 of 21 impact on the dependent variable. Therefore, the low renewable energy participation in the energy matrix causes a limited effect on combating the increase of gases that cause premature deaths from outdoor air pollution. p p Finally, the positive impact of the independent variables (LN2O and LPM2.5) on the dependent variable (LDOAP) was not found in the literature. Indeed, the possible explanation for this impact could be linked to the low participation of green energy sources in the energy matrix. As mentioned above, in the EU, the share of renewable energy sources in the energy matrix is only 22.4%. Therefore, the low percentage of renewable energy in the energy matrix limits green energy sources’ capacity to mitigate the N2O and PM2.5 in some sectors. The low impact of renewable energy sources on premature deaths from outdoor air pollution reﬂects this limitation. Moreover, the low participation of electric vehicles in the ﬂeet in the EU countries also limits the capacity of the electriﬁcation of road transport to mitigate these gases mentioned above. Additionally, the low impact of electriﬁcation of road transport on premature deaths from outdoor air pollution reﬂects this limitation. However, it is necessary to realise more studies to conﬁrm these impacts and explanations. The negative effect of the independent variable (LCH4) on the dependent variable (LDOAP) was also not found in the literature. The possible reason for this impact could be associated with several initiatives that the EU countries have adopted in the last years to mitigate methane emissions. Therefore, the EU countries have set out actions across different sectors, focusing on energy, agriculture, and waste to mitigate the CH4. In the EU, 53% of anthropogenic methane emissions come from the agricultural sector, 26% come from waste, and 19% come from energy [8]. Indeed, to mitigate the methane emissions from the agriculture sector, the EU has been promoting sustainable livestock management, including circular approaches, supporting feed additives, and promoting sustainable and balanced diets with less red meat. Hence, the nega tion of transport 5. Discussions From the waste sector, the EU has been encouraging landﬁll operators to manage landﬁll gas, for example, by using it to generate energy. Furthermore, in the energy sector, the EU has gradually abandoned the use of fossil fuels [8]. However, although CH4 can mitigate premature deaths from outdoor air pollution, its impact is negligible. The probable reason for this small impact could be related to the speed at which each country in the EU adopts these initiatives to mitigate methane emissions. This section has presented the possible explanations for the results found in the empir- ical analysis. Nevertheless, this analysis contributes to the literature with a macroeconomic analysis of the heterogeneous impact of the electriﬁcation of road transport on prema- ture deaths from outdoor air pollution. However, more studies are necessary to deepen knowledge about this topic. New macroeconomic studies should be directed to identify the relationship between the electriﬁcation of road transport and the consumption of renewable energy sources and air pollution reduction to conﬁrm the results found. The following section will present the conclusions and the policy implications. 6. Conclusions and Policy Implications The present study addressed the potential positive externalities of road electriﬁcation on the mitigation of premature deaths, a relationship that has not been explored by the social science literature to the best of our knowledge. The main argument of our proposal is that the reduction of air pollution—especially in urban areas, as already extensively reported by the literature—reduces human exposure to pollutants (e.g., CO, CO2, and NO2) that may trigger illnesses, such as respiratory diseases, and, therefore, will play a role in reducing deaths at premature ages. To tackle this issue, we estimated the following two models: a moments quantile regression (MM-QR) and an ordinary least squares (OLS) with ﬁxed effects. The goal of the models was to verify whether the penetration of electric vehicles (alongside other control variables for economic growth, renewable energy consumption, and air pollution) signiﬁcantly reduced premature mortality due to air pollution. The panel data included all EU-27 countries, plus the United Kingdom and Norway, from 2010 to 2020. World Electr. Veh. J. 2022, 13, 155 19 of 21 19 of 21 The results indicated that renewable energy consumption and the penetration of electric vehicles have a statistically signiﬁcant negative effect on premature deaths due to air pollution. This result corroborates our core hypothesis that road electriﬁcation might reduce premature mortality. However, as we expected, nitrous oxide and PM2.5 have a positive signiﬁcant coefﬁcient, indicating that a higher level of these pollutants—such as in highly urbanised and industrial areas—increases premature mortality. Although signiﬁcant, the coefﬁcients of both models indicated only a modest impact on these variables. However, one may notice that they point towards the expected direction, especially considering the low penetration of electric vehicles in the analysed period. p y p In short, our results reinforce the importance of designing policies for promoting road electriﬁcation. Following the Paris Agreement (2016), many countries around the globe committed to implementing policies to reduce emissions of pollutants and promote an active role in promoting energy transition. The plans to achieve these goals are expressed in the nationally determined contributions (INDC) submitted by countries as an expression of their will. In December 2020, the European Commission (EC), on behalf of the European Union, submitted an update of the EU NDC. 6. Conclusions and Policy Implications In the document, the EC commits to reducing their global emissions levels and, in particular, to cut the emissions of vehicles (light and heavy duty) by at least 30% before 2030. In addition, the document highlights Directive (EU) 2019/1161 (the promotion of clean and energy-efﬁcient road transport vehicles) as part of this plan. p p While the promotion of large-scale renewables and distributed electricity generation focuses on reducing global levels of greenhouse gas emissions—which, in the long term, may also impact health and premature deaths—local policies and standards could have local mid-run effects. The local effects include the reduction of direct exposure to pollutants, which can be particularly harmful to people who already live with respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD), especially those living near roads with high trafﬁc or in highly dense urban areas. In addition, further research could be carried out to identify whether the proximity of roads with heavy trafﬁc could receive a greater externality from the electriﬁcation of vehicles. Finally, the direct impact at the local level could be an incentive for synergies between climate change policies and health policies. The perceivable impacts on individual lives are important for increasing the acceptance of decarbonisation policies and costs. Our research raises the possibility and importance of dealing with both subjects together. 7. Limitations and Future Research The main limitations to the generalisation of results are twofold, namely (i) the short period under analysis (11 years), and (ii) the low penetration of electric vehicles. Further- more, deaths from outdoor pollution are essentially a local phenomenon. This characteristic introduces another limitation, as follows: aggregated data can blur the intensity of the link between the electriﬁcation of road transportation and premature mortality due to outdoor pollution. Indeed, aggregated data can mask very different situations. Finally, cross-validation is absent, given the novelty of the link between the penetration of electric vehicles in the literature and premature mortality. Indeed, this study was carried out in a set of mainly rich countries sharing a political framework prone to compromise. How does this phenomenon behave in transition economies? The logical step for further research is extending the analysis to cities and industrial zones. These places are particularly affected by outdoor pollution. Consequently, the electriﬁcation of the transportation sector is more pressing and can capture the proximity of pollution with premature mortality. In addition, changes in human behaviour, such as the expansion of tourism, inﬂuence exposure to outdoor pollution. These changes can be another promising ﬁeld of research. Replacing fuels used in internal combustion engines with green hydrogen and exploring the possibility of adding a percentage of hydrogen to gas vehicles are other possibilities of research in the ﬁeld of premature mortality due to outdoor pollution. World Electr. Veh. J. 2022, 13, 155 20 of 21 20 of 21 Recent years have seen intense technological progress in batteries and the efﬁciency of electric vehicles. These factors, coupled with approaching technological maturity, are impacting outdoor pollution in many ways that are not just due to electric vehicles. Disen- tangling these individual contributions can shed light on other dimensions of the linkage between the electriﬁcation of societies and premature mortality due to pollution. Finally, improving the quality of econometric models by including other variables that help to identify transport electriﬁcation’s contribution to premature mortality due to outdoor pollution, or exploring whether the variables have different parameters depending on the quantiles of premature mortality, or else if the coefﬁcients themselves evolve, is a vast area for further research. Author Contributions: E.K.: Writing—Original draft, and Investigation. M.K.: Conceptualisation, Writing—Original draft, Supervision, Validation, Data curation, Investigation, Formal analysis, and Visu- alisation. J.A.F.: Writing—review and editing, Supervision, Funding acquisition, Project administration, and Investigation. M.T.: Writing—Original draft, and Investigation. 7. Limitations and Future Research A.M.: Writing—Original draft, and Investigation. All authors have read and agreed to the published version of the manuscript. Funding: CeBER: R&D unit funded by national funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., project UIDB/05037/2020. Funding: CeBER: R&D unit funded by national funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., project UIDB/05037/2020. Data Availability Statement: Data available on request from the corresponding author. Data Availability Statement: Data available on request from the corresponding author. Conﬂicts of Interest: The authors declare that they have no known competing ﬁnancial interest or personal relationships that could have appeared to inﬂuence the work reported in this paper. Conﬂicts of Interest: The authors declare that they have no known competing ﬁnancial interest or personal relationships that could have appeared to inﬂuence the work reported in this paper. References 1. New South Wales (NSW) Government. Outdoor Air Pollution. 2022. 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https://openalex.org/W1889647689	https://inria.hal.science/hal-01529786/document	English	null	Ontology-Driven Dynamic Discovery and Distributed Coordination of a Robot Swarm	Lecture notes in computer science	2,012	cc-by	5,323	To cite this version: Niels Bouten, Anna Hristoskova, Femke Ongenae, Jelle Nelis, Filip De Turck. Ontology-Driven Dy- namic Discovery and Distributed Coordination of a Robot Swarm. 6th International Conference on Autonomous Infrastructure (AIMS), Jun 2012, Luxembourg, Luxembourg. pp.2-13, 10.1007/978-3- 642-30633-4_2. hal-01529786 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01529786 https://inria.hal.science/hal-01529786v1 Submitted on 31 May 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. 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 Ontology-driven Dynamic Discovery and Distributed Coordination of a Robot Swarm Niels Bouten, Anna Hristoskova, Femke Ongenae, Jelle Nelis, and Filip De Turck Ghent University - Department of Information Technology - IBBT Gaston Crommenlaan 8/201, B-9050 Ghent, Belgium {niels.bouten,anna.hristoskova,femke.ongenae, jelle.nelis,filip.deturck}@intec.ugent.be http://ibcn.intec.ugent.be Abstract. Swarm robotic systems rely heavily on dynamic interactions to provide interoperability between the diﬀerent autonomous robots. In current systems, interactions between robots are programmed into the applications controlling them. Incorporating service discovery into these applications allows the robots to dynamically discover other devices. However, since most of these mechanisms use syntax-based matching, the robots cannot reason about the oﬀered functionality. Moreover, as contextual information is often not included in the matching process, it is impossible for robots to select the most suitable device under the current context. This paper aims to tackle these issues by proposing a framework for semantic service discovery in a dynamically changing environment. A semantic layer was added to an existing discovery protocol, oﬀering a semantic interface. Using this framework, services can be searched based on what they oﬀer, with services best suiting the current context yielding the highest matching scores. Keywords: Service Discovery, Semantics, Context-awareness, Distributed Planning, Swarm Robotics 1 Introduction Heterogeneous system designers have to cope with the lack of standardisation that exists between diﬀerent devices. Programmers are therefore often obliged to incorporate statically programmed interactions, deteriorating the overall ﬂexi- bility. Service-Oriented Architectures (SOA) [5] are a popular approach towards attaining higher versatility and ﬂexibility in networked environments. Devices oﬀer their services over the network, allowing their functionality to be easily discovered using a service discovery protocol. Most discovery mechanisms however, perform service matching solely based on the syntax resemblance between requested and oﬀered descriptions [15]. This often leads to poor results, since the requested description can be semantically similar but syntactically diﬀerent from the oﬀered descriptions (e.g., go and move to which are synonyms with diﬀerent syntax), or syntactically similar but with a diﬀerent meaning (e.g., object meaning a goal and object meaning a thing). Another drawback is of course that syntax matching does not consider rela- tions between the diﬀerent concepts in the descriptions. Semantic descriptions overcome these shortcomings by using ontologies to capture semantics of and relations between the diﬀerent concepts. Applying conventional discovery mechanisms in a robotic setting requires to overcome some challenges imposed by the speciﬁc properties of mobile robots. Using a central repository like most discovery mechanisms do, is not viable in a swarm robotics application since there is no guarantee that the repository will remain available due to the mobility properties. Moreover, since robots only have limited resources and the matchmaking of semantic services scales exponential with the number of instances, a robot is not able to process all incoming matching requests in reasonable time when serving as a central repository [9]. Another shortcoming of existing mechanisms, is the absence of context eva- luation during the matching process, i.e. current location, consumed resources and current tasks. A robot requesting help on a certain location, will prefer a robot which current location is closer to the goal to execute the task. Robots acting as service providers also need to deal with their limited resources when oﬀering services. Based on the current status, a robot could decide to no longer oﬀer a speciﬁc service since it would overload the system. Taking into account the current context of both the requesting robot and the service provider during the matching process would enable more accurate and resource eﬃcient service discovery. 1 Introduction This paper suggests a diﬀerent approach by letting the provider of the services match the incoming requests to their oﬀered services. In this way, each device only has to match a limited number of services with low delay. By oﬀering matching as a service, the number of semantic descriptions exchanged between devices is considerably smaller than when each device collects all descriptions of every other device in the swarm. Furthermore, context information, such as the current location, is easily injected into the matching process, without the need to disseminate all context information of each service provider. The outline of this paper is as follows: ﬁrst, a brief overview of related work is presented in Section 2. In Section 3, the proposed framework for ontology-driven discovery and coordination of a robot swarm is discussed. Section 4 describes the details concerning the implementation of the aforementioned framework. The evaluation results are presented in Section 5. Section 6 concludes this paper. 2 Related Work Ontologies are used to incorporate semantics in service descriptions, by mod- elling the domain knowledge in terms of concepts and relationships between them [7]. Semantic Web services are often used to overcome the interoperability issues between diﬀerent robot platforms. Several frameworks exist for deﬁning, matching, invoking and monitoring of services [11–13]. However, these platforms are focussed on Web services oﬀered by Web servers and consumed by desktop and laptop computers over the Internet. To apply these frameworks in a robotic setting several issues need to be overcome, such as the local context of both the service requestor and provider, the limited resources available on mobile devices and the inherent distributed nature of swarm robotic systems. To overcome the high delays caused by the computationally intensive seman- tic matching for central repositories, the use of semantic caches was proposed by Stollberg et al. [20]. This is a feasible solution when dealing with static environ- ments where little context information has to be processed during the matching of services. In a robotic setting however, where rapid context changes are com- mon, the caching of semantic requests and their respective matching results would lead to less accurate and even unusable results. VOLARE [18] proposes a Service-Oriented Architecture for mobile devices, taking into account the cur- rent context. This solution is mainly focussed on making services located on a Web server available to mobile devices. EASY [16] tries to reconcile the compu- tationally expensive semantic matching and the limited resources of robots and embedded systems by encoding semantic descriptions and organising them into service caches. This allows faster semantic matching without overloading the de- vices. As mentioned before, the use of caching causes diﬃculties to incorporate the current context into the matching process. Other projects focus more on the semantic discovery aspect in swarm robotic applications. In Geminga [1], a robot periodically announces its available services which are stored in a local repository located on each robot. Every robot has to match his service request locally with the services in the repository. When context information needs to be included in the matching process, the current context of each robot has to be obtained, causing delays proportional to the num- ber of robots in the swarm in both the service matching and the context retrieval process. ROBOSWARM [23] makes use of a central mediator [8], responsible for maintaining the service repository and serving matching requests. 2 Related Work S-Ariadne [16] uses a set of repositories located in a P2P overlay network. This speeds up the matching process, but still requires context dissemination throughout the swarm. Other technologies focus more on semantic service composition [6, 22], com- posing new complex services out of existing services. In most of these projects, the emphasis lies on the generation of the semantic descriptions for these ser- vices by matching the outputs of a service to the oﬀered inputs of the services. In this paper, existing composite service descriptions are used, but the binding of a service type to a speciﬁc service is done dynamically, taking into account the current context. 3 Framework Design Details The proposed framework, presented in Figure 1, consists out of a Service Man- ager, a Context Manager and a Robot Control component. The services oﬀered by the robot are accessible through the Service Manager, which is responsible for matching incoming requests with the local repository and monitoring service invocations. The Robot Control component executes the invoked services and is able to ﬁnd, match and execute remote services through the Service Manager. The Robot Control component periodically reports contextual information to the Context Manager. This component keeps track of the current context and semantically annotates the received data, which can then be used by the Service Manager to evaluate matching results under the current context. Service Manager Context Manager Robot Control Match Request Invoke Service Matched Services Invocation Status Annotated Context Context updates Request Service Execution Execute Service Swarm Monitor Environment Apply Changes Environment Robot Application Fig. 1. Framework overview presenting the mechanism for oﬀering the robot function- ality (Robot Control) as services to the other robots in the swarm through the Service Manager and the monitoring of the current environment state by the Context Manger. Context Manager Context updates Annotated Context Execute Service Robot Control Service Manager Request Service Execution Match Request Invoke Service Matched Services Invocation Status Monitor Environment Swarm Environment Fig. 1. Framework overview presenting the mechanism for oﬀering the robot function- ality (Robot Control) as services to the other robots in the swarm through the Service Manager and the monitoring of the current environment state by the Context Manger. In order to attain the required degree of ﬂexibility and interoperability, each robot oﬀers its functionality as a set of services which are discoverable by its peers. Each of these services is stored in a local repository, accompanied by its corresponding semantic description containing the semantic representation of the services’ Inputs, Outputs, Preconditions and Eﬀects. Figure 2 gives an overview of the interactions between the diﬀerent components of the Service Manager. The Service Repository, keeps track of the availability of each atomic service as well as the composite services. Additionally to these robot-speciﬁc services, each of the devices also needs to oﬀer a common MatchMaker service, which is responsible for matching incoming service requests and local context injection (2). 3 Framework Design Details A conventional Service Discovery mechanism is used to discover the MatchMaker services of other devices, which can then be invoked to perform semantic matching with the services in the local repository of that device (3). Each of the discovered robots will match the requested description with the semantic descriptions available in the local repository taking into account the current context (4) and return the results, if any (5). The matching algorithm is explained in more detail in Section 4.3. The requesting robot can then choose the best suited service based on the returned matching results. The inputs are then transformed in order to match the ones stated in the semantic description SIM SD SD RobotA RobotB SD RobotC 3 3 4 5 5 7 9 8 1 Semantic service request 2 Local matching and context injection 3 Discovery of semantic services 4 Context-aware matching with local Service Repository 5 Matching Results 6 Mapping of semantic inputs to service description 7 Semantic service invocation 8 Service execution 9 Results of execution SD M SIM MatchMaker Service Discovery Service Execution Manager Semantic Service M M 4 Service Repository M 1 2/6 Fig. 2. Interactions during the semantic service discovery process of the oﬀered service (6). After this, the service is invoked (7) and monitored (9) by the Execution Manager. SIM SD RobotA SD RobotC 3 4 5 7 9 8 M M 1 2/6 RobotC Fig. 2. Interactions during the semantic service discovery process of the oﬀered service (6). After this, the service is invoked (7) and monito by the Execution Manager. Fig. 2. Interactions during the semantic service discovery process of the oﬀered service (6). After this, the service is invoked (7) and monitored (9) by the Execution Manager. Fig. 2. Interactions during the semantic service discovery process of the oﬀered service (6). After this, the service is invoked (7) and monitored (9) by the Execution Manager. 4.1 Robot Ontology Since the proof of concept scenario, discussed in more detail in Section 5.1, takes place in an environment with a heterogeneous robot swarm and a multiplicity of networked devices, a specialised ontology is constructed. Bearing in mind the rapid innovation in the ﬁeld of robotic devices, the ontology is designed to be easily expandable while taking into account the limited resources robots have to reason about semantics. Figure 3 shows how a distinction is made between physical entities and general properties. The physical entities are then split up into Components and Devices. This classiﬁcation is based on the fact that Devices can execute certain tasks, while Components can not do this without being part of a Device. The class Component has two subclasses: Actuators grouping components able to make changes to the environment and Sensors who are able to measure changes or properties of the environment. The Properties describe physical attributes such as Location and Memory. In order to model the current context, each individual will have some properties containing information about its state, such as a MobileRobot having a Location-property. 4.2 Service Description, Discovery and Invocation UPnP [21] is chosen as the service discovery protocol, speciﬁcally the Cling pro- tocol stack [2] is used. UPnP oﬀers automatic service discovery, allowing for Entity hasLocomotionSystem Component Device Sensor Actuator Robot Locomotion System MobileRobot hasLocation SubClass ObjectProperty DataProperty Property Location Double hasXLocation Thing Memory Fig. 3. Fragment of the robot ontology displaying a MobileRobot containing a Loco- motionSystem modelled by a ObjectProperty-relation and the instantiation of a X- Coordinate of the Location-Property by a DataProperty-relation. Thing SubClass ObjectProperty DataProperty Property Entity Component Device Location hasXLocation Actuator Robot Double hasLocation MobileRobot Fig. 3. Fragment of the robot ontology displaying a MobileRobot containing a Loco- motionSystem modelled by a ObjectProperty-relation and the instantiation of a X- Coordinate of the Location-Property by a DataProperty-relation. devices to dynamically join and leave the swarm. Moreover it supports monitor- ing of the execution process of a service via an eventing mechanism. A common way to describe semantic information is by using an ontology, representing ob- jects and their relations. For the construction of the swarm ontology, OWL [17] is chosen as semantic language. This decision is made after reviewing the oﬀered functionality of OWL-S [14], a service ontology based on OWL. OWL-S can be used to semantically describe the inputs, outputs, preconditions and eﬀects of services and reason about them. Originally OWL-S is aimed at Web Services only, but little eﬀort is necessary to extend the grounding (the link between the semantic description and the WSDL description) to be interoperable with UPnP and other discovery protocols. 4.3 Service Matchmaking Algorithm Fail If none of the above conditions hold. Exact If r is an equivalent concept to o. Exact matches yield the highest scores, while for Plugin and Subsume matches the score is dependent on the number of levels between both semantic concepts by turning the parameter levelscoring on. Each graph G′ = (R, O, E′) can then be evaluated based on the weights of the diﬀerent edges. The edge with the min- imum weight min(w′) : e′ ∈E′ determines the overall degree of match x for each graph G′. The graph having the highest value for x is the best match, which is returned. Each of the matching scores for the IOPE’s, along with the scores for the context evaluation are returned to the requesting application. 4.3 Service Matchmaking Algorithm The matchmaking algorithm is used to link a semantic service request to the each service in the Repository, by mapping their respective Inputs, Outputs, Precon- ditions and Eﬀects (IOPE’s) and calculating a score representing their degree of resemblance. The implemented matching algorithm uses bipartite graph match- ing [3] to determine the degree of interoperability between the service request and the oﬀered services in the local repository. Some modiﬁcations had to be made to the original algorithm to include the current context into the matchmak- ing process. Preconditions and eﬀects are split up into context-dependent (i.e. when the evaluation of the precondition is aﬀected by the current context) and context-independent preconditions, this is done by the creator of a service. The parameters of context-dependent preconditions and eﬀects are matched to the semantic representations of the current context using the same bipartite graph matching algorithm as for inputs and outputs. Those matched parameters are then substituted by their respective current context-values and the obtained ex- pression is evaluated. Depending on the condition, this leads either to a boolean or a numeric value, which is included in the ﬁnal matching report of the service. For example the evaluation of the distance to a given target (i.e. expressed as a Location) returns a numeric value, while the condition that a certain locomotion system (e.g., a LocomotionSystem) has to be present returns a boolean value. The bipartite graph matching algorithm matches each concept of the IOPE’s of the requested description to those of the most suited concept of the oﬀered ser- vice description. For example, for the output matching, this is done by creating a bipartite graph G = (R, O, E), where R is the set of requested outputs, O the set of oﬀered outputs and E the set of edges such that each e ∈E has one vertex in R and one in O. A matching of a graph G is a subgraph G′ = (R, O, E′) such that no two edges e1, e2 ∈E′ share the same vertex. A matching G′ is complete if each vertex r ∈R is adjacent to exactly one o ∈O. The corresponding edge e′ connecting r to o is allocated a weight w′ describing their degree of match: Exact If r is an equivalent concept to o. Plugin If r is a superclass of o, or r subsumes o. Subsume If o subsumes r. 5.1 Application of Swarm Robotics for Search and Rescue Performance comparison of centralised versus distributed matchmaking Fig. 4. Performance comparison of centralised versus distributed matchmaking 5.1 Application of Swarm Robotics for Search and Rescue A search and rescue scenario is adopted during the functional testing where a swarm of robots is responsible for searching survivors in a burning building. The robots diﬀer in functionality: some are able to detect human beings, others are capable of shooting a video on a speciﬁc location. Additionally to the robot swarm, a collection of networked devices such as cameras, TV’s and laptops are at hand. The goal of the swarm is to cooperatively locate the survivors and create an impression of their situation. The scenario is demonstrated using iRobot Roombas [10] controlled by a ALIX2D3 computer, connected via the serial interface of the Roombas and equipped with a wireless antenna. A camera is mounted on top of some of these robots. The functional tests are then split up into several scenarios. The semantic context-aware matching and selection is demonstrated by letting three devices oﬀer somewhat similar services. Two devices oﬀer a service that matches the re- quest but one of them is closer to the goal location, yielding a higher score for the context evaluation. The selection algorithm evaluates these matches and chooses the robot closest to the goal to execute the service. A second scenario tests the execution monitoring and the selection of a backup service. The robot closest to the goal location is selected to execute the service, but during execution some problem occurs, causing the Execution Manager to detect a failed execution. In reaction to that, the second service is selected and executed, showing the desired backup behaviour. The third scenario emulates the search and rescue use case where a swarm of robots is ordered to scan and report a burning building. This composite service (i.e. consisting out of 3 services: locating the survivors, shoot- ing a video on these locations and streaming to the ﬁremen outside) is oﬀered by the respective robots. The selected robot searches for suitable robots or devices to fulﬁl each of these subtasks and monitors their executions. For each of the services, robots and devices are ranked based on their current context and the best suited robot is chosen (i.e. the camera robot closest to a certain survivor is selected to go and monitor the situation). 0! 10! 20! 30! 40! 50! 60! 0! 10! 20! 30! 40! 50! Matchmaking time (s)! Number of services per robot! Distributed matching! Centralised matching! Fig. 4. 5.3 Impact of Included Features on Matchmaking Performance and Accuracy The OWLS TC benchmark [19] is used to measure the inﬂuence of the fea- tures (i.e. levelscoring, proﬁlechecking, precondition and eﬀects checking) of the Matchmaker on the matchmaking time and the accuracy of the corresponding results. This benchmark only includes inputs and outputs in the matching pro- cess. For this reason, only the levelscoring can be evaluated for both the accuracy and performance measurements. For evaluating the inﬂuence of the respective features on the performance, the average matching time for a set of 27 requests on a repository of 1000 services is measured. The accuracy of the Matchmaker is evaluated by counting the number of true positives (tp) and true negatives (tn), summing up to the number of correctly classiﬁed matches, and the number of false positives (fp) and false negatives (fn), totalling the number of misclassiﬁed matches. The precision and recall values were then calculated as tp tp+fp and tp tp+fn respectively. A high precision value is desired, since only suited services will be useful to be executed. The Fβ-score, calculated as in (1), is the harmonic mean of precision and recall. A smaller value for β puts more emphasis on the precision than on recall. The inﬂuence of each parameter on the performance is displayed in Figure 5, while the eﬀect of scoring according to the degree of matching on the accuracy is shown in Figure 6. (1 + β2) ∗ precision ∗recall β2 ∗precision + recall (1) (1) Level scoring By turning this parameter on, not only the fact that the re- quested parameter type is a sub or super type of the oﬀered parameter type is taken into account, also the distance between these two types is measured to calculate the corresponding score. Turning level scoring on reduces the number of false positive matches from 24% to 8% and the number of false negative matches from 32% to 12%, increasing recall from 62% to 70% and precision from 81% to 88%. The F1 and F0.5 values increase from 70% to 78% and from 77% to 84% respectively. This increased accuracy comes at a cost of a 16% higher matching delay. Level scoring By turning this parameter on, not only the fact that the re- quested parameter type is a sub or super type of the oﬀered parameter type is taken into account, also the distance between these two types is measured to calculate the corresponding score. 5.2 Centralised Versus Distributed Matching Several tests are conducted measuring the corresponding matchmaking time rel- ative to the number of services in the repository. As shown in Figure 4, the matchmaking time shows a linear relationship with the repository size. The graph also displays the diﬀerence in matchmaking time between the centralised and the distributed approach. For the centralised matchmaker, all services of all robots are stored on one single robot. This leads to a matchmaking delay pro- portional to the number of robots in the swarm. With 10 robots, each oﬀering 10 services, the matchmaking in the centralised approach takes 10 times as much time as when each robot does local matchmaking on its own services. These tests are executed under several assumptions, where in the centralised approach, the robot already has the complete semantic repository and the context information of each robot. In a realistic scenario the dissemination of all context information of each robot to the central robot would cause extra delay, and even increase the total delay incurred in the centralised approach. These high delays can lead to inaccurate matching since the context could already have changed when the matching results become available. 6 Conclusions and Future Work In this article, the viability of ontology-driven and context-aware discovery in a swarm robotics setting is studied in detail. Using semantics to annotate the service descriptions as well as the current context yields more accurate service matching, resulting in a more eﬃcient use of the available services. Using ex- isting technologies, a discovery framework was developed where each robot is responsible for matching incoming service requests to its local repository. This distributed approach, where each swarm member performs a part of the match- ing task, induces considerably lower delays than in the centralised approach. Considering a robot swarm where each robot oﬀers a similar amount of services, the matching delay caused by the centralised approach is ten times higher than in the distributed matching approach. Moreover, since in the distributed ap- proach, each robot is aware of its local context, no extra delays are incurred by the context dissemination process as is the case with centralised matching. The proposed framework also allows to execute composite services in a dynamic way, where the executor for each atomic service is selected based on the current con- text. Future work includes the dynamic generation of composed services based on the available semantic services as well as the current context. A ﬁrst step was taken towards oﬀering semantic matching as a service, but some scalability is- sues remain when the number of robots and oﬀered services increases drastically. Extending the matchmaker to allow semantic grouping of services based on their functionality could overcome these issues. Furthermore, since it concerns com- putationally extensive matching on mobile devices, energy consumption should be taken into account. During the evaluation, it turned out that there are no benchmarks available for evaluating the accuracy of precondition and eﬀects matching, the development of such benchmarks would be beneﬁcial. 5.3 Impact of Included Features on Matchmaking Performance and Accuracy Turning level scoring on reduces the number of false positive matches from 24% to 8% and the number of false negative matches from 32% to 12%, increasing recall from 62% to 70% and precision from 81% to 88%. The F1 and F0.5 values increase from 70% to 78% and from 77% to 84% respectively. This increased accuracy comes at a cost of a 16% higher matching delay. Proﬁle checking It is possible to ﬁlter services based on their proﬁle deﬁnition. A hierarchy is created to model relations between several proﬁles. For this test, a distinction is made between RoboticServices and ComputerServices. When searching for a mobile robot for the execution of some task, the services catalogued as ComputerServices are excluded. Turning the proﬁle checking on reduces the matching time by 30%. 0! 50! 100! 150! 200! 250! level scoring! proﬁle ﬁltering! precondition checking! effect checking! Matchmaking time (ms)! on! off! Fig. 5. Impact of selected features included during matchmaking on matching time 0%! 10%! 20%! 30%! 40%! 50%! 60%! 70%! 80%! 90%! 100%! True Positives! True Negatives! False Positives! False Negatives! Precision! Recall! F1! F0.5! levelscoring on! levelscoring off! Fig. 6. Impact of selected features included during matchmaking on matching accuracy 0! 50! 100! 150! 200! 250! level scoring! proﬁle ﬁltering! precondition checking! effect checking! Matchmaking time (ms)! on! off! Fig. 5. Impact of selected features included during matchmaking on matching time Fig. 5. Impact of selected features included during matchmaking on matching time 0%! 10%! 20%! 30%! 40%! 50%! 60%! 70%! 80%! 90%! 100%! True Positives! True Negatives! False Positives! False Negatives! Precision! Recall! F1! F0.5! levelscoring on! levelscoring off! Fig. 6. Impact of selected features included during matchmaking on matching accuracy Fig. 6. Impact of selected features included during matchmaking on matching accuracy ) / 7. Gruber, T.: Ontology deﬁnition, http://tinyurl.com/tomgruber. References 1. Baer, P. A., Weise, T., Geihs, K.: Geminga: Service Discovery for Mobile Robotics. Systems and Networks Communications, 2008. ICSNC ’08. 3rd International Con- ference on , vol., no., pp.167-172, 26-31 Oct. 2008 doi: 10.1109/ICSNC.2008.29. 2. Bauer, C.: Cling UPnP, http://teleal.org/projects/cling/. 3. Bellur, U., Kulkarni, R.: Improved Matchmaking Algorithm for Semantic Web Services Based on Bipartite Graph Matching. Web Services, 2007. ICWS 2007. IEEE International Conference on , vol., no., pp.86-93, 9-13 Jul. 2007 doi: 10.1109/ICWS.2007.105. 4. Donoho, A., Costa-requena, J., Mcgee, T.: UPnP Device Architecture 1.1. Archi- tecture. Oct. 2008. 5. Durvasula, S., Guttmann, M., Kumar, A., Lamb, J.: SOA Practitioners Guide, Part 2, SOA Reference Architecture. Combined Eﬀort. 1-52 2006. 6. Fujii, K., Suda, T.: Dynamic service composition using semantic information. In Proceedings of the 2nd international conference on Service oriented computing (IC- SOC ’04). ACM, New York, NY, USA, 39-48. DOI=10.1145/1035167.1035174 ) / 7. Gruber, T.: Ontology deﬁnition, http://tinyurl.com/tomgruber. 8. Haseeb, A., Matskin, M., Kungas, P.: Mediator-Based Distributed Web Services Discovery and Invocation for Infrastructure-Less Mobile Dynamic Systems. Next Generation Web Services Practices, 2008. NWESP ’08. 4th International Conference on , vol., no., pp.46-53, 20-22 Oct. 2008. doi: 10.1109/NWeSP.2008.23 9. Hristoskova, A., Moeyersoon, D., Van Hoecke, S., Verstichel, S., Decruyenaere, J., De Turck, F., Dynamic composition of medical support services in the ICU: Platform and algorithm design details. Computer Methods and Programs in Biomedicine, v 100 n 3 p 248 264 Dec 2010 doi: 10 1016/j cmpb 2010 03 019 v.100 n.3, p.248-264, Dec. 2010. doi: 10.1016/j.cmpb.2010.03.019. 10. IRobot: iRobot Corporation: Home Page, http://www.irobot.c 11. Kapahnke, P., Klusch, M.: Semantic web service selection with SAWSDL-MX. Proceedings of the International Workshop on Service Matchmaking and Resource Retrieval in the Semantic Web (SMR2 2008) at ISWC 2008, pp. 318 2008. ( ) 12. Kaufer, F., Klusch, M.: WSMO-MX: A Logic Programming Based Hybrid Service Matchmaker. Web Services, 2006. ECOWS ’06. 4th European Conference on , vol., no., pp.161-170, Dec. 2006. doi: 10.1109/ECOWS.2006.39 13. Lopes, A.: SEA: A Semantic Web services context-aware execution agent. Proceed- ings of the AAAI Fall Symposium on Agents and the Semantic Web, 2005. 14. Martin, D., Burstein, M., Hobbs, J., Lassila, O., McDermott, D., McIl- raith, S., Narayanan, S., Paolucci, M., Parsia, B., Payne, T., Sirin, E., Srinivasan, N., Sycara, K.: OWL-S - Semantic Markup for Web Services, http://www.w3.org/Submission/OWL-S/. // / / / 15. References Meshkova, E., Riihijrvi, J., Petrova, M., Mhnen, P., A survey on resource discovery mechanisms, peer-to-peer and service discovery frameworks. Comput. Netw., Vol. 52, No. 11. Aug. 2008, pp. 2097-2128. doi:10.1016/j.comnet.2008.03.006 16. Mokhtar, S., Preuveneers, D., Georgantas, N., Issarny, V., Berbers, Y.: EASY: Eﬃcient semAntic Service discoverY in pervasive computing environments with QoS and context support. Journal of Systems and Software, Volume 81, Issue 5, May 2008, Pages 785-808, ISSN 0164-1212. doi: 10.1016/j.jss.2007.07.030 17. OWL Working Group: OWL 2 Web Ontology Language Document Overview, 1-12 Oct. 2009. 18. Papakos, P., Rosenblum, D.S., Mukhija, A., Capra, L.: VOLARE: Adaptive Web Service Discovery Middleware for Mobile Systems. In Proceedings of ECEASST. 2009. 19. SemWebCentral: OWL-S Service Retrieval Test Collection: Project Info, http://www.semwebcentral.org/projects/owls-tc/. 20. Stollberg, M., Hepp, M., Hoﬀmann, J.: A caching mechanism for semantic web service discovery. The Semantic Web. Lecture Notes in Computer Science, Volume 4825, 480-493, Jan. 2007. doi:10.1007/978-3-540-76298-0 21. UPnP: UPnP Forum, http://www.upnp.org/. 22. Urbieta, A., Barrutieta, G., Parra, J., Uribarren, A.: A Survey of Dynamic Service Composition Approaches for Ambient Systems. Proceedings of the 2008 Ambi-Sys workshop on Software Organisation and MonIToring of Ambient Systems. 2008 23. Vallivaara, I., Kemppainen, A., Makela, T., Haverinen, J., Roning, J., Martinez, D. M.: Roboswarm, http://roboswarm.eu/. 24. Voyage: Voyage Linux — x86 Embedded Linux, http://linux.voyage.hk/.
https://openalex.org/W3034441070	https://iris.unica.it/bitstream/11584/243936/1/Employability%20and%20Competences%20Innovative%20Curricula%20for%20New%20Professions%202018.pdf	English	null	The Role Model within Professional Training. The Complexity and Flexibility of Education Professions	Studies on adult learning and education/Studies on Adult Learning and Education	2,018	cc-by	197,552	Studies on Adult Learning and Education Studies on Adult Learning and Education Paolo Federighi (University of Florence) Vanna Boffo (University of Florence) Employability & Competences Innovative Curricula for New Professions edited by Vanna Boffo Monica Fedeli FIRENZE Employability & Competences Innovative Curricula for New Professions edited by Vanna Boffo Monica Fedeli FIRENZE Employability & Competences Innovative Curricula for New Professions edited by Vanna Boffo Monica Fedeli FIRENZE UNIVERSITY PRESS Employability Employability & Competences Innovative Curricula Studies on Adult Learning and Education – 8 – Studies on Adult Learning and Education Editorial Board Gianfranco Bandini (University of Florence) Paul Bélanger (Universitè du Québec, Montréal) Pietro Causarano (University of Florence) Giovanna del Gobbo (University of Florence) Regina Egetenmeyer (Julius-Maximilians Universität Würzburg) Balàzs Nemèth (Pécsi Tudományegyetem – University of Pécs) Petr Novotny (Masarykova Univerzita, Brno) Ekkehard Nuissl von Rein (Technische Universität Kaiserslautern) Aleksandra Pejatovic (Univerzitet u Beogradu) Simona Sava (Universitatea de Vest din Timişoara) Maria Slowey (Dublin City University) Francesca Torlone (University of Florence) Employability & Competences Innovative Curricula for New Professions edited by Vanna Boffo Monica Fedeli Employability & Competences : innovative Curricula for New Professions / edited by Vanna Boffo, Monica Fedeli. – Firenze : Firenze University Press, 2018. (Studies on Adult Learning and Education ; 8) http://digital.casalini.it/9788864536729 ISBN 978-88-6453-671-2 (print) ISBN 978-88-6453-672-9 (online) Graphic design: Alberto Pizarro Fernández, Pagina Maestra Employability & Competences : innovative Curricula for New Professions / edited by Vanna Boffo, Monica Fedeli. – Firenze : Firenze University Press, 2018. (Studies on Adult Learning and Education ; 8) http://digital.casalini.it/9788864536729 ISBN 978-88-6453-671-2 (print) ISBN 978-88-6453-672-9 (online) Graphic design: Alberto Pizarro Fernández, Pagina Maestra This essay has been sponsored by Progetto Prin 2012 prot. 2012LATR9N, titled To design innovative programs for higher education, to promote a personalized learning, to build on job competencies, to value talents to create new work-opportunities. Positive strategies in Higher Education to support young adults during their employment emergency, as a response to socio-economic crisis and as a citizenship action. All the chapters included in this volume have been subjected to peer review process. Cooperativa ITC, Interpreti e Traduttori in Cooperativa (Rome, Italy) oversaw the proofreading of the volume and the english version of Bruno Rossi’s, Vanna Boffo’s and Paolo Federighi’s essays. Peer Review Process All publications are submitted to an external refereeing process under the responsibility of the FUP Editorial Board and the Scientific Committees of the individual series. The works published in the FUP catalogue are evaluated and approved by the Editorial Board of the publishing house. For a more detailed description of the refereeing process we refer to the official documents published on the website and in the online catalogue of the FUP (www.fupress.com). Peer Review Process All publications are submitted to an external refereeing process under the responsibility of the FUP Editorial Board and the Scientific Committees of the individual series. The works published in the FUP catalogue are evaluated and approved by the Editorial Board of the publishing house. For a more detailed description of the refereeing process we refer to the official documents published on the website and in the online catalogue of the FUP (www.fupress.com). Firenze University Press Editorial Board A. Dolfi (Editor-in-Chief), M. Boddi, A. Bucelli, R. Casalbuoni, M. Garzaniti, M.C. Grisolia, P. Guarnieri, R. Lanfredini, A. Lenzi, P. Lo Nostro, G. Mari, A. Mariani, P.M. Mariano, S. Marinai, R. Minuti, P. Nanni, G. Nigro, A. Perulli, M.C. Torricelli. Employability & Competences Innovative Curricula for New Professions This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0: https://creativecommons.org/licenses/by/4.0/legalcode). This book is printed on acid-free paper CC 2018 Firenze University Press Università degli Studi di Firenze Firenze University Press via Cittadella, 7, 50144 Firenze, Italy www.fupress.com Printed in Italy All publications are submitted to an external refereeing process under the responsibility of the FUP Editorial Board and the Scientific Committees of the individual series. The works published in the FUP catalogue are evaluated and approved by the Editorial Board of the publishing house. For a more detailed description of the refereeing process we refer to the official documents published on the website and in the online catalogue of the FUP (www.fupress.com). Firenze University Press Editorial Boardi A. Dolfi (Editor-in-Chief), M. Boddi, A. Bucelli, R. Casalbuoni, M. Garzaniti, M.C. Grisolia, P. Guarnieri, R. Lanfredini, A. Lenzi, P. Lo Nostro, G. Mari, A. Mariani, P.M. Mariano, S. Marinai, R. Minuti, P. Nanni, G. Nigro, A. Perulli, M.C. Torricelli. A. Dolfi (Editor-in-Chief), M. Boddi, A. Bucelli, R. Casalbuoni, M. Garzaniti, M.C. Grisolia, P. Guarnieri, R. Lanfredini, A. Lenzi, P. Lo Nostro, G. Mari, A. Mariani, P.M. Mariano, S. Marinai, R. Minuti, P. Nanni, G. Nigro, A. Perulli, M.C. Torricelli. This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4 0 h // i /li /b /4 0/l l d ) This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0: https://creativecommons.org/licenses/by/4.0/legalcode). (CC BY 4.0: https://creativecommons.org/licenses/by/4.0/legalcode). This book is printed on acid-free paper CC 2018 Firenze University Press Università degli Studi di Firenze Firenze University Press via Cittadella, 7, 50144 Firenze, Italy www.fupress.com Printed in Italy CC 2018 Firenze University Press Università degli Studi di Firenze Firenze University Press TABLE OF CONTENTS TABLE OF CONTENTS INTRODUCTION: THE PROJECT FROM BEGINNING TO END AND BEYOND Monica Fedeli, Vanna Boffo INTRODUCTION: THE PROJECT FROM BEGINNING TO END AND BEYOND Monica Fedeli, Vanna Boffo XI PART I RESULTS OF RESEARCH THROUGH THE 5 PATHWAYS CHAPTER I THE DEVELOPMENT OF CALLING DURING COLLEGE: A 3-YEAR INVESTIGATION The Role of Mentors on the Development of Calling in Students: a 3-Year Investigation 5 Anna Dalla Rosa, Michelangelo Vianello, Barbara Barbieri The Pursuit of Happiness: a Reflection on the Calling Construct 19 Barbara Barbieri CHAPTER II ENGAGING STUDENTS IN HIGHER EDUCATION AND FACULTY DEVELOPMENT The Student Voice in Higher Education and the Implications of Promoting Faculty Development 25 Monica Fedeli The Design of Work-Related Teaching & Learning Methods: Case Studies and Methodological Recommendations 37 Daniela Frison The Student Voice in Higher Education and the Implications of Promoting Faculty Development 25 Monica Fedeli The Design of Work-Related Teaching & Learning Methods: Case Studies and Methodological Recommendations 37 Daniela Frison VI EMPLOYABILITY & COMPETENCES CHAPTER III THE INTERNSHIP EXPERIENCE AS A LEARNING MOMENT IN HIGHER EDUCATION Supporting Informal Learning in Higher Education Internships 51 Claudio Melacarne Higher Education and Work-Related Learning from Professionalism to Professionality 65 Maura Striano CHAPTER IV GUIDANCE AS A PEDAGOGICAL TOOL Guiding at University. The Protagonists’ Point of View 77 Francesco Lo Presti Self-Telling to Orientate Oneself 91 Bruno Rossi Guidance Specificity in Educational Research. Criticisms and Area of Intervention in Higher Education 103 Antonia Cunti CHAPTER V EMPLOYABILITY AND TRANSITIONS Employability and Transitions: Fostering the Future of Young Adult Graduates 117 Vanna Boffo Non-linear Paths in Transitions through the Labour Market 129 Paolo Federighi PART II THE PERSPECTIVES OF THE PEDAGOGICAL COMMUNITY CHAPTER I DIDACTICS The Voice of Teachers Involved in School-Work Alternance Programmes 151 Concetta Tino CHAPTER III THE INTERNSHIP EXPERIENCE AS A LEARNING MOMENT IN HIGHER EDUCATION Supporting Informal Learning in Higher Education Internships 51 Claudio Melacarne Higher Education and Work-Related Learning from Professionalism to Professionality 65 Maura Striano 65 CHAPTER IV GUIDANCE AS A PEDAGOGICAL TOOL GUIDANCE AS A PEDAGOGICAL TOOL Guiding at University. The Protagonists’ Point of View 77 Francesco Lo Presti Self-Telling to Orientate Oneself 91 Bruno Rossi Guidance Specificity in Educational Research. Criticisms and Area of Intervention in Higher Education 103 Antonia Cunti CHAPTER V EMPLOYABILITY AND TRANSITIONS EMPLOYABILITY AND TRANSITIONS Employability and Transitions: Fostering the Future of Young Adult Graduates 117 Vanna Boffo Non-linear Paths in Transitions through the Labour Market 129 Paolo Federighi PART II THE PERSPECTIVES OF THE PEDAGOGICAL COMMUNITY CHAPTER I DIDACTICS The Voice of Teachers Involved in School-Work Alternance Programmes 151 Concetta Tino 151 VII TABLE OF CONTENTS Promoting 21st century skills in Higher Education through collaboration and activities 163 Nadia Sansone, Donatella Cesareni, Ilaria Bortolotti A Student Voice Approach in Work-Related Learning. From Lesson-Learned in Secondary School to Suggestions for Higher Education 175 Glenda Galeotti, Gilda Esposito Professionalization of Teachers and Problematization Processes 193 Daniela Maccario INTEREURISLAND: from Field Research to a Possible Model for Internationalizing University Social Responsibility Experiences 205 Nicola Andrian CHAPTER II TRAINEESHIP The Role Model within Professional Training. The Complexity and Flexibility of Education Professions 223 Giordana Szpunar, Barbara Barbieri The ‘Second Level’ Education Professional: a Traineeship Programme Fostering a Proactive Approach to Employability 237 Cristina Palmieri, Marina Barioglio, Andrea Galimberti, Maria Benedetta Gambacorti-Passerini, Tania Morgigno Professional Development Tutoring: Comparing Italian and International Models 249 Alessandra Romano Insights from the Fields: the Role of Reflection in ‘Learning How to Learn’ 265 Anna Salerni, Silvia Zanazzi Educators in Training and Writing: Perception, Experiences, Problems 281 Patrizia Sposetti VII TABLE OF CONTENTS Promoting 21st century skills in Higher Education through collaboration and activities 163 Nadia Sansone, Donatella Cesareni, Ilaria Bortolotti A Student Voice Approach in Work-Related Learning. From Lesson-Learned in Secondary School to Suggestions for Higher Education 175 Glenda Galeotti, Gilda Esposito Professionalization of Teachers and Problematization Processes 193 Daniela Maccario INTEREURISLAND: from Field Research to a Possible Model for Internationalizing University Social Responsibility Experiences 205 Nicola Andrian CHAPTER II TRAINEESHIP The Role Model within Professional Training. The Complexity and Flexibility of Education Professions 223 Giordana Szpunar, Barbara Barbieri The ‘Second Level’ Education Professional: a Traineeship Programme Fostering a Proactive Approach to Employability 237 Cristina Palmieri, Marina Barioglio, Andrea Galimberti, Maria Benedetta Gambacorti-Passerini, Tania Morgigno Professional Development Tutoring: Comparing Italian and International Models 249 Alessandra Romano Insights from the Fields: the Role of Reflection in ‘Learning How to Learn’ 265 Anna Salerni, Silvia Zanazzi Educators in Training and Writing: Perception, Experiences, Problems 281 Patrizia Sposetti VIII EMPLOYABILITY & COMPETENCES CHAPTER III GUIDANCE AND CALLING Interpretations. Market, Work, Training 295 Pierluigi Malavasi Fostering Personal Knowledge and Competences in Higher Education Guidance Processes 305 Sergio Bellantonio Nursing Students’ Future Employment Preferences and the Real Demands of Patients using the Health Services. A Qualitative Study on a Group of Final-Year Students at the University of Padua 315 Natascia Bobbo, Silvia Lazzaro Guidance Models and Practices Adopted Internationally to Promote the Exploration of Skills Relating to the Employability of Students with Disabilities. A First Meta- Analysis 327 Valentina Paola Cesarano, Marianna Capo, Maria Papathanasiou, Maura Striano Telling Transversal Competences… to be Professionally Promoted 341 Marianna Capo, Valentina Paola Cesarano, Maria Papathanasiou, Maura Striano The Group as an Education Resource in Guidance Processes 365 Alessandra Priore Career Calling: Lights and Shadows 375 Marco Schiavetta, Sonia Startari CHAPTER IV EMPLOYABILITY AND TRANSITIONS CHAPTER III GUIDANCE AND CALLING 295 * Monica Fedeli, PhD, Associate Professor of Teaching and Learning, and Organizational Development, Department of FISPPA, University of Padua, Italy. Email: monica.fedeli@unipd.it; Vanna Boffo, PhD, Associate Professor of General Pedagogy, Department of Education and Psychology, University of Florence, Italy. Email: vanna. boffo@unifi.it. CHAPTER IV EMPLOYABILITY AND TRANSITIONS Boosting Entrepreneurship Capability in Work Transitions 389 Massimiliano Costa, Andrea Strano Transfer of Non-Academic Skills in Academic Context: towards a Sustainable Employability 399 Maria Luisa Iavarone, Fausta Sabatano Boosting Entrepreneurship Capability in Work Transitions 389 Massimiliano Costa, Andrea Strano Transfer of Non-Academic Skills in Academic Context: towards a Sustainable Employability 399 Transfer of Non-Academic Skills in Academic Context: towards a Sustainable Employability 399 Maria Luisa Iavarone, Fausta Sabatano IX TABLE OF CONTENTS Employability-Oriented Curriculum: Strategies and Tools to Train Young Graduates. The PRIN EMP&Co. Project 411 Gaia Gioli Doctorates and Employability: New Perspectives for Doctoral Education 425 Cristina Lisimberti Employability and Competence. But for which Europe? 435 Stefano Polenta The Role of Higher Education in a Changing World: Why Employability Matters 447 Carlo Terzaroli The Value Proposition of Organizations for Young Graduates and their Employability 459 Francesca Torlone Employability and Transitions towards Work: MSc Degree Programme in Educational Planning and Human Resource Development, Catholic University of the Sacred Heart of Brescia 471 Alessandra Vischi INNOVATION AND KNOWLEDGE TRANSFER OF THE RESEARCH. FINAL REMARKS 481 Vanna Boffo, Monica Fedeli LIST OF AUTHORS 487 INTRODUCTION: THE PROJECT FROM BEGINNING TO END AND BEYOND Monica Fedeli (University of Padua), Vanna Boffo (University of Florence)* The present volume is the result of a three year PRIN Research proj- ect called Employability and Competences, which was developed in Italy in order to respond to the need for innovation and modernization in higher education. A consortium of universities (Padova, Firenze, Siena, Napoli Parthenope) conducted the research within the higher education system starting March 2014 and ending March 2017. The volume collects a rel- evant number of contributions of scholars that take part to the last con- ference in Florence, organized in March 2017. i This book had a significant impact, since it is calling into question the long tradition of teaching (transmission) in Italian universities, the inno- vation of teaching and learning in higher education and the challenge of employability consulting the Italian scientific community around four major foci of the research: didactics, traineeship, guidance and calling, and employability and transitions. p y y The purpose of the research was to generate new strategies, models, methodologies to modernize the traditional Italian university system, to create space for teaching and learning and to connect the university pro- grams with the professional communities in order to improve employ- ability after the economic crises of 2008. Guiding research questions for the book included: How can we in- novate the teaching and learning in higher education in our traditional context? How can we listen to the needs of the students and connect them with those of the labor world? How can we promote work-related teaching and learning in our courses? How can we manage the transi- tions from the university to the labor market, how can we train faculty to respond to these new challenges and students to be active and engage them during the college? XII MONICA FEDELI, VANNA BOFFO These questions were explored by five research groups of four Uni- versities (Padova, Firenze, Siena and Napoli Parthenope). Surveys and qualitative research designs developed with interviews, focus groups and observation. More than 20.000 students responded to the surveys and were involved in the studies. This book is organized around two main parts. Part I discusses the related research themes and how they help to es- tablish a strong rationale for the PRIN project. INTRODUCTION: THE PROJECT FROM BEGINNING TO END AND BEYOND g p j In chapter I Michelangelo Vianello, together with Anna Dalla Ro- sa e Barbara Barbieri, presents a three-wave longitudinal study, that in- vestigated the role of mentoring on calling development, in the second chapter Barbara Barbieri discusses some reflections and results arose from the study on the calling construct presented by the colleagues of Uni- versity of Padova. In chapter II Monica Fedeli and Daniela Frison introduced innova- tive constructs of teaching: student voice, work-related teaching and learning and the related implications in higher education. The aim is to share with the community the results of the research, how we can listen the voice of our students and how we can design work-related practices for promoting employability in the classroom. Some case studies, data analysis and methodological recommendations in both contributions are presented to give clear indications of some implications for practice. Claudio Melacarne, and Maura Striano present the internship and the importance of supporting this kind of situated and embedded learning experience for students and how it can generate awareness about their future professional choices. Francesco Lo Presti, Bruno Rossi and Antonella Cunti focus their chapters on guidance as a pedagogical tool, and considers how guidance can develop meaningful paths for young people at the beginning of their university career. The main areas of interventions, the strengthens and weaknesses of the practices are discussed as well as how to improve some of these approaches to be more successful. f pp In the last chapter of Part I Vanna Boffo and Paolo Federighi deal with the last segment of the chain of higher education: employability and transitions, in particularly how to promote employability in higher education and how to support students during the transition from uni- versity to work and to attempt to match and connect the competences taught at university with those needed by the labor market. i These topics were discussed during the final conference organized in Florence with the contributions of international scholars invited to add global perspectives into the research and to create and stimulate the community to go beyond these results and to develop new ideas on the basis of the research results collected in the last three years. XIII INTRODUCTION Part II of the book is a rich collection of descriptive and empirical studies on the main topics of the project. INTRODUCTION: THE PROJECT FROM BEGINNING TO END AND BEYOND We asked colleagues of the Italian pedagogical community to present their contributions in order to enlarge the perspectives and with the aim of improving, innovating and changing our higher education system. This aim is encouraged in the report on Improving the Quality of Teaching and Learning in Europe’s Higher Education Institutions (European Commission, 2013) which states: higher education institutions need to create environments and feedback mechanisms and systems to allow students’ views, learning experience, and their performance to be taken into account (p. 28). p (p ) Another key recommendation is the involvement of students and business in curricula design, such that: «curricula should be developed and monitored through dialogue and partnerships among teaching staff, students, graduates and labor market actors, drawing on new methods of teaching and learning, so that students acquire relevant skills that enhance their employability» (p. 41). Furthermore, there should be an emphasis on providing counseling and mentoring services helping en- sure that make their way to graduation and beyond. This part of the book is divided in four sections and each section deals with a theme re- lated to the project. p j Chapter I Didactics is a collection of studies on the didactics in the higher education. Valentina Grion, Concetta Tino, Nadia Sansone, Do- natella Cesareni, Ilaria Bortolotti, Glenda Galeotti, Gilda Esposito, Dan- iela Maccario, and Nicola Adrian present some issues related to teaching in classroom, and to promote service learning, teachers’ development and innovation involving students and teachers together. Chapter II Internship deals with informal learning setting and train- eeship. Giordana Szpunar, Barbara Barbieri, Manuela Gallerani, Cristina Palmieri, Andrea Galimberti, Tania Morgigno, Marina Barioglio, Ma- ria Benedetta, Gambacorti Passerini, Alessandra Romano, and Patrizia Sposetti. This section is a collection of practices and studies developed in higher education and schools in order to give value to the informal experiences of situated and embedded learning, considering the role of students and teachers and how they can create partnership with organi- zations and labor market. Chapter III Guidance & Calling is dedicated to the educational guid- ance and how to promote educational development during the guidance process. Furthermore, this section explores the new concept of ‘calling’ and its role in education and for the development of students’ skills in order to improve their persistence in higher education and facilitate their entrance in the labor market. INTRODUCTION: THE PROJECT FROM BEGINNING TO END AND BEYOND Pierluigi Malavasi, Sergio Bellantonio, Natascia Bobbo, Silvia Laz- zaro, Valentina Paola Cesarano, Marianna Capo, Maria Carolina Galdo, Maura Striano, Alessandra Priore, Marco Schiavetta, Sonia Startari, and XIV MONICA FEDELI, VANNA BOFFO Barbara Barbieri present studies and practices related to guidance and calling developed in different fields of higher education from the medi- cal to the international setting and with students with disability that give us a rich range of insights to take into consideration in our pedagogical community for future research.f y Chapter IV Employability and Transition discusses from different perspectives the need to manage and improve the work transitions in higher education in order to foster employability. Massimiliano Costa, Andrea Strano, Maria Luisa Iavarone, Fausta Sabatano, Gaia Gioli, Cris- tina Lisimberti, Stefano Polenta, Carlo Terzaroli, Francesca Torlone, and Alessandra Vischi offer the readers classroom and informal learn- ing experiences and practices. They provide concrete suggestions how to develop or implement new strategies for supporting students in the transitions and creating employability skills during college. Policies and university career services are taken into consideration as well as part of the discussion for the area. In conclusion a personal note and acknowledgments is provided about the people who managed, supported and developed this project with us. We mention first the colleagues, with whom we had and still have the privilege to work; prominent scholars and friends who had never given up during this long journey that was rich of new insights, discoveries in term of research, but also difficulties and weaknesses. Francesco Lo Presti, Claudio Melacarne and Michelangelo Vianello and ourselves. We were in charge of the realization of the project and won the challenge to follow the research from the beginning to the end. This was a great opportunity for our career development. We grew up together united by the passion for research, with a constant attention to the results and the pleasure found in working hard and together. All this makes our community stronger and capable to share, builds informal relationships and develops a sense of empathy that became an instrument of creating better research and learning opportunities. Each of us was the leader of a research team, in which other colleagues, young scholars, and PhD students collaborate and make all that we reached possible! European Commission 2013, Report to the European Commission on Improving the quality of teaching and learning in Europe’s higher education institutions, Publications Office of the European Union, Luxembourg. INTRODUCTION: THE PROJECT FROM BEGINNING TO END AND BEYOND Finally, our sincere thanks to all the colleagues of the entire pedagogi- cal community that contributed in different ways to the development of the project and to the authors of this book. Most of them went through different phases of the project and share with us their studies and their consideration in many occasions. i y Looking back, we feel more confident, that we will manage other projects together, looking beyond… We really want to create new op- portunities and challenges to work together and to develop communi- ties of scholars that are interested in thinking critically how to innovate the educational field and give young people the opportunity to grew up in a more engaging educational environment! XV INTRODUCTION References European Commission 2013, Report to the European Commission on Improving the quality of teaching and learning in Europe’s higher education institutions, Publications Office of the European Union, Luxembourg. European Commission 2013, Report to the European Commission on Improving the quality of teaching and learning in Europe’s higher education institutions, Publications Office of the European Union, Luxembourg. * Anna Dalla Rosa, PhD, Post-doc Researcher in Industrial and Organizational Psychology, University of Padua, Italy. Email: anna.dallarosa@unipd.it; Michelangelo Vianello, PhD, Associate Professor of Work and Organizational Psychology, University of Padua, Italy. Email: michelangelo.vianello@unipd.it; Barbara Barbieri, PhD, Assistant Professor of Psychology of Human Resources, Department of Social Sciences and Institutions, University of Cagliari, Italy. Email: barbara.barbieri@unica.it. Anna Dalla Rosa (University of Padua), Michelangelo Vianello (University of Padua), Barbara Barbieri (University of Cagliari)* Abstract: In a three-wave longitudinal study, we investigated the role of men- toring on calling development. The results suggest that the mere presence of a mentor is associated with higher levels of calling and the development of a call- ing is fostered by the mentors’ calling orientation, yet it is restrained by mentors’ job and career orientation. Keywords: calling, longitudinal design, calling development, mentorship. 2.1 The role of the social environment in calling development There are only three published studies that investigated the role of oth- ers in the development of a calling, and all these studies focused on the development of a calling for the music domain (Dobrow, Tosti-Kharas 2012; Dobrow 2006, 2013). Dobrow (2006) found that parents’ involve- ment in the arts had a positive effect on students’ initial calling for the music domain. Dobrow and Tosti-Kharas (2012) investigated the rela- tionship between calling and students’ receptivity to the advice, provided by a mentor, which discourages them from pursuing a professional path in their calling domain. They found that calling reduces the effect of the discouraging advice, so that students with a higher calling are more likely to ignore negative career-related advice provided by their private music teacher. This result was replicated in a cross-sectional study that involved a sample of business students. In 2013, Dobrow investigated the role of social comfort in the devel- opment of a calling. Social comfort in the music domain measures the extent to which students enjoy spending time with other musicians, and it was found to have a positive effect on initial calling and a small nega- tive effect on its development over time. Finally, Dalla Rosa, Vianello, and Anselmi (Unpublished manuscript), found that social support pro- vided by friends, family and a special person helps students to develop their calling. Taken together, these results suggest that a calling is not only an intraindividual phenomenon. There is evidence of a connection between the intimate experience of having a calling and relationships with others and the social context (Dobrow, 2006, 2013; Dobrow, Tos- ti-Kharas 2012). Indeed, participants in qualitative studies commonly mentioned the supportive role of others as a factor that influences the emergence of a calling (Duffy et al. 2012; French, Domene 2010). The effect of social comfort and support on calling suggests that the enjoy- ment and pleasure in being around others, and the presence of people with whom students can discuss their problems and who are willing to comfort and encourage them fosters calling development. However, we do not know whether these positive effects on calling are due to the mere presence of people willing to support or whether these persons may also represent a role and attitude model. 2. Theoretical backgrounds 2. Theoretical backgrounds 1. Introduction A calling is a multi-dimensional construct that describes affective, motivational, spiritual, and identity-related facets of the relationship be- tween individuals and specific domains in life or work. Viewing one’s career as a calling is critical to the individual success and central in pro- moting positive work-related outcomes (Dobrow, Heller 2014; Dobrow, Tosti-Kharas 2011, 2012; Duffy, Allan, Autin, Douglass 2014; Duffy, Douglass, Autin, Allan 2014; Hirschi, Herrmann 2012, 2013; Praskova, Hood, Creed 2014). Theoretical and empirical contributions suggest that a calling changes over time (Dobrow 2013; Duffy, Manuel, Borger, Bott 2011; Vianello, Galliani, Dalla Rosa, Anselmi, Unpublished manuscript) and involves an «ongoing process» (Duffy, Dik 2013) in which individu- als evaluate the purpose and meaningfulness of their job activities and the quality of the interaction with the context. Despite an emerging in- terest in calling, little is known about the origin of calling and wheth- er the social environment influences its development. Previous research suggested that the social environment may influence individuals’ attitude toward work and help people live out their calling (Cardador, Dane, Pratt 2011; Guo et al. 2014; Harzer, Ruch 2012). Trusted sources of informa- tion and experienced individuals may play an important role in the de- velopment of calling by providing a role and an attitude model (Ragins, Cotton, Miller 2000). ANNA DALLA ROSA, MICHELANGELO VIANELLO, BARBARA BARBIERI 6 THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENT 7 2.2 The role of a mentor in career development Specifically, a mentor helps finding meaning in work activities, encour- ages a protégé to associate work with what they think is important in life, and might promote «a sense of transcendence throughout the work process by appealing to his or her sense of calling» (Weinberg, Locander 2014: 395). A mentor with high levels of calling might help a person to find meaning in an activity, to understand the deeper aspects of work and, consequently, provide the opportunity to develop a sense of calling. Having a calling gives meaning and purpose to a life role (Dik, Duffy 2009; Dobrow, Tosti-Kharas 2011; Praskova et al. 2014), and it is related to well-being and positive work outcomes (Dalla Rosa, Vianello, Galliani 2017). Therefore, positive work attitude, work meaningfulness and work- place spirituality are all constructs that are theoretically related to calling hence we expect to find evidence of a relationship between mentorship and calling. In addition, a mentor can be perceived as a role model. This implies that protégés could carry on imitating and assimilating values and attitudes of their mentor (Bell 1970; Kaufmann, Harrell, Milam, Woolverton, Miller 1986). This may also be true for protégés’ perception of having a calling. Having a calling gives meaning and purpose to a life role (Dik, Duffy 2009; Dobrow, Tosti-Kharas 2011; Praskova et al. 2014), and it is related to well-being and positive work outcomes (Dalla Rosa, Vianello, Galliani 2017). Therefore, positive work attitude, work meaningfulness and work- place spirituality are all constructs that are theoretically related to calling hence we expect to find evidence of a relationship between mentorship and calling. In addition, a mentor can be perceived as a role model. This implies that protégés could carry on imitating and assimilating values and attitudes of their mentor (Bell 1970; Kaufmann, Harrell, Milam, Woolverton, Miller 1986). This may also be true for protégés’ perception of having a calling. 2.1 The role of the social environment in calling development p The study conducted by Dobrow and Tosti-Kharas in 2012 was the first to focus on the role of a mentor and suggested that having a strong calling is associated with greater willingness to ignore negative career advice on time. However, this study did not investigate the specific ef- fect of having a mentor in the development of a calling. Thus, the role of the social context in the development of calling is still unclear. 2.2 The role of a mentor in career development Research on career attitude and commitment highlighted the impor- tance of others, especially family, peers and mentors, on career develop- ment. Some scholars have suggested that a mentor may positively influence the work attitude of a protégé (Eby, Allen, Evans, Ng, DuBois 2008; Ragins et al. 2000), may help to find a meaning in work (Rosso, Dekas, Wrzesniewski 2010) and may support the development of workplace spirituality (Buzzanell 2009; Reave 2005; Weinberg, Locander 2014). l p y ( g ) First, mentoring was found to influence the way people experience a work role. Research comparing people with and without a mentor showed that the presence of a reference and a trusted person leads to greater ca- reer and job satisfaction, career commitment and involvement, positive job attitude and motivation (Ragins et al. 2000; Payne, Huffmann 2005; Chao 1997; Eby et al. 2008).i y ) Workplace spirituality can be defined as «the recognition that employ- ees have an inner life that nourishes and is nourished by meaningful work that takes place in the context of community» (Ashmos, Duchon 2000: 137). Weinberg and Locander (2014) suggest that a mentor provides not only psychological and vocational support, but can also provide spiritual support, encouraging the development of protégé workplace spirituality. Specifically, a mentor helps finding meaning in work activities, encour- ages a protégé to associate work with what they think is important in life, and might promote «a sense of transcendence throughout the work process by appealing to his or her sense of calling» (Weinberg, Locander 2014: 395). A mentor with high levels of calling might help a person to find meaning in an activity, to understand the deeper aspects of work and, consequently, provide the opportunity to develop a sense of calling. f Workplace spirituality can be defined as «the recognition that employ- ees have an inner life that nourishes and is nourished by meaningful work that takes place in the context of community» (Ashmos, Duchon 2000: 137). Weinberg and Locander (2014) suggest that a mentor provides not only psychological and vocational support, but can also provide spiritual support, encouraging the development of protégé workplace spirituality. 4.1 Participants The sample was mainly composed of women (63.8%, 65.8%, and 68% females at T1, T2, and T3, respectively). Participants’ ages ranged be- tween 18 and 69 (MT1 = 23.37; SDT1 = 5.39; MT2 = 23.47; SDT2 = 4.82; MT3 = 24.02; SDT3 = 4.50). Among the students who took part to the three data collection 28% (n = 123 out of 434) declared that they had a mentor at times 1, 2 and 3; 27% declared that they did not have a men- tor at times 1, 2 and 3 (n = 118 out of 434). In our sample, a mentor is often a professor at high school (10.7%; 8.7%; 7% respectively at T1, T2 and T3) or at College (8.6%; 9.9%; 9.6% respectively at T1, T2 and T3), a friend or a relative (16.5%; 9.8%; 15.1 respectively at T1, T2 and T3). 4. Materials and method Data were collected in three waves using a non-experimental on- line survey. The second and third wave respectively occurred 12 and 24 months after the first data collection. A sample of 5886 bachelor’s and master’s students enrolled at four Italian universities was involved in the first data collection (T1), 1700 students took part in the second data col- lection (T2), and 881 took part in the third data collection (T3). The analyses presented in this study were performed on the sample of 434 students who participated at all the three waves. 3. Hypotheses This study investigated the role of a mentor on calling development fo- cusing on two factors: (1) the effect of the mere presence of a mentor, and ANNA DALLA ROSA, MICHELANGELO VIANELLO, BARBARA BARBIERI 8 (2) the effect of mentors’ orientation toward work on their protégés’ call- ing. First, we hypothesize that students with a mentor have a higher level of calling than students who do not have a mentor (HP1). In addition, we expect the level of calling of students with a mentor to increase over time, and to remain stable or to decrease over time for students without a mentor (HP2). Second, we expect that mentors’ orientation toward work influence their protégés’ calling over time, making them more similar (HP3). Accord- ing to the values of openness and reproducibility (Open Science, <https:// osf.io/tvyxz/wiki/home/>), these hypotheses and analysis plans have been already registered and are publicly available at <https://osf.io/2wcky/>. THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS 9 Student and mentor orientation toward work. Students were provided with three paragraphs (Work-Life Questionnaire; Wrzesniewski et al. 1997) describing work as a job (e.g. people only interested in the material ben- efits of working), a career (e.g. people mainly interested in achievement and power), or a calling (e.g. people work for personal satisfaction and fulfillment), and asked which one best matched their orientation to work. We then asked them to indicate if they have a mentor (a person with ex- perience, who is a wise guide, a reference model and a trusted advisor; Noe 1988; Ragins et al. 2000) and to describe the mentor’s orientation toward work using the same scale. 4.2 Measures Calling. We used the 22-item Unified Multidimensional Calling Scale (Vianello, Dalla Rosa, Anselmi, Unpublished manuscript), which mea- sures seven facets of calling: passion, purposefulness, sacrifice, pervasive- ness, prosocial orientation, transcendent summons, and identity. Identity was not assessed at Time 1. A detailed presentation of the measure is re- ported at https://osf.io/zc8ha. THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS 5.1 Hypotheses 1 and 2: effects of the mere presence of a mentor 5.1 Hypotheses 1 and 2: effects of the mere presence of a mentor First, we tested the effect of the mere presence of a mentor on calling level and development. Figure 1 show interaction and main effects of the presence of a mentor on calling facets. We found a significant main ef- fect of mentor at T1 on passion (F(1,419) = 8.20, µ2 = .02), transcendent summons (F(1, 380) = 11.54, µ2 = .03), sacrifice (F(1,415) = 4.64, µ2 = .01) and pervasiveness (F(1, 420) = 7.88, µ2 = .02). Thus, students with a mentor showed different levels of calling depending on the presence of a mentor at Time 1. Specifically, within each time, students with a mentor have higher levels of calling than students without a mentor. We then analyzed the difference in the level of calling between the group of stu- dents with a mentor and the group without a mentor. Results suggest that students with a mentor at Time 1, 2 and 3 have higher levels of passion, sacrifice, transcendent summons, prosocial orientation, purposefulness, identity and pervasiveness than students without a mentor during the en- tire data collection (t test ranged from t(239) = -2.99 to t(231) = -4.87). Regarding the effects of the mere presence of a mentor on calling de- velopment, we found a significant two-way interaction between the pres- ence of a mentor at T2 and time on Sacrifice, F(2,418) = 6.36, µ2 = .03, and Pervasiveness, F(2, 419) = 4.05, µ2 = .02. The significant interaction of time and presence of a mentor means that the groups’ level of calling chang- es over time and in different ways depending on the presence of a mentor. Specifically, sacrifice and pervasiveness decrease significantly over time only for students with a mentor at T1 and T2 who lose it at T3 (paired t-tests for sacrifice: tT2-T3(31) = 2.46, p = .02; and pervasiveness: tT2-T3(31) = 2.11, p = .04). However, this result was observed on a small sample of 33 students. if First, we tested the effect of the mere presence of a mentor on calling level and development. Figure 1 show interaction and main effects of the presence of a mentor on calling facets. ANNA DALLA ROSA, MICHELANGELO VIANELLO, BARBARA BARBIERI ANNA DALLA ROSA, MICHELANGELO VIANELLO, BARBARA BARBIERI 10 and mentors influence each other over time. Models were estimated us- ing MPlus 6.11 (Muthén, Muthén 1998–2012). To choose the best-fitting model between alternatives, we used the chi-square test of close fit, the difference in comparative fit index (CFI; Bentler, 1990), and the standard- ized root mean squared of residuals (SRMR). We interpreted a difference in CFI and SRMR greater than .01 as evidence that the least parsimo- nious model should be chosen (Chen 2007; Cheung, Rensvold 2002). 5. Results 5. Results 4.3 Analytical Procedure To test the effect of the mere presence of a mentor on calling (hy- potheses 1 and 2), generalized linear models for repeated measures were adopted. The dependent variables were the facets of calling (within sub- ject variables). The independent variables were the presence of a men- tor at T1, T2 and T3 (between-subject variables with 2 levels: with and without a mentor). A full factorial design was estimated with the main effects of both the within (time) and between subjects (presence of a mentor) factors, and the interaction terms between presence of mentor and time. In addition, we performed t-tests and post-hoc analyses in or- der to test whether the dependent variables significantly increased over time within each group, and if the differences in the level of dependent variables were significant within time and groups.f gi g p To test the effects of mentors’ orientation toward work on students’ experience of having a calling (Hypothesis 3) we estimated and compared four alternative nested panel models (Little, Preacher, Selig, Card 2007; Selig, Little 2012). In each model, we specified alternative longitudinal relationships between mentor’s orientation (job, career and calling) and students’ calling (seven facets) and orientation toward work (job, career, calling). Model 1 was a baseline autoregressive model (no lagged effects) which estimated the effect of a construct on itself over time and the cor- relations between different constructs evaluated at the same time point. The second model added the cross-lagged paths from mentor orienta- tion toward work at T1 and T2 to students’ orientation toward work and calling respectively at T2 and T3. The third model added to Model 1 the cross-lagged path from students’ calling and orientation toward work at T1 and T2 to mentor’s orientation toward work respectively at T2 and T3. In this model, the opposite of Model 2, measures of student calling and calling orientation influences their mentor’s orientation toward work. This alternative is possible, for example, if students chose a mentor accord- ing to their orientation toward work. Finally, the fourth model estimated all the cross-lagged structural patterns to test the hypothesis that students THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS Finally, there is a signifi cant interaction eff ect between time and the presence of a mentor at T1, 2 and 3 on passion, F(2,418) = 3.38, µ2 = .02. Passion increases over time for students who had an unstable pres- ence of a mentor over time (tT1-T2(187) = -2.44, p = .02; tT1-T3(190) = -3.16, p = .002). if p We did not found signifi cant interaction eff ects on transcendent sum- mons, however we observed that it decreases signifi cantly over time for students with a mentor (tT1-T2(116) = 3.74, p < .001) and with an unstable presence of a mentor (tT1 T2(173) = 3.37, p < .001; tT1 T3(173) = 2.87, p < .001). We did not found signifi cant interaction eff ects on transcendent sum- mons, however we observed that it decreases signifi cantly over time for students with a mentor (tT1-T2(116) = 3.74, p < .001) and with an unstable presence of a mentor (tT1-T2(173) = 3.37, p < .001; tT1-T3(173) = 2.87, p < .001). Finally, purposefulness increases for students with a mentor (tT1-T2(122) = -4.05, p < .001; tT1-T3(122) = –3.52, p = .001), for students without a mentor (tT1-T3(117) = -2.04, p = .04), and for the group with an unstable presence of a mentor (tT1-T2(189) = -5.00, p < .001; tT1-T3(188) = -4.19, p < .001). p ( T1-T2( ) p T1-T3( ) p ) Finally, purposefulness increases for students with a mentor (tT1-T2(122) = -4.05, p < .001; tT1-T3(122) = –3.52, p = .001), for students without a mentor (tT1-T3(117) = -2.04, p = .04), and for the group with an unstable presence of a mentor (tT1-T2(189) = -5.00, p < .001; tT1-T3(188) = -4.19, p < .001). p ) These results provided a mixed picture of whether the presence of a mentor is associated with an increase or decrease in calling over time. Sacrifi ce and pervasiveness tend to decrease signifi cantly when students lose their mentor. Both prosocial orientation and passion increase over time for students who have an unstable presence of a mentor over time. ure 1 – Interaction and main eff ects of the mere presence of a mentor on students’ calling. Figure 1 – Interaction and main eff ects of the mere presence of a mentor on students’ calling. 5.1 Hypotheses 1 and 2: effects of the mere presence of a mentor We found a significant main ef- fect of mentor at T1 on passion (F(1,419) = 8.20, µ2 = .02), transcendent summons (F(1, 380) = 11.54, µ2 = .03), sacrifice (F(1,415) = 4.64, µ2 = .01) and pervasiveness (F(1, 420) = 7.88, µ2 = .02). Thus, students with a mentor showed different levels of calling depending on the presence of a mentor at Time 1. Specifically, within each time, students with a mentor have higher levels of calling than students without a mentor. We then analyzed the difference in the level of calling between the group of stu- dents with a mentor and the group without a mentor. Results suggest that students with a mentor at Time 1, 2 and 3 have higher levels of passion, sacrifice, transcendent summons, prosocial orientation, purposefulness, identity and pervasiveness than students without a mentor during the en- tire data collection (t test ranged from t(239) = -2.99 to t(231) = -4.87). Regarding the effects of the mere presence of a mentor on calling de- velopment, we found a significant two-way interaction between the pres- ence of a mentor at T2 and time on Sacrifice, F(2,418) = 6.36, µ2 = .03, and Pervasiveness, F(2, 419) = 4.05, µ2 = .02. The significant interaction of time and presence of a mentor means that the groups’ level of calling chang- es over time and in different ways depending on the presence of a mentor. Specifically, sacrifice and pervasiveness decrease significantly over time only for students with a mentor at T1 and T2 who lose it at T3 (paired t-tests for sacrifice: tT2-T3(31) = 2.46, p = .02; and pervasiveness: tT2-T3(31) = 2.11, p = .04). However, this result was observed on a small sample of 33 students. if p We found two significant interaction effects on prosocial orientation, specifically between the presence of a mentor at T2 and 3 and Time, F(2,417) = 3.45, µ2 = .02, and between time and the presence of a mentor at T1 and 3, F(2,417) = 3.23, µ2 = .02. Specifically, prosocial orientation increases over time for the group of students with an unstable presence of a mentor over time (tT1-T2(188) = -2.46, p = .01; tT1-T3(188) = -3.27, p < .001). 11 THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS Figure 1 Interaction and main eff ects of the mere presence of a mentor on students calling. 12 ANNA DALLA ROSA, MICHELANGELO VIANELLO, BARBARA BARBIERI Different lines represent groups of students with a mentor (n = 123), without a mentor (n = 118) and with a non-stable presence of a mentor across the data collections (n = 193). Bars represent 95% confidence inter- val. Across time, students with a mentor showed higher levels of passion and sacrifice than students without a mentor. Pervasiveness was higher at Time 1 for students with a mentor compared to the other groups. Tran- scendent Summons was lower at Time 1 for students without a mentor compared to the other groups. 5.2 Hypothesis 3: The longitudinal effect of mentors’ orientation on protégés’ orientation and calling 5.2 Hypothesis 3: The longitudinal effect of mentors’ orientation on protégés’ orientation and calling To test the third hypothesis, we analyzed the effect of mentor’s orien- tation toward work on student’s orientation and calling. Table 1 shows model comparisons and fit indices for the four competing models. The models have a moderate fit to the data; CFI is around .90, with RMSEA lower than .06 and SRMR lower than .13. One possible reason for the non-excellent fit is that the paths between different dimensions of call- ing over time are not estimated. Since the focus of the analysis was the effect of mentors’ orientation, it was decided to not modify the models and to focus on comparisons. Table 1 – Results of Nested-Models comparisons. ΔCFI ΔRMSEA ΔSRMR Δχ2 Δdf p Winner Model 1 vs. 2 -.01 -.002 .024 102.38 60 <.001 Model 2 Model 1 vs. 3 -.006 -.003 .004 88.15 57 .005 Model 1 Model 1 vs. 4 -.014 -.005 .029 186.18 116 <.001 Model 4 Model 2 vs. 4 -.004 -.003 .005 83.803 56 .01 Model 2 Note. Model 1 - autoregressive: χ2(456) = 1064.80; CFI = .896; RMSEA = .055; 95% CI [.051-.06]; SRMR = .13. Model 2 - Mentor influences protégé: χ2(396) = 962.41; CFI = .906; RMSEA = .057; 95 CI [.053-.062]; SRMR = .106. Model 3 - Protégé influences Mentor: χ2(399) = 976.64; CFI = .902; RMSEA = .058; 95% CI [.053-.062]; SRMR = .126. Model 4 - Reciprocal causation model: χ2(340) = 878.61; CFI = .91; RMSEA = .06; 95% CI [.056-.065]; SRMR = .101. Table 1 – Results of Nested-Models comparisons. Note. THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS Model 1 - autoregressive: χ2(456) = 1064.80; CFI = .896; RMSEA = .055; 95% CI [.051-.06]; SRMR = .13. Model 2 - Mentor influences protégé: χ2(396) = 962.41; CFI = .906; RMSEA = .057; 95 CI [.053-.062]; SRMR = .106. Model 3 - Protégé influences Mentor: χ2(399) = 976.64; CFI = .902; RMSEA = .058; 95% CI [.053-.062]; SRMR = .126. Model 4 - Reciprocal causation model: χ2(340) = 878.61; CFI = .91; RMSEA = .06; 95% CI [.056-.065]; SRMR = .101. The chi-square difference tests between Models 1, 2, and 4 were sig- nificant, and the differences in CFI and/or SRMR were larger than .01. Model 2 and 4 fit the data better than Model 1. On the contrary, the differences in CFI and SRMR suggested that Model 3, which specifies student’s calling as a predictor of mentor’s orientation toward work, did not fit the data better than Model 1 and was then rejected. i j Models 2 and Model 4 were then compared in order to identify which types of causal relationship, mentor to protégé or reciprocal, better de- THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS 13 scribes our data. Model 4 (reciprocal causation) was favored over Model 2 by the test of close fi t, but not by the diff erences in CFI and SRMR, which were lower than .01. Hence, we accepted Model 2 as the most parsimonious. This result suggests that mentors’ orientation toward work infl uence student’s calling and orientation. Figure 2 reports the non-null eff ects of mentor on students’ calling. Figure 2 – Model 2: Statistically signifi cant paths from mentors’ orientation toward work on students’ levels of calling and calling orientation. Covariances and autoregressive paths are not depicted for clarity. O. = Orientation toward work. The eff ect of a mentor on calling is mainly due to the career and job orientation toward work. Mentor’s job orientation negatively infl uences students’ passion, sacrifi ce, and pervasiveness at T2, and negatively in- fl uences students’ transcendent summons, purposefulness, and calling orientation at T3. The mentor’s job orientation has a positive eff ect on students’ job orientation. Having a mentor who is interested in materi- al benefi ts from work reduces student’s passion, willingness to sacrifi ce, pervasiveness, purposefulness and transcendent summons. THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS In addition, having a mentor with a job orientation promotes the same attitude on students and discourage a calling orientation toward work. M t ’ i t ti t T1 h ti ff t t d t The eff ect of a mentor on calling is mainly due to the career and job orientation toward work. Mentor’s job orientation negatively infl uences students’ passion, sacrifi ce, and pervasiveness at T2, and negatively in- fl uences students’ transcendent summons, purposefulness, and calling orientation at T3. The mentor’s job orientation has a positive eff ect on students’ job orientation. Having a mentor who is interested in materi- al benefi ts from work reduces student’s passion, willingness to sacrifi ce, pervasiveness, purposefulness and transcendent summons. In addition, having a mentor with a job orientation promotes the same attitude on students and discourage a calling orientation toward work.f g g Mentor’s career orientation at T1 has a negative eff ect on students sacrifi ce and pervasiveness, and promotes students career orientation at T2. Having a mentor interested in career and success, promotes in the protégé the same interest in career and success, while it reduces willing- ness to sacrifi ce and pervasiveness. 14 ANNA DALLA ROSA, MICHELANGELO VIANELLO, BARBARA BARBIERI Mentor’s calling orientation has a positive significant effect on stu- dents’ identity at T2 and passion at T3. So, when mentors are passion- ate, when they live out their work as a vital part of their life, students tend to develop higher identification and passion in their calling domain. 6. Discussion This study is, to our knowledge, the first investigation of the effect of mentoring on the development of calling. In agreement with hypothesis 1, we found that students with a mentor show a higher calling than stu- dents without a mentor within three time points. Second, the presence of a mentor was expected to increase the level of calling over time (Hy- pothesis 2). Our results did not provide a clear support for this hypoth- esis. The mere presence of a mentor is related to both an increment and decrement in calling: it is possible that individuals vary much on their calling development (Vianello, Galliani et al., Unpublished manuscript) and that other variables intervene in explaining how the presence of a mentor influences changes in calling over time.f l g g To test the third hypothesis we analyzed the effect that a mentor’s orientation has on the students’ sense of calling and orientation. A men- tor was expected to shape the protégé’s sense of work as a calling, a job, or a career, because there is evidence that a mentor influences their pro- tégé’s attitudes (Allen, Eby, Poteet, Lentz, Lima, 2004; Eby et al. 2008), and because people look to others for cues regarding how to think and behave (Social Learning Theory; Bandura 1971; Social Information Pro- cessing Theory; Salancik, Pfeffer 1978). The results support the presence of a longitudinal effect of a mentor on a student’s calling and attitude to- ward work. The model with a mentor’s job, career and calling orientation as predictors of students’ calling (Model 2) better represented the data. Results suggest that having a mentor interested in career advancement or financial gain inhibits the development of a calling. Indeed, mentor job and career orientation emerged as strong negative predictors of pro- tégé calling. If examples of people with a calling are not available, stu- dents might be less willing to find or look for their calling. On the other hand, the calling orientation of a mentor is associated to an increase in students’ identification in and passion for the calling domain. Therefore, it seems that the role of a mentor can be more useful if it allows an in- dividual’s calling to be expressed in a particular job, profession, role or area of life, rather than orientating the calling to a particular job or role. THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS 15 with a job or career orientation toward work may prevent the develop- ment of a calling. Thus, we do not know why students with a mentor have a higher level of calling than students without a mentor. One pos- sible explanation is that students with higher levels of calling are more likely to look for a mentor. This theoretical account was not analyzed in this study, and represents one of the open questions that may be ad- dressed in future research. Allen T.D., Eby L.T., Poteet M.L., Lentz E. and Lima L. 2004, Career benefits associated with mentoring for protégés: a meta-analysis, «Journal of applied psychology», LXXXIX (1), 127-136. 6. Discussion g g p j Even if these results are only tentative, they contribute to the litera- ture on calling in many ways. Having a mentor is associated with high- er initial calling and mentor’s orientation influences a protégé’s calling orientation and levels of calling facets toward work. Having a mentor THE ROLE OF MENTORS ON THE DEVELOPMENT OF CALLING IN STUDENTS 7. Limitations and future directions Further analyses are needed to clarify some results. First, we did not investigate thoroughly the development of calling for students who lost and/or found their mentor during the data collection (unstable presence of a mentor). Thus, further research is needed in order to extend our re- sults. Second, we did not investigate whether variables such as gender, duration of and satisfaction with the mentoring relationship, the type of mentorship relation (formal or informal), and the quality of the men- toring (role modeling, vocational and psychological support), intervene in explaining the influence of a mentor on students’ calling orientation. Literature on mentorship suggests that gender is a key factor in influ- encing people’s choices, quality of relationship and the effectiveness of a mentorship (Ragins, Cotton 1991; Scandura, Williams 2001). Formal and informal mentoring relationships (Ragins et al. 2000; Kram 1985) have different origins and different developments; we might expect an informal mentor to be more effective in terms of influencing a student’s attitude toward work than a formal mentor. In addition, how much a person considers the mentor as a role model or the level of psychologi- cal and vocational support provided by the mentor may explain the as- sociation between mentor’s and protégé’s calling and orientation toward work that we found. As applied to mentoring relationships, role modeling can be seen as a form of relational identification (Mitchell, Eby, Ragins 2015); role modeling involves the protégé identifying with the mentor and the protégé internalizing valued aspects of the mentor into his or her self concept (Kram 1985). Third, a critical point in these results is that mentor orientation toward work was assessed only by students. Thus, a study involving both mentors and protégés is needed. 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THE PURSUIT OF HAPPINESS: A REFLECTION ON THE CALLING CONSTRUCT Barbara Barbieri (University of Cagliari)* Barbara Barbieri (University of Cagliari)* It is precisely upon this point that I will try to reason: on the implications of what we should (as academics) 20 BARBARA BARBIERI always keep in mind when we are called to think about career paths for students, and how it could be important to help students develop a call- ing, by putting them in the conditions of making an informed and pas- sionate choice about their future working life. In fact, the topic is central for those research fields that focus on creating value within organizations, within a wider scenario which is that of the knowledge economy, where the gaze is increasingly shifted from human capital (what you know) to psychological capital (who you are) as a possible competitive advantage for organizations, in which the search for ‘talents’ becomes a necessary condition to remain competitive in the marketplace. Talent is a keyword within the theme of the calling, because one of the questions about this construct is just this: is it a gift or a talent? always keep in mind when we are called to think about career paths for students, and how it could be important to help students develop a call- ing, by putting them in the conditions of making an informed and pas- sionate choice about their future working life. In fact, the topic is central for those research fields that focus on creating value within organizations, within a wider scenario which is that of the knowledge economy, where the gaze is increasingly shifted from human capital (what you know) to psychological capital (who you are) as a possible competitive advantage for organizations, in which the search for ‘talents’ becomes a necessary condition to remain competitive in the marketplace. Talent is a keyword within the theme of the calling, because one of the questions about this construct is just this: is it a gift or a talent? h b h h l h There is no unambiguous answer to this in the literature, however it seems more interesting, for the sake of reasoning, to remain within the dimension of talent, since a talent requires exercise and an area of action in which its practice is defined. As has already been said, the authors of this study have proposed a multidimensional and integrated definition of the Calling Construct, which describes the relationship between individuals and a specific do- main in life or work. Barbara Barbieri (University of Cagliari)* Abstract: This brief chapter arises from reflections on the research studies re- lating to the Calling construct, carry out by colleagues from the University of Padua. Basically this is a reasoning on the implications of which we should (as academics) always keep in mind when we are called to think about career paths for students, and how it could be important to help students to develop a Calling, putting them in the conditions of making an aware and passionate choice about their future working life. Keywords: calling; multidimensional construct, vocational behavior, students career, University of Padua. This brief chapter arises from reflections on research studies relating to the Calling Construct presented by colleagues from the University of Padua (Dalla Rosa, Galliani, Vianello 2014; 2017; Dalla Rosa, Vianello, Anselmi 2017) at the Final Event of the Project Employability & Compe- tencies. Innovative Curricula for New Professions. Certainly, as my colleagues have suggested, from a theoretical point of view, a calling is a complex multidimensional construct that describes spiritual, motivational, affec- tive, and identity-related facets of the relationship between individuals and a specific domain in life or work (Wrzesniewski et al. 1997; Dik, Duffy 2009; Elangovan et al. 2010; Dalla Rosa, Galliani, Vianello 2016). The longitudinal research carried out by colleagues on the development of a calling – the sample consisted of Italian college students – during three years of university, highlighted, very clearly, that calling is far from being a stable construct such as a personality trait, but is extremely dy- namic. In addition, ‘being called’ is not a condition for engaging in do- main-related activities or for developing a clear idea of our professional future, but quite the opposite, if anything: the more students have a clear idea of their future and the more they engage in learning activities, the more they will develop a calling. The findings underlined some impor- tant practical implications for both professors and university governance, namely, that if we foster students’ engagement and if we let them explore many different domains, we help them feel passion for a domain and find a meaning in their work and their life. THE PURSUIT OF HAPPINESS: A REFLECTION ON THE CALLING CONSTRUCT 21 research on vocational behaviour, and to provide vocational guidance. Furthermore, in the light of the above, it would be interesting to under- stand what facilitates the development of a calling in relation to its so- cial and structural setting, for example, the opportunities offered by its environment (both in training and work environments). From an organizational point of view, if we consider a calling an outcome, it might be interesting to explore it within the framework of Organizational Support Theory (Eisenberger et al. 2001), where some objective social conditions can facilitate the emergence of a calling, for example in new hires. instead, within the Job Demands-Resources Model (Bakker, Demerouti 2007), it would be interesting to explore the role of the calling as a personal resource, able to, at least hypothetically, mod- erate or mediate the relationship between perceived or required costs by the organization as well as certain dimensions of stress and burnout. y g Lastly, another intriguing perspective in which to explore calling is the relationship between motivation and the Regulatory Mode Theory (Pierro, Kruglanski, Higgins 2006) on goal-pursuit. It would be inter- esting, within this framework, to understand the relationship between a calling and goal attainment, namely the function of a calling between the steady pursuit of an object, and flexibility in the lens remodelling. To conclude, I would like to emphasize this: the importance of the methodological and theoretical systematization concerning the Call- ing Construct, carried out by colleagues from the University of Padua, which underscores not only the complexity of the construct but also the necessity to pursue, in further researches, a more shared definition of what a calling is, to clarify its place in a nomological network of similar constructs; the importance of a calling in the practical implications for professional choices. Barbara Barbieri (University of Cagliari)* Hence, although it is a construct whose dimen- sions are individual, and in turn, these dimensions can be considered as individual personal resources, it should however be explained within a social relationship, for the above reasons.i p Indeed, the findings of this research underline that a calling is an outcome of something, rather than a predictor of something else. In this regard, it seems important to emphasize what Hall & Chandler (2005) have pointed out regarding the definition and domain (subjective vs. objective) of calling, namely, the need to abandon an ‘either/or’ logic. Instead, it is the interdependence between the two domains that is the central question, to wit, the relationship between subjective and objec- tive, or between individual and organization. Though a calling is some- thing we ‘feel’, if we move from a transcendent dimension, it can be ‘felt’ only if it is tied to the dimension of doing, or can be ‘discovered’ only through doing, or ‘found’ within the process of attributing meaning to acting, which drives us to pursue a goal. Within this relationship be- tween subjective and objective, the finding of the Calling Study tells us that the greater the engagement, the higher the chances of developing a calling. Therefore, it could be said that a calling is a talent that emerges. g g g Despite being rooted in the well-established theoretical and empiri- cal tradition of vocational psychology, and in the more recent sphere of positive psychology, research into the Calling Construct is still in its pre- liminary stages (Dalla Rosa, Galliani, Vianello 2014). A growing interest in its theoretical implications and potential appli- cations (e.g. career counseling) make this construct important both in THE PURSUIT OF HAPPINESS: A REFLECTION ON THE CALLING CONSTRUCT References Bakker, A.B. and Demerouti E. 2007, The Job Demands-Resources model: State of the art, «Journal of Managerial Psychology», XXII (3), 309-328. Dalla Rosa A, Vianello M. and Anselmi P. 2017, What does it mean to have a calling? A construct validation of the Integrated Multidimensional Calling Scale. DOI:10.17605/osf.io/86ink. Dalla Rosa A., Galliani E.M. and Vianello M. 2014, Theories and Measures of Occupational Calling: A Review and Research Agenda, «Educational reflective practices», 2, 99-121. Dalla Rosa A., Galliani E.M. and Vianello M. 2017, An integrative model of Career Calling and meta-analyses of its nomological network, in Boffo V., Fedeli M., Lo Presti F., Melacarne C., Vianello M. (eds.), Employability, Teaching and Learning, and Guidance: New Strategies in Higher Education, Pearson, London, 1-49. 22 BARBARA BARBIERI Dik B.J. and Duffy R.D. 2009, Calling and vocation at work definitions and prospects for research and practice, «The Counseling Psychologist», XXXVII (3): 424-450. DOI: 10.1177/0011000008316430. ( ) Eisenberger R., Armeli S., Rexwinkel B., Lynch P.D. and Rhoades L. 2001, Reciprocation of perceived organizational support, «Journal of Applied Psychology», LXXXVI (1), 42. Elangovan A.R., Pinder C.C. and McLean M. 2010, Callings and organizational behaviour, «Journal of Vocational Behavior», LXXVI (3), 428-440. DOI: 10.1016/j.jvb.2009.10.009. Hall D.T. and Chandler D.E. 2005, Psychological success: When the career is a calling, «Journal of Organizational Behavior», XXVI (2), 155-176. Pierro A., Kruglanski A.W. and Higgins E.T. 2006, Regulatory mode and the joys of doing: Effects of ‘locomotion’ and ‘assessment’ on intrinsic and extrinsic task-motivation, «European Journal of Personality», XX (5), 355-375. Wrzesniewski A., McCauley C., Rozin P. and Schwartz B. 1997, Jobs, careers, and callings: People’s relations to their work, «Journal of Research in Personality», XXXI (1), 21-33. DOI: 10.1006/jrpe.1997.2162. Monica Fedeli (University of Padua)* Monica Fedeli (University of Padua)* Abstract: This chapter presents a literature overview of the student voice in higher education, the main results from a three-year research project conducted at five Italian universities with the aim of investigating students’ voices in higher education and how they offer insight into fostering Faculty Development. Keywords: teaching and learning methods, student voice, Faculty Development, professional development. * Monica Fedeli, PhD, Associate Professor of Teaching and Learning, and Organizational Development, Department of FISPPA, University of Padua, Italy. Email: monica.fedeli@unipd.it. 1. Introduction The report on Improving the Quality of Teaching and Learning in Eu- rope’s Higher Education Institutions (European Commission 2013) de- livered a strong message that highlighted key issues for universities in Europe. The report states: «Higher education institutions need to create environments and feedback mechanisms and systems to allow students’ views, learning experience, and their performance to be taken into account» (p. 28). When considering this recommenda- tion in relation to Italian Universities, despite their long and pres- tigious history of scholarship and research, the teaching approaches predominantly reflect traditional transmission model practices (e.g., Pratt et al. 1998), with little direct and active participation by students in course design and curriculum development. This lack of partici- pation is reflected in the classroom by an over-reliance on didactic teaching practices, highly formal student/faculty relationships, and little access for student input in course curricula. Counter to this historical tradition and like other universities in Europe, there is a growing interest in Italy for a better understanding of what faculties are really doing in the classroom and the degree of innovation that might actually be taking place. To respond to this need for didac- tic innovation and the modernization of teaching, a consortium of universities (Padua, Florence, Siena, Naples Parthenope, and Rome La Sapienza) developed the PRIN EMP&Co project, and the team from the University of Padua investigated the teaching and learning 26 MONICA FEDELI methods at Italian Universities, starting from the students’ perspec- tive. Based on the results of the survey, which provide a clear-cut im- age of the students’ perceptions and highlight how traditional higher education teaching is still based on lecturing, formal relationships, and on practices in which the instructor is more of a «Sage on the Stage» than a «Guide on a Side» (Morrison 2014), we have been en- couraging the implementation of Faculty Development opportuni- ties for instructors from the universities involved in the EMP&Co project since 2014. p j The strong message of the European Commission and the pecu- liarly Italian context encourages the development of discussions of and research into higher education teaching. One of the more rel- evant needs that has emerged is to break the students’ silence, to lis- ten to their views on classroom teaching, and to include them in the innovation process. 2. The Student Voice: an overview Too often students in higher education are not listened to. They passively attend lectures and are not encouraged to express their voices and actively participate. The student voice is one of the main theo- retical frameworks of this EMP&Co project. Notionally, it should be framed within learner-centred teaching, an umbrella concept, seen as a «new way of understanding, interpreting, or viewing some- thing» (Meyer, Land, 2005 372). This represents not only a different approach to teaching, but a new understanding of learning that in- cludes paying serious attention to the students’ needs and appreciat- ing their experiences and points of view in the classroom. (Blackie, Case, Jawitz 2010; Spalding 2014; Weimar 2013). J p g ) Students are the most important actors of the learning process, and therefore including their perspectives in the planning and pro- gramming of teaching and the curriculum is essential. At the same time, instructors can improve their teaching as they listen to their students as partners in the learning process. Implicit in this are the assumptions that students’ feedback and evaluation have an impact on the transformation of teaching practices, and that students are more involved if they can participate and take an active part in the process. Despite the importance of the student voice, the higher educa- tion literature on this construct is very limited. What is known is that student-faculty partnerships are rooted in beliefs that highlight the importance of considering: students’ insights to make teaching engaging; the value of listening to their voices in designing teaching THE STUDENT VOICE IN HIGHER EDUCATION 27 and curricula; and the potential of changing perspectives for both to become better teachers and students. (Cook-Sather et al. 2014). Ben- efits for staff, students, and institutions include enhanced engagement, motivation, and learning; metacognitive awareness and a sense of identity; teaching and classroom experiences; student-staff relation- ships and development of a range of graduate attributes (Brockett 2015; Cook-Sather et al. 2014; Dirkx 2006; Spalding 2014; Taylor 2007). When students take responsibility and an active role in the learning process, they become more aware of what is being learned, changing their role from passive actors to active ones and fostering a related reorientation of the academic staff (Baxter-Magolda 2006; Cook-Sather et al. 2014). 3. The Student Voice: suervey design 3. The Student Voice: suervey design 3. The Student Voice: suervey design The research realized as part of the EMP&Co project (Boffo, Fedeli et al., eds.) was the most extensive survey of data explor- ing the students’ voice in higher education in Italy. The question- naire was submitted to over 50,000 students attending a variety of courses throughout the participating universities. It offers a unique national perspective of adult and higher education on a scale rarely presented and its potential is likely to lead to significant innova- tions in teaching and learning methods in both adult and higher education in Italy.ii y The findings of the study were organized around five focus areas: 1. Course organization; 2. Creation of a participatory environment; 3. Methods and resources for teaching and learning; 4. Feedback and assessment; 5. Work-related learning and university business dialogue. Each area was represented by different items, 35 in total, asking students what percentage of instructors (0%-25%, between 25% and 50%, between 51% and 75%, more than 75% and ‘I don’t know’) were engaged in these learner-centred teaching practices. Each focus area is discussed below. 2. The Student Voice: an overview i ) Although there are many benefits, engaging the student voice does require the Faculty to take some risks and transform their role as educators (disciplinary content experts) to include their role as a facilitator, and work collaboratively with the students (Bovill, Cook- Sather, Felten 2011; Nygaard et al. 2013) with particular attention to students’ interests and needs (Gentile 2014; Quinn, Owen 2016). ( Q ) Even though there is a growing interest in teaching innovations in higher education, the student voice as a construct and a practice is not given the necessary attention when developing instructional practices (Smyth, McInerney 2012). Typically, teaching is related to the instructor’s design and students have little influence on teach- ing approaches and curriculum development. (Bovill, Cook-Sather et al. 2015). Instructors are resistant to change and encouraging stu- dents’ engagement. Furthermore, instructors in Italy generally have more formal relationships with students, which can discourage a frank and open climate for discussion. Furthermore, some of our classrooms accommodate a considerable number of students, up to two or three hundred, so that it can be even more difficult to build a rapport in this situation. Moreover, institutional norms and practices in the Italian higher education system (managed by the government and the universities): for example, the considerable number of exam sessions, the free- dom of the students to attend many courses or not, an over-reliance of certain programmes on a fixed curriculum and the institutions’ overall lack of flexibility, do nothing to help create a collaborative teaching environment. Alternative strategies to promote innovation in our situations must be sought and applied to this context. This will be the challenge to face to create our own ‘Italian-style’ inno- vation and modernization. The next paragraph will present some of the survey results col- lected within the EMP&Co project and analysed through the lens of student voice literature. 28 MONICA FEDELI 5. Creation of a participatory environment The second set of questions was related to the class setting and the relational climate concerning the degree of questioning, discussing, and reflection on students’ experiences and prior knowledge in each Faculty. This section consisted of (4) questions titled Creation of a par- ticipatory environment (α =.799). These questions aimed to explore the students’ perceptions of their participation in the classroom and whe- ther their ideas or proposals were taken into consideration by the in- structors and other classmates. 6. Methods and resources for teaching and learning The teaching methods surveyed in this section focused on active learning. Active learning is defined as «any instructional method that en- gages students in the learning process» (Coryell, 2016). Italy has a long tradition with instructors (Morrison, 2014), predominantly lecturing and demonstrating little interest in encouraging discussion and the shar- ing of student experiences. The aim of these items (9) was to investigate the methods and resources that the instructors devised for their classes (α =.756). These methods included group work or individual work, a flipped classroom, the sharing of studies and research on the topic, and adaptation of the type of explanation or lecture to the students’ level and skills. In contrast, the survey also asked students about the extent of PowerPoint use and how the instructors used it (e.g., reading the slides aloud, or integrating with real examples). Another focus was the deliv- ery/availability of new and varied resources on some of the proposed topics for students to explore more deeply. THE STUDENT VOICE IN HIGHER EDUCATION THE STUDENT VOICE IN HIGHER EDUCATION 29 4. Course organization The first area of the survey aimed to explore the introductory part of the course and its organization (α =.801). The items (7) in- vestigated whether the faculty explained the programme at the be- ginning of the course, and presented the related contents. This is generally the first information to be shared with students and even- tually discussed and negotiated. The other questions were related to the textbooks and resources in use for the course and the reason for the choice, so that the students have a better understanding and can support the instructors and their proposals. Furthermore, it is im- portant that the instructor explain what the students need to know, and which skills they must develop during and after the course. The last two questions of this set were addressed to the students’ needs and desires, to understand whether there were any additions need- ed to the programme presented and if the instructor was willing to change the programme based on the considerations that emerged in the discussion with the students. 8. Work-Related Learning and University-Business dialogue The last and fifth area (α =.837), asked students to express their perceptions on work-related teaching and learning methods to pro- mote the dialogue with work and business partners. This approach is defined in American literature as Work-Related Learning (WRL) (Dirkx 2011) and in Australian literature as Work-Integrated Learning (WIL) (Cooper, Orrell, Bowden 2010; Gardner, Barktus 2014). This area of the questionnaire consisted of seven questions that aimed to understand how instructors foster/encourage a dialogue with busi- ness and work partners in their teaching, whether they explain the connections between course contents and related professions, and if they consider and encourage students to reflect on the ethical aspects of professions and their utility. Students were also asked whether the instructors invite representatives of the job market to their classes, or had ever proposed activities or projects in cooperation with organi- zations and professional communities. 7. Feedback and assessment The fourth area investigated the activities of feedback and assess- ment (α = .678). This set of questions aimed to better understand the students’ perceptions and how visible the processes of feedback and assessment were. The questions (8) investigated whether the instruc- tors clearly communicated the methods and criteria of the assessment process in class. In addition, we asked students whether the instructors promoted self- and peer-evaluation in the group along with timely feedback during the activities they proposed in the classroom. The last two questions focused on the exam and how it was organized, if it was a rigidly pre-set type of exam or divided into separate parts to give students different tests and opportunities. 30 MONICA FEDELI 8. Work-Related Learning and University-Business dialogue THE STUDENT VOICE IN HIGHER EDUCATION 31 teaching. Furthermore, the current national evaluation for faculty career advancement in Italian Universities is based exclusively on re- search ranking and very little attention is paid to the teaching and other organizational or administrative tasks. In addition, instructor- student relationships at the University are very formal, distant, and hierarchical. Most of the professors focus more on their performance in the classroom and less on the students’ interest. Students’ percep- tions highlight a low level of engagement and collaboration between students and instructors as promoted by the student voice. Consistent with this perspective, assessment and feedback are integrated in the learning and teaching process. (Fedeli, Frison, Grion 2016). These data confirm that these processes are still managed only by the in- structors, who are leading the teaching and assessment process with- out listening to the students or negotiating some of the criteria to be assessed with them. 9. The Student Voice: making sense of the findings The survey was completed by 3,760 students (2,453 females equal to 65.2% and 1,307 males equal to 34.8%) from five Italian Univer- sities. After a test run at La Sapienza University of Rome and Siena University between December and January 2015 (Creswell, 2008), the questionnaire was administered to a sample of students enrolled during the 2014-2015 academic year in Bachelor’s Degree or Master’s Degree Courses, and the former regulation primary education pro- grammes, attending the last year of the legal term of the course, plus those attending the third year of all Master’s courses. Each was sent an invitation to complete the questionnaire on the Moodle platform. The survey data were downloaded into SPSS and Excel and analysed. The findings of this study were rather significant, reflecting a univer- sity system with a long and rich scholarly tradition with instructors who are somewhat resistant to innovative teaching approaches. The dominant teaching approach continues to be the practice of lectur- ing, with the Faculty fostering little interaction with students in the form of class discussions and group activities. Moreover, there is an obvious lack of attention in providing regular and consistent infor- mal and formal feedback to students, promoting peer- and self-eval- uation, and lastly, very few instructors are fostering dialogue with professional communities or promoting employability. i The survey was completed by 3,760 students (2,453 females equal to 65.2% and 1,307 males equal to 34.8%) from five Italian Univer- sities. After a test run at La Sapienza University of Rome and Siena University between December and January 2015 (Creswell, 2008), the questionnaire was administered to a sample of students enrolled during the 2014-2015 academic year in Bachelor’s Degree or Master’s Degree Courses, and the former regulation primary education pro- grammes, attending the last year of the legal term of the course, plus those attending the third year of all Master’s courses. Each was sent an invitation to complete the questionnaire on the Moodle platform. p p g p y y One way to make sense of the findings is to recognize that the Italian University system has a very long tradition of teacher-centred THE STUDENT VOICE IN HIGHER EDUCATION 10. Implications for Faculty Development and the Student Voice 10. Implications for Faculty Development and the Student Voice A recent significant result of this study was the creation of a Faculty Development Programme at the University of Padua. This project, called Teaching4Learning@Unipd, began in November 2016, and was a direct result of the survey, with the aim of train- ing the Faculty to promote students’ participation in their courses. In addition, there was also a growing interest in de-privatizing (Adam, Mix 2014), teaching through the development of Facul- ty learning communities that encouraged sharing beliefs and val- ues among colleagues, and supporting each other in innovating their teaching. The first learning communities were starting to form, introducing the use of an informal peer-observation pro- cess among instructors. This programme aims to encourage greater awareness of the deep assumptions about teaching and learning and to offer the opportu- nity to learn new methods and techniques that encourage student participation and involvement. Bit by bit, policies, public relations, and mission statements can be revised to promote new strategies with the intention of innovating teaching approaches and students’ participation in teaching and learning at universities. The instructors involved in this project are self-selected, strongly interested and motivated to participate, with a significant propensity to sharing their own experience with other colleagues. A group of experts at the University of Padua are working with a variety of departments, and are planning Faculty Development programmes. In the last two years, the results have been extremely 32 MONICA FEDELI positive. Eight out of 32 departments have offered training to mixed groups (assistant-, associate-, and full professors) of 25-30 instructors including a one-week long training session for instructors from all the departments, a group of 28 young instructors who took part in a residential summer school and talked and worked together to in- novate their teaching practices and share their thoughts and discuss their assumptions on teaching and learning. Small groups of instructors who volunteer to come to the train- ing sessions are starting to consider teaching an important devel- opmental factor for their community. They collaborate, and share their teaching practices with colleagues. This is a synergistic pro- cess among faculty they share first-hand experiences, practices, and emotions about teaching in which trusting relationships are developed leading to further de-privatization of teaching. THE STUDENT VOICE IN HIGHER EDUCATION THE STUDENT VOICE IN HIGHER EDUCATION 33 monthly meeting with all the deans of the 32 departments, regular updates of Faculty Development efforts are shared. A first attempt at a reward system has been developed by issuing a ‘badge’ (a kind of certificate) for those instructors who take part in a Faculty Develop- ment training programme of at least 25 hours. f Despite these efforts, the change process is slow and incremental, particularly among individual faculty. Instructors and students are of- ten resistant to new ways of teaching. More work needs to be done to help the Italian university to make sense of the nature of the insti- tutional context and how the traditional culture of teacher-centred teaching is a significant barrier to more innovative learner-centred approaches to teaching. 10. Implications for Faculty Development and the Student Voice Consis- tent also with the research on transformative learning (Mezirow 1990, 2000; Taylor 2007) concerning significant personal change among adults, it is «trustful relationships that allow individuals to have questioning discussions, share information openly, and achieve mutual and consensual understanding» (Taylor 2007: 179). Mul- tiple groups are starting to collaborate and create faculty learning communities (Cox 2004; Daly 2011; Nugent et al. 2008; Schlitz et al. 2009); learning communities (Mackenzie et al. 2010; Sherer, Shea, Kristensen 2003) of both interdisciplinary and disciplinary forms and differing in rank. In the next month, the Faculty Development programmes will be evaluated both as to how they were received by the Faculty and the related impact on classroom practices. This will be a first attempt to see whether there has been integration of innovative practices in the classroom. It is interesting to monitor them and understand how to improve and share insights and ideas throughout the process. p g g p This process of innovation is being promoted by the University management, which is starting to invest resources in developing or- ganizational measures to respond to the need for change of some in- structors, and to think how to develop policies to give more relevance to the teaching process in career evaluation. i The University is intending to allocate resources to finance more training and to create a reward system for those instructors who present projects that encourage student-instructor partnerships, and a higher evaluation of the teaching based on students’ perceptions. Furthermore, instructional technology courses are being organized and offered to all instructors to promote the use of technologies in teaching, introducing blended courses and technological tools. g g g The intention is to create a system that places a higher priority on teaching and make it a part of career advancement policies. In a 11. Conclusion The aim of this chapter was to share with the readers some of the results of the research developed within the PRIN EMP&Co project, in particular, the findings of the University of Padua unit, which was in charge of investigating teaching methods in our Italian univer- sity system. Further effort was made to highlight, through an over- view of the student voice, the importance of listening to students and thereby encourage actions that consider their perceptions and some of their expressed needs. p The results of the research gave staunch support to the promotion of Faculty Development at the University of Padua with the aim of finding our own ‘Italian way’ to transform teaching and learn- ing. The Faculty Development programme Teaching4Learning@ Unipd began based on the evidence of the survey, study of the lit- erature, and an investigation of national and international cases and situations, Efforts are continuing to find more formal and informal occasions to share the findings of this research project at confer- ences, workshops, seminars, and discussion circles at both national and international levels. These actions and results provide strong support in encouraging instructors to pay greater attention to the students’ voice. This project, because of its highly competitive na- ture, gives the Faculty Development initiative greater credibility among colleagues at Italian Universities. Finally, the findings of the project have been shared with the didactic commission of the uni- versity, scientific committees, and other university bodies. Finally, the many studies and publications on the findings have supported our willingness and conviction that it is time to teach in a differ- ent way, time to engage the students’ attention more in Italian and European Universities. 34 MONICA FEDELI References Adams S.R. and Mix E.K. 2014, Taking the lead in Faculty Development: Teacher Educators Changing the Culture of University Faculty Development through Collaboration, «AILACTE», 11, 37-56. Baxter M. 2006, Intellectual Development in the College Years, «Change: The Magazine of Higher Learning», XXXVIII (3), 50-54. 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Daniela Frison (University of Padua)* Daniela Frison (University of Padua)* Abstract: What kind of Work-Related activities/programmes do Italian teach- ers propose to their students? What are common elements considered in the de- signing of Work-Related activities by those proposing them? A multiple case study research programme was chosen as a method to explore the current Italian situation regarding Work-Related teaching and learning methods in Higher Education (Coll et al. 2008; Dirkx 2011) and to define some possible methodo- logical recommendations to encourage the design of Work-Related Learning activities/programmes. Keywords: work-related learning, higher education, case study, university- business dialogue. business dialogue. * Daniela Frison, PhD, Postdoc Researcher, Department of FISPPA, University of Padua, Italy. Email: daniela.frison@unipd.it. 1. Introduction The purpose of this paper is to explore and identify some possible methodological recommendations to encourage the design of work- related learning activities/programmes in higher education, to pro- mote links between theoretical contents and the professional world. The research was grounded on the National Project Employability and Competencies, and began in 2013 with the aim of exploring current teaching and learning methods in the Italian higher education system (Boffo, Fedeli, Lo Presti, Melacarne, Vianello 2017). 1.1 The theoretical and methodological background As for the theoretical and methodological background, the research is part of a scheme to modernize the European Higher Education sys- tem which emphasizes the need to enhance the quality of Tertiary Education through the involvement of the business world (European Commission 2011), and to encourage more interactive learning envi- ronments and partnerships with the job market (Commission of the European Communities 2006, 2009). 38 DANIELA FRISON This European focus on university-business cooperation and teach- ing and learning methods to link education and work, refer to what is called in North American literature Work-Related Learning (Dirkx 2011) and in Australian literature as Work-Integrated Learning (Cooper, Orrell, Bowden 2010; Gardner, Barktus 2014). Both terms concern the integration between formal and professional contexts, to create meaningful benefits for students, organizations, and other stakehold- ers (Gardner, Barktus 2014). ( , ) A previous literature review (Frison, Fedeli, Taylor 2015) allowed the research group to clarify terms, theoretical orientations, and prac- tical implications regarding Work-Related Learning. On the one hand, the term refers to a period spent in a professional environment with different purposes and objectives such as study, the development of generic or technical skills, and the exploration of possible career op- tions and job opportunities (Coll, Eames, Paku, Lay 2008). On the other hand, it is more widely related to «a complex educational phe- nomenon. It is at once a set of educational missions (e.g. careers edu- cation), a range of activities (work experience), a collection of topics (understanding credit and work) and a repertoire of teaching and learning styles» (Huddleston, Stanley 2012: 11). Furthermore, adopt- ing a broader definition offered by Dirkx (2011), Work-Related Learn- ing (WRL) is defined as the process of learning that may occur «in educational preparation programs apart from the workplace, in for- mal and informal learning within the workplace, and in further ed- ucation and professional development programs offered outside the workplace» (p. 294). i p (p ) The definitions mentioned suggest a connection with the formal context and the strategies and opportunities of a link between edu- cation and the world of work developed in the educational setting. They allude to an open category that encompasses work-related pro- grammes − for example internships − as well as didactic strategies to support students to better understand the relationships of theory- practice, education-profession (Litchfield, Frawley, Nettleton 2010). 1.1 The theoretical and methodological background Based on a literature overview and multiple definitions of Work- Related (or Integrated) Learning (Frison, Fedeli, Taylor 2015), the project EMP&Co – as it is informally called – carried out a first ex- ploration of current WRL in Italy. A questionnaire was proposed to a convenience sample of Italian students enrolled in the 2014-2015 academic year, to explore how they perceive the use and competence of the teaching and learning methods proposed by their instructors (Fedeli, Frison, Grion 2017). Of the five areas of the survey, one was specifically devoted to work-related teaching and learning methods. The students’ opinions referred to a scarce percentage of teachers who invite company speakers to classes or propose assignments or thesis The definitions mentioned suggest a connection with the formal context and the strategies and opportunities of a link between edu- cation and the world of work developed in the educational setting. They allude to an open category that encompasses work-related pro- grammes − for example internships − as well as didactic strategies to support students to better understand the relationships of theory- practice, education-profession (Litchfield, Frawley, Nettleton 2010). i Based on a literature overview and multiple definitions of Work- Related (or Integrated) Learning (Frison, Fedeli, Taylor 2015), the project EMP&Co – as it is informally called – carried out a first ex- ploration of current WRL in Italy. A questionnaire was proposed to a convenience sample of Italian students enrolled in the 2014-2015 academic year, to explore how they perceive the use and competence of the teaching and learning methods proposed by their instructors (Fedeli, Frison, Grion 2017). Of the five areas of the survey, one was specifically devoted to work-related teaching and learning methods. The students’ opinions referred to a scarce percentage of teachers who invite company speakers to classes or propose assignments or thesis THE DESIGN OF WORK-RELATED TEACHING & LEARNING METHODS 39 projects in direct cooperation with the professional world. Conse- quently, the EMP&Co survey revealed a university system cut off from the world of work and professions, as well as a very low level of connections among all disciplines even if strongly encouraged by the aforementioned European documents dedicated to the so-called «university-business dialogue» (Commission of the European Com- munities 2006, 2008, 2009). 1.2 Research design Considering this general gap, to go more deeply into this topic, the research group became interested in mapping work-related activities/ programmes proposed by Italian teachers in relation to their classes to: 1) identify common elements considered by those proposing, p g p p y 1) identify common elements considered by those proposing, y y p p g 2) trace methodological recommendations to encourage the design of work-related learning activities/programmes. 2) trace methodological recommendations to encourage the design of work-related learning activities/programmes. A multiple case study research design was chosen as a qualitative methodology (Bassey 2003; Stake 2008), which allowed an in-depth understanding of the issues of interest. Indeed, as Yin states, the case study research design is more suitable when the goal is to provide and answer ‘how’ questions that aim to explain a certain phenomenon investigated in its real-life context (2009). Furthermore, case studies are a common methodological approach used in research into Work- Related Learning (WRL) because of the highly contextualized nature of these programmes (Coll, Chapman 2000). Therefore, the research group has been researching and mapping multiple instrumental cases (Stake 1995; Yin 2009), such as cases se- lected to better understand the issue. To explore WRL, instrumen- tal cases are different activities/programmes related to the academic courses that involve the professional world, to explore and analyse ‘how’ instructors propose and manage them, to identify the common elements taken in account, and to formulate possible methodologi- cal recommendations. 1.3 Instruments and procedure Multiple sources of information are involved in the investigation of the selected cases (Creswell 2013; Yin 2009). First, semi-structured interviews were identified as an appropriate method to collect instruc- tors’ practices and experiences related to WRL. Second, documents, reports, and papers relating to the selected activities/ programmes were analysed, when available. 40 DANIELA FRISON Due to the ‘micro’ dimension of the WRL activities/programmes proposed in line with instructors’ personal interests and initiatives, a snowball technique was used to identify the cases (Morgan 2008). Indeed, snowball sampling is wholly suitable when cases or repre- sentatives of a population are difficult to locate, as in this case. A first small pool of instructors mentioned other cases according to the eli- gibility criteria (direct contact with the world of work, direct con- nection with a course or a study programme). The research group started from the University of Padua and from the educational area to then open to other disciplinary areas and universities (Siena and Florence, both involved in the aforementioned EMP&Co project). p j ) Semi-structured interviews, audiotaped and transcribed, explored the following main themes and issues that emerged from the litera- ture overview on WRL (Frison, Fedeli, Taylor 2015): • Purpose of the activities/programmes; • Integration: the process of bringing together formal learning and the world of work; • Partnership: type of partnership between organizations and university; f • Support: type of support offered to students during WRL activities/ programmes; • Assessment: assessment forms and instruments; • Learning: the “kind” of learning encouraged by WRL; • Faculty needs to improve WRL. 1.4 Participants 1.4 Participants As mentioned, a convenience group of instrumental cases provid- ed a snapshot of possible WRL activities among a range of academic disciplines. So far, 3 universities have been involved in the explor- atory study and 10 Faculties as shown below: y y • University of Padua (7) from: Education (2), Chemical (1) and In- dustrial (1) Engineering (1), Mathematics (1), Psychology (1), So- ciology (1) • University of Siena (2) from: Education (1), Sociology (1) • University of Florence (1) from: Education. 1.5 Themes and results 1.5 Themes and results 1.5 Themes and results Several key stakeholders can be involved in WRL activities/pro- grammes, such as faculties, academics, employers, professional ac- counting bodies, and the government (Abeysekera 2006) as well as, of course, students. Furthermore, there are several issues that need to be considered and clarified in relation to these stakeholders in order THE DESIGN OF WORKRELATED TEACHING & LEARNING METHODS 41 to design successful WRL programmes (Abeysekera 2006; Cooper, Orrell, Bowden 2010). to design successful WRL programmes (Abeysekera 2006; Cooper, Orrell, Bowden 2010). 1.6 Purpose of the activities/programmes and level of integration The activities/programmes mapped can be divided into three main groups based on their work-related objectives and approaches (Fig. 1), from a weaker relationship with the world of work (Level 1) to a stronger and more direct one (Level 3): g ( ) Level 1 g Level 1 Problem-based or project-based activities strongly anchored in the theoretical contents of the course but with reference to real problems, contexts, cases, and real-life situations (2 cases) (Prince, Felder 2006). These activities are carried out in the classroom, and no direct rela- tionship with the workplace is expected; p Level 2 Level 2 Assignments to be carried out in real contexts (e.g. within the workplace) and collaborative research (5 cases) to develop research proj- ects in cooperation with organizations and starting from real prob- lems proposed by them (Frison 2016; Munari 2011); p p Level 3 Structured programmes based on a formal partnership with or- ganizations, such as workplace activities and programmes (3 cases) based on a longer period spent in the workplace, outside the formal academic setting. e 1 – The ‘work-related’ content of the activities/programmes mapped. Figure 1 – The ‘work-related’ content of the activities/programmes mapped. Figure 1 – The ‘work-related’ content of the activities/programmes mapped The level of ‘integration’ of these activities may assume two dif- ferent meanings. On the one hand, integration can be considered a process of intertwining theory and practice, theoretical contents, and professional issues (Cooper, Orrell, Bowden 2010; Litchfi eld, Fraw- ley, Nettleton 2010). On the other, integration may be considered the process of recognizing work-related activities/programmes with- in the formal curriculum. In both cases, as Abeysekera (2006) stated, curriculum alignment is one of the key issues related to WRL. Since an important objective of these programmes is to increase students’ employability, the content of the curriculum should embrace em- 42 DANIELA FRISON ployability skills (Duignan, 2002; Fallows, Steven 2000). To reach this goal, the course should encourage analysis and interpretation of theoretical concepts, proposing assignments and tasks that need to be solved with an incomplete set of information, as in real situations (Fallows, Steven 2000). This aspect of the integration of work-related activities/programmes and their purposes is strictly linked to a further dimension, that of ‘support’. The literature suggests that students require support before, during, and after their work-related experiences and this can acquire different forms. 1.6 Purpose of the activities/programmes and level of integration It may be simply administrative assistance or some- thing more, such as educational support (Cooper, Orrell, Bowden 2010; Frison 2016; Martin et al. 2012; Munari 2011). In most of the cases mapped (7/10), the instructors provided a theoretical background related to their courses, but no dedicated support was offered. Despite this lack of official educational assistance, the instructors were willing to informally support students in facing organizational issues, when requested. Only in the 3 more structured work-related programmes based on a formal partnership with an organization, or more than one, was dedicated educational support expected and provided to deal with the challenges posed by professional contexts and dynamics. Figure 2 – University/organizations partnership formality and structure. 1.7 Partnerships It is not possible to have work-integrated learning without strong partnerships between industry and educational institutions, as Coo- per, Orrell and Bowden (2010) underline. A partnership ensures the representation of different interests and the identification of strengths and issues, as well as the strategies to reach common goals together (Cooper, Orrell, Bowden, 2010). As the authors highlight, a partner- ship exists at the individual practitioner and institutional level and may be transitory or ongoing, formal or informal, and involve single or multiple workplaces (Cooper, Orrell, Bowden 2010). p p ( p ) As for the instrumental cases mapped, we can identify two main partnership dimensions: 1) f l i f l p p p As for the instrumental cases mapped, we can identify two main partnership dimensions: 1) formal vs. informal; 2) structured vs. unstructured. Indeed, the relationship between the university and organizations may be wholly informal, based on a direct and informal contact be- tween the single instructor and the organizations’ representatives, without an official agreement between the two institutional actors. Or else the university-organization relationship may be wholly for- mal, as in the case of programmes based on an institutional agree- ment (e.g. internships or work-experiences). THE DESIGN OF WORKRELATED TEACHING & LEARNING METHODS 43 1.8 Assessment The concept of integration mentioned above refers to another cru- cial dimension of WRL: the assessment of these activities and their recognition in terms of grades or credits (Abeysekera 2015; Cooper, Orrell, Bowden 2010) based on the formal recognition of learning through practice. As Abeysekera points out, «academics could work with practitioners to jointly design learning outcomes and assessment techniques acceptable to both parties» (2015: 82), as well as being use- ful to students to understand their level of skills and competencies, beyond knowledge. Furthermore, as Martin and colleagues underline, these programmes encourage the combined use of both formative and summative assessment (2012). «Informal formative approaches are var- ied, ranging from the use of technologies such as emails, blogs and other e-journals, through to the use of more formal approaches such as reflective journals. Summative assessment provides a meaningful opportunity to assess, in particular, report writing and oral presen- tations both of which can be learning outcomes» (Martin et al.: 32).i g ( ) Considering the cases analysed, only one bases the final grade of the study course completely on the assessment of work-related activ- ity: a final group project report. (1) In the other instrumental cases, the final grade is the result of integration between an assessment of work-related experiences (through reports and/or oral presentations) and traditional exams (6). Sometimes neither form of assessment nor integration is expected for work-related activities (3), and the final grade is based only on traditional oral or written exams. THE DESIGN OF WORKRELATED TEACHING & LEARNING METHODS Additionally, the university-organization relationship may be for- mally structured, step by step, with planned phases and meetings, co- herently arranged, and focused on timed objectives. If not, it may be unstructured, based on informal meetings according to the instruc- tors’ openness and willingness. With regard to these two coordinates, the 10 cases explored are located as shown below (fi g. 2). In only one case (Education area), is the partnership wholly informal and unstructured. This is the case of a problem-based learning activity proposed to the students during a methodology of educational research course. Most of the mapped cases (5) are based on an informal relationship between the instruc- tor of reference and the organization/s involved. In any case, the in- structor provides the students with a clear pathway to cover, made up of agreed phases and milestones to reach common objectives. The Mathematics, Psychology, and Engineering cases (3) present a strong- ly formal and structured partnership based on an institutional agree- ment between the university and the organization/s. In this case, the work-related activities refer to formal projects that involved students from the whole university (Industrial Engineering and Psychology) and the students attending a study course directly linked to the in- ternship experience, within a Bachelor’s Degree study programme (Mathematics). In every case, this institutional relationship was led by a teacher who oversaw the project as coordinator and took care of the organizations’ requests as well as the students’ needs. Figure 2 – University/organizations partnership formality and structure. Figure 2 – University/organizations partnership formality and structure. E = Education; En = Engineering; M = Mathematics; P = Psychology; S = Sociology E = Education; En = Engineering; M = Mathematics; P = Psychology; S = Sociology 44 DANIELA FRISON 1.8 Assessment 1.10 Faculty needs As the literature underlines, academics are the stakeholder most involved in designing WR activities/programmes (Abeysekera 2006; Reeders 2000). An Australian survey refers to the contents of train- ing provided to academics on this topic, such as policy formulation, programme design, the preparation of students, sourcing of positions, assessment, student supervision, student mentoring, industry relation- ship, and programme evaluation (Reeders 2000). p p g ( ) As for the needs pointed out by the interviewees, they underlined difficulties related to the assessment of WR activities. They would ask for strategies and tools to assess students and to go beyond a traditional assessment form focused on knowledge rather than on skills and com- petencies. The connection between these activities contextualized to the workplace and the study course still remains the first challenge to face with. This challenge is strictly related to an effort to define in- tended learning outcomes, the choice of teaching and learning meth- ods to achieve them, and the assessment strategies and tools necessary. Finally, interviewees highlight the need to receive support from the institution to manage relations with the organization, to create a wider network of available organizations and move from a personal initia- tive to an institutional strategy of partnership with the world of work. • opportunity for self-awareness and awareness of the world. Work-related activities/programmes can become a learning opportu- nity for teachers, too. In accordance with the literature, through WRL teachers can establish links with a wide range of employers and, through this, can ensure that their teaching is up to date (Fraser, Deane 2002). d d h d l d h f h h Indeed, the interviewees underlined that for them the projects were an opportunity: pp y • to learn about business needs and new research topics, and for di- alogue and exchange • to learn about business needs and new research topics, and for di- alogue and exchange • to strengthen relationships with organizations; • to re-think teaching and learning methods and design; • to change the role of instructor as Charon, as a guide; • to design experiential didactics; • to have fun and interact with other students. 1.10 Faculty needs 1.9 Learning The literature analysed focuses on the experiential (Kolb 1984) and situated (Lave, Wenger 1990) dimensions of work-related learning. Indeed, the students learn through experiential learning in the workplace, by do- ing and interacting with professionals facing genuine professional prob- lems and situations, where theoretical knowledge can be put into practice. In the cases mapped, according to the faculty’s perspectives, what kind of learning is encouraged by work-related experiences? The in- terviewees mentioned: • emotional, experiential, inductive learning; • learning of soft skills (team-working, problem-solving, public speaking, negotiation, leadership); • learning on the job, situated learning; • self-directed learning;l • multilevel learning (reflective, metacognitive, emotional, organi- zational, scientific); THE DESIGN OF WORK-RELATED TEACHING & LEARNING METHODS 45 Abeysekera I. 2006, Issues Relating to Designing a Work-integrated Learning Program in Accounting, «Asia-Pacific Journal of Cooperative Education», VII (1), 7-15. 2. Discussion and conclusion The challenge of enhancing WRL in Higher Education is a co- nundrum that has been well detailed by European documents (Com- 46 DANIELA FRISON mission of the European Communities 2006, 2008, 2009) and the international literature (Cooper, Orrell, Bowden 2010; Gardner, Barktus 2014; Dirkx 2011; Frison, Fedeli, Taylor 2015). Beyond that starting point, this exploratory study has begun to reveal strengths and weaknesses in the proposal, design, and management of work- related activities/programmes. Firstly, support appears to be a crucial issue. WR activities should provide time and space for support, a sort of ‘help service’ that wel- comes administrative and organizational problems, offering the stu- dents a ‘learning guide’ to cope with critical situations. Reflective spaces and tools, monitoring meetings, and peer-tutoring meetings have this goal, and the challenge is to make them compulsory and sustainable for both the students and the faculty. y Secondly, the change of attitude of the instructor from sage on the stage to guide on the side (Morrison 2014) is another central ele- ment. WR activities relate to an experiential and active learning process, where the students become more active. Based on WR ac- tivities/programmes, the instructors can bring into the classroom authentic research material and real problems, connecting these to theoretical contents, stimulating critical thinking and creative solutions, proposing simulations that can reproduce real-life and work contexts. This point relates to a third dimension, that of recognizing learn- ing through practice and its assessment, which are two crucial aspects of WR strategies to consider part of the curriculum, the formal one – as an academic plan – and the hidden one – referring to norms, values, and beliefs embedded in the curriculum, classroom life, and in the faculty’s approach (Reeders 2000). y pp ( ) Finally, the faculty’s approach to WRL and dedicated teaching methods is strongly bund up with the support offered to instructors to foster WR strategies, in terms of specific training to design WR activities/programmes or administrative services to assist them in the management phase. The cases mapped are based on a personal in- structor’s choice to encourage connection to the professional world. The challenge, as mentioned above, is to move from an individual choice to an academic strategy that recognizes and encourages learn- ing in and through the workplace. 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K. 2003, Case study research design and methods, Sage, Los Angeles. * Claudio Melacarne, PhD, Associate Professor of General and Social Pedagogy, Department of Education, Humans Sciences and Intercultural Communication, University of Siena, Italy. Email: claudio.melacarne@unisi.it. Claudio Melacarne (University of Siena)* Abstract: This paper addresses several critical issues relating to the organization of internships at universities, dealing above all with ‘curricular’ internships, i.e., those apprenticeship experiences included as mandatory in university courses. Starting from the idea of ‘informal learning’, the paper shows the strong con- nection between new workplace needs and the potentiality of pedagogical and reflective approaches in designing internship experiences. Keywords: higher education, reflective practices, informal learning. 1. Introduction In recent years, universities have been called upon to respond to numerous invitations: the new targets involved in higher education, the invitation to internationalize study courses, the request to work on students’ acquisition of soft skills and, not least in terms of impor- tance, the need to adopt teaching strategies that can foster dialogue and cooperation between formal knowledge and professional knowledge, both in person and online. University education is thus urged, with increasing weight, to configure itself as an ‘expanded’ place capable of integrating teaching methods and techniques that support the stu- dents’ learning process beyond the classroom walls and the physical spaces of the university (Walton, Matthews 2017). For example, many studies suggest that it is no longer enough to train students by transmitting organized knowledge. The importance of socializing the new generations to established cultural or scien- tific knowledge is not in discussion. Timeless, general, and universal knowledge is accompanied by a further need to examine the knowl- edge generated in specific local situations linked to temporal events. g g pi p Several studies have highlighted four general types of disconti- nuity between academic learning and the nature of cognitive activ- ity outside universities, schools, and formal educational contexts in general. These contributions can help identify certain aspects of the concept of knowledge that universities still share with a good part of the school system, for example (Bertagna 2006). 52 CLAUDIO MELACARNE Universities focus on individual performance, while outside mental work is often shared socially. Universities aim to encourage thinking without support, while mental work in everyday life usually includes cognitive tools. University courses often cultivate only symbolic thought, while daily mental activity is directly involved with objects and situations. Finally, there is a tendency, even in higher education, to teach skills and general knowledge, while outside, specific skills for an individual situation dominate (Resnick, Levine, Teasley 1991). ( y ) As much research has shown, in Italian universities the domi- nant form of learning is individual, and students are judged by what they do for themselves. The main part of the activity is constructed on individual work: home study and individual interventions in the classroom. Many activities outside the university are, by and large, shared socially; in the same way, work always takes place within so- cial systems, and the ability of each person depends very much on what others are doing. 1. Introduction At university, the greatest merit is directed at activities of ‘pure thought’, what individuals are able to do with- out the external support of textbooks and notes, calculators, or other complex tools. Universities, like schools, tend to value independent thought, without the use of material and cognitive tools. In contrast, most outside mental activities are intimately connected with tools, and the emerging cognitive activity is formed and dependent on the type of tool available. In the professional world, thinking is connected to the material world. University learning is essentially based on sym- bols, with the risk that connections with real events and objects may be lost. Finally, the university is mainly anchored to the objective of teaching skills and generic, widely usable, theoretical principles. Gen- erality and transferability are the added values of a higher education and, to be competent in the professional world, people must develop appropriate forms of knowledge adequate to the situation. pp p g q Therefore, a broad range of data leads one to think that what people do at university is difficult to transfer to external practical contexts, and suggests that both the structure of knowledge and the social conditions of practical activities may differ more from what is achieved through formal education than previously thought (Resn- ick, Levine, Teasley 1991: 69).f What do these differences suggest about the relationship between universities and competence in work and daily life? At least two con- siderations: on the one hand, the need to discuss possible strategies to contain and reduce what many studies define as ‘the encapsulation of scholastic learning’, i.e., the problem of ‘academically’ training first- class students who, however, are unable to transfer what they have learned at university into daily practice. On the other, the opportu- nity to appreciate practice as a combination of knowledge and ac- SUPPORTING INFORMAL LEARNING IN HIGHER EDUCATION INTERNSHIPS 53 tions and to pose the problem of learning the knowledge intrinsic to actions. In the first case, we are faced with a problem of transform- ing university teaching. In the second, of supporting and designing curricula or settings in which students can ‘learn from experience’ or ‘learn by doing’, supporting informal learning both inside and out- side university experiences. Marsick & Watkins (1990) dedicated some of their most exten- sive research into describing, defining, and developing a more com- prehensive understanding of informal and incidental learning within organizations. 1. Introduction The authors start from a comparative description. In contrast to formal learning, informal and incidental learning refers to the natural opportunities for learning that occur every day in a person’s life, when the person controls his/her learning» (p. 350). Marsick categorized the types of informal and incidental learning to include: self-directed learning, social learning, mentoring, coaching, and networking, learning from mistakes and trial and error» (p. 350). Watkins developed a theoretical framework or model to understand how the process of informal learning takes place. Thus, Watkins de- fined incidental learning as a “sub-category of informal learning […] a by-product of some other activity, such as task accomplishment, interpersonal interaction, sensing the organization, trial and error experimentation, or even formal learning” (Marsick, Watkins 1990: 12). Informal Learning means reaching out to the person in the next cubicle and cultivating relationships through networking, coaching, and mentoring. It also takes place in varied self-directed ways by reading reports, newsletters, and memos, and by conducting research. This paper aims to describe how internship activities could increase students’ employability (Boffo, Federighi, Torlone 2015), supporting informal learning processes and organizational learning. 2. University, workplaces, and social knowledge Higher education systems have undergone a momentous change that has irreversibly transformed its nature, goals, and scientific, educational, and organizational practices. In Europe, and partic- ularly in Italy, new working scenarios and new knowledge needs have underlined the critical factors and contradictions of univer- sity curricula and governance strategies that are all too often fixed on a theoretical-disciplinary logic. Repeatedly, there is no parallel attention to pertinence with outgoing professionals, when there is a need to give the right amount of room to all the disciplinary ar- eas in the courses. It is not difficult to trace experiences planned more on self-referential than workplace-oriented criteria. Thus, the 54 CLAUDIO MELACARNE challenge of producing important, relevant knowledge for social, organizational, and working contexts becomes increasingly vital for universities, on top of spreading investigative strategies that can produce located knowledge.i There is still a significant gap and much misalignment between the world of work and the university, as there is between the university and students’ need for personal and professional development. Plan- ning courses that can intercept emerging, challenging learning needs in relation to current working scenarios by talking with stakeholders, are further commitments that characterize current academic policies. Some aspects of the new university set-up could be summed up by a few dichotomies: user-client, general-located, vertical-transverse. g User-client: Students are no longer merely subjects using a ser- vice, but are the possessors of wider, more complex interests than in the past. Parents’ expectations, students’ professional ambitions, personal attitudes, and critical factors and fragilities all contribute to people’s expectations from university. More so than in the past, universities today must respond to questions of knowledge, but also of care, support, specialization, and integration. If we look at the profiles of current university students, we find that they have partly changed their status. They have become student-clients, with more awareness of what the organization must guarantee in terms of learning and services; they have different learning needs, have knowledge-gathering tools that can give value to services through universities’ national and international rankings, they pay more attention to a balanced evaluation of the costs and benefits (taxes vs. occupation, distance from home vs. services offered, cultural vivacity vs. safety). 2. University, workplaces, and social knowledge y y) General-located: Those who work in university environments know that it is not easy to change the attachment that teachers have to an idea of general and universal knowledge, that can suit any course or any classroom. Many academic communities share unique meaning systems where a view of education as a job of knowledge delivery remains central and where the student’s learning is mainly seen as an individual process independent of any kind of social involvement. From a view of knowledge as a skill that must be exercised and then evaluated in a decontextualized way, the idea that knowledge is lo- cated and therefore anchored in contexts, practices, and material and immaterial located restrictions, becomes central. Vertical-transverse: Both the economic world and the European Union (EU), have supported various initiatives to help development of transverse skills that are useful for staff to carry out active citi- zenship projects and increase social inclusion and employment. Es- sential tools in these directions were the identification of key skills SUPPORTING INFORMAL LEARNING IN HIGHER EDUCATION INTERNSHIPS 55 in 2006, and a European reference framework on qualifications and academic certificates in 2008. The university is thus impelled to plan programmes that can support the acquisition of strictly specialized or technical-professional abilities, and ‘soft’ or ‘transverse’ skills. p Like any other organization, the directions of innovation that are taking over Italian universities are not straightforward (Raelin 2000), or even expected. Alongside routines that have difficulty in changing, are promising views that interpret the university’s priorities in diverse ways, by cohabiting and expanding. New awareness has emerged from: a) the use of research as a transformational and collaborative process; b) the enhancement of professional knowledge; c) the professional- ization of knowledge; d) training professionals whose skills are not simply rooted in knowledge of the subject, but also in the students’ own informal learning. Knowing how to work in a group, problem- solving, knowing how to face improvisation and uncertainties that are a part of working practices, being a leader or more simply, an ability to write a report, are just some of the skills that universities are trying to offer across the board to the subject sectors and specific professional areas. p These new areas of interest outline promising openings so that universities can learn from their own experience and from the kind of critical incidents that occur and have been experienced in recent years. 2. University, workplaces, and social knowledge It could be said that we are in a phase in which universities are questioning the instruments, routines and premises that govern their strategies: governance, research, teaching, and relations with the world of employment. We have seen are still seeing a critical, reflec- tive validation process (Boud, Cressey, Docherty 2006) for systems of meaning and activities embedded in the university organizational system (Yorks, Marsick 2000). Who can plan a course today with- out taking into consideration an outgoing professional figure, ask- ing what the organizational routines contain that no longer works? Planning requires that the leadership, and often the entire academic community, question its own usual methods for working and inter- preting problems. Urged on by university reform and the changes in economic contexts, new spaces for discussions have opened up that have re- quired the adoption of different codes for speaking, sharing, and resolving problems that are apparently no more than ‘technical’. Students and their families have become more central in teaching- learning processes. What was routine a few years ago, is today the subject of negotiation, for identifying attractive professional pro- files, and for planning sustainable study courses that can offer an education in a position to win over clients, research commission- ers, and project partners. 56 CLAUDIO MELACARNE 3. Supporting Informal Learning in Internship Experiences • An opportunity for socialization and participatory knowledge of working situations. g • An active orientation tool, given that they allow direct knowledge and experience in a work context and thus help students make fu- ture career choices. g • An active orientation tool, given that they allow direct knowledge and experience in a work context and thus help students make fu- ture career choices. Today, these are an integral part of various study courses to be car- ried out with the support and accompaniment of a tutor. Beyond a general appreciation of internships, we are now coming to terms with an experience which, over time, has revealed structural problems. 3. Supporting Informal Learning in Internship Experiences Regardless of the theoretical options at stake, the university needs to answer several questions. How does one learn a profession in the current cultural and social climate? What knowledge does a pro- fessional called to interact with current work scenarios need? How to train a professional who will need to change jobs many times? How to re-design learning paths that allow the acquisition of skills useful for insertion into multiple and uncertain professional worlds? The link between universities and the professional world, in addi- tion to calling into question the meeting/exchange between these two universes, also requires consideration of the university’s rela- tionship with employment. The problem that prompted and fuelled the survey set out below arises from the realization that training professionals who are capable of living in new work scenarios is a commitment that challenges us as university teachers and decision-makers within university gover- nance systems. Managing and organizing effective internship experi- ences can be a first effective response to the new needs of businesses and students, the latter often being involved in potentially promising activities with little attention to the monitoring and support processes implemented by universities. This paper addresses several critical issues relating to the orga- nization of internships in university education, dealing specifically with ‘curricular’ internships, i.e., those apprenticeship experienc- es included as mandatory in university courses. Not surprisingly, the increasingly widespread instances of innovation in university teaching testify to the crisis of a teaching model centred mainly on lessons. and on the idea that meaningful knowledge must be conveyed by the teacher. In other words, teaching based only on symbolic mediators, where one speaks, and the others listen, that lacks the active participation of students in knowledge construc- tion processes, and where research is not a widespread educational tool. Today more than ever, professionals are primarily required to be problem-solvers, to produce that situated knowledge useful for acting in certain contexts. g Within this new framework, internships are increasingly:f • An educational opportunity, thanks to the chance offered to everyone to study and interact with work practices while attempting to situate the knowledge gained in university studies. g g y • An opportunity to learn practical knowledge. Through internships, knowledge and learning should be situated in a context of authentic experience. SUPPORTING INFORMAL LEARNING IN HIGHER EDUCATION INTERNSHIPS 57 • An opportunity for socialization and participatory knowledge of working situations. 5. Situated Learning as informal education 5. Situated Learning as informal education In some empirical research conducted in collaboration with Wenger, Lave (1991) described some everyday practices of several groups and individuals with the intention of highlighting how learn- ing is not merely a process of participation, but also involves certain social aspects and restrictions imposed by the community. For ex- ample, he describes how young Mayan girls often have a ‘midwife’ relative who introduces them to the expertise necessary for per- forming this task. Some women become midwives by participating in the practices of the ‘experts’, mothers or grandmothers, observ- ing and implicitly learning what to do during childbirth, about the remedies and cures to provide in case of illness, the expectations of the new-born’s parents, the social scripts that legitimize this practice, the nursing practices for newborns that are the responsibility of the community or caregivers. In this sense, we are using the term ‘situ- ated learning’ to go beyond the definition of ‘learning-by-doing’, of natural learning. Lave uses this construct to reveal that ways of think- ing about and solving problems and the use of complex forms of ab- straction are linked to the specific environment in which individuals live, and are connected to the particular context in which they occur and are realized. It is not possible to become midwives in the Mayan communities analysed by Lave without legitimization by the com- munity and without the support of an expert. Learning is synonymous with participation in practices in which it is possible to learn by observing how more expert people perform a task, in contexts that ensure both a relationship and the possibility of gradually participating in the experience. From this perspective, learning a business, acquiring a skill, or developing abilities, involve a process of participation, of becoming a member of a community. This means that learning is situated in the social interaction area, in life contexts. Thus, the use of the situated learning construct does not seem applicable to all experiences, since it implies a gradual in- sertion into a practice characterizing a community which, in turn, depends on the willingness of the community itself to facilitate ac- cess through legitimization. The most important contribution of this focus was that it highlighted that learning is a process connected to social conditions of accessibility to knowledge embodied in practice. 4. The pedagogy of workplaces and learning processes Internships play a key role in helping students enter current pro- fessional scenarios (Matthew, Taylor, Ellis, 2012) and in supporting the acquisition of real skills. Much of the knowledge that is useful for students in practising a profession and entering the ‘swamp of professional practice’ is, in fact, only partially obtainable from for- mal educational settings, in a classroom or through participation in workshops and seminars. Despite the significant overhauling of uni- versity teaching through the adoption of more participatory and ac- tive approaches (Fedeli, Giampaolo, Coryell 2013), for example by promoting students’ acquisition of soft skills or instrumental learning, practical knowledge remains embodied in the professional commu- nity and only through special training instruments can it become a resource for people and communities (Wenger 1998; Fabbri 2007). f h h h l d d p p g It is from this perspective that internships included in university curricula have been considered the most promising placement tools to meet the supply and demand of work, to foster the development of professional identity and acquire tools useful for solving business problems (Billett et al. 2008). p ( ) How can students be given the ability to confront and solve prob- lems similar to those they will encounter in life and the workplace? How can students entering professional communities that are not ‘naturally’ configured as learning contexts be assisted? From an analysis of national and international literature, there are two paradigms that have laid the first foundations for envisaging an up-to-date interpretation of how students learn during the intern- ship experience. The first has its roots in studies of learning as a so- cial phenomenon. The second comes from research fields that are more careful to emphasize the critical-emancipatory dimensions of learning processes. 58 CLAUDIO MELACARNE SUPPORTING INFORMAL LEARNING IN HIGHER EDUCATION INTERNSHIPS SUPPORTING INFORMAL LEARNING IN HIGHER EDUCATION INTERNSHIPS 59 Apprenticeship is an important emerging construct, because it has emphasized that it is not enough ‘to participate in an experience to learn’. Social and environmental factors come into play that can hinder or support the entry and participation of a novice into a community. F l W d L id ifi d l h f For example, Wenger and Lave identified at least three success factors for participation to generate learning in an organizational context: For example, Wenger and Lave identified at least three success factors for participation to generate learning in an organizational context: • A member of the community must legitimate the novice to take part in ‘non-trivial’ practices. p • A member of the community must take responsibility for supporting (explaining, illustrating, describing) the practices in which the nov- ice is participating. p • A member of the community must take responsibility for supporting (explaining, illustrating, describing) the practices in which the nov- ice is participating. • The learning trajectory must be organized starting from simple tasks to arrive at increasingly complex engagements. • The learning trajectory must be organized starting from simple tasks to arrive at increasingly complex engagements. Apprenticeship can be described as a particular form of partici- pation in the life of a community that can also be configured as an opportunity for learning a) if the novice can legitimately take part in those significant work practices that are central to, and charac- terizing of a community (Lave, Wenger, 1991; Wenger 1998); b) if the novice is supervised by an expert (Rogoff 1990); and c) if the novice is put in the condition of gradually entering into the com- plexity of the experience and, above all, can find training spaces to reflect on the work practice (Mezirow, Taylor 2009; Fabbri 2007). Apprenticeship is that particular form of participation through which a novice learns a profession by observing and taking part in an ac- tivity managed by one or more experts (Lave, Wenger 1991). These studies suggest that we should not look at internship merely as an application experience. pp p Internship as an experience that takes place in a work context, it is not automatically an experience for professional growth. 5. Situated Learning as informal education y g p More recently, the ‘situated learning’ construct has also been as- sociated with the term ‘apprenticeship’, used as a metaphor for de- scribing learning processes that do not necessarily take place in work contexts, but in different social contexts, from the family to the peer group, and from schools to non-profit organizations (Pontecorvo, Ajello, Zucchermaglio 2005). SUPPORTING INFORMAL LEARNING IN HIGHER EDUCATION INTERNSHIPS The problem is not only that of not letting our students experience insignificant forms of participation (making photocopies, dealing with adminis- trative matters, handling routines with low innovative value), but of providing tools to study work practices, to become increasingly cen- tral in organizational routines, to intercept the most promising paths within the organizations hosting them. Staying within the apprenticeship metaphor, internship becomes an experience organized by someone (the University) to allow stu- dents to experience a situation that ensures legitimate and peripheral participation. Internship is an opportunity for students to pass from the status of novice to that of a semi-expert, through involvement in increasingly complex work practices and educational tutorship (Bel- lingreri 2015). However, no student can be left alone in the organi- zation of these experiences of entry, stay and exit from organizations, 60 CLAUDIO MELACARNE and certainly not if this experience is part of an educational offering in a university course. SUPPORTING INFORMAL LEARNING IN HIGHER EDUCATION INTERNSHIPS 61 ing from pre-critical to critical forms of learning solely through the internship experience. There is a need to design settings in which the experience can materialize in awareness, reflection, and vali- dated forms of thought. 6. From low skills to critical reflective thinking The reflective approach, albeit starting from different theoretical and empirical studies, shares some basic assumptions with the participatory approach: learning is a form of participation, is a social phenomenon, and is generated by routines or patterns/perspectives of meaning. However, the adoption of a reflective approach to the study of work practices, educational practices, or training intervention methodolo- gies, introduces a further distinction. This not only means partici- pating in a legitimate and peripheral way that makes an experience a learning opportunity, but is an opportunity for the person to reflect on the experience afterwards that makes any experience a potential learning opportunity. The reflective approach, especially in its crit- ical-emancipatory guise, shifts the focus from ‘experiencing’ to ‘re- flecting on it afterwards’.l It is by reflecting afterwards that it is possible to become aware and learn to understand how we acted, why, and with respect to which of our convictions (Mezirow 1991). The literature provides many suggestions and indications useful in promoting reflective learning. Three seem particularly interesting: • Constructing settings capable of ensuring a positive dialectic. d h d f l h l f • Adjusting the commitment required from people to the limits of the context, the organization, or the professional community.l g p y • Allowing broader reflection on the experience, including the emo- tional reprocessing of the experience (Mezirow, Taylor 2009). In the light of these considerations, internships become an ex- perience which to design afterwards reflective activities useful for students to validate their epistemic, socio-linguistic, and psycho- logical perspectives: What did I think on that particular occasion? Why did I express that opinion? With respect to which personal convictions did I act? How did I feel? What sources did I use to form that idea?l Studies dealing with reflective practices suggest paying particular attention to both support aimed at facilitating the gradual partici- pation of students in internship experiences, and educational super- vision aimed at students’ acquisition of perspectives that are more inclusive, discriminating, personal, open, and emotionally available to change and reflection (Mezirow 1991). Also, and especially in this case, no university student can autonomously succeed in pass- References Agashae Z., Bratton J. 2001, Leader-follower dynamics: developing a learning environment, «Journal of Workplace Learning», XIII (3), 89-102. Alcalde D.M. 2005, Proquest Dissertations and Theses, The University of Nebraska, Nebraska. Bellingreri A. 2015, Imparare ad abitare il mondo. Senso e metodo della relazione educativa, Mondadori Education, Milano. Bertagna G. 2006, Pensiero manuale. 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SUPPORTING INFORMAL LEARNING IN HIGHER EDUCATION INTERNSHIPS Maura Striano (University of Naples Federico II)* Abstract: Within a complex economic, political, and social scenario that requires high-level competencies as well as increasingly active citizenship, the European and international agenda for higher education needs to be renewed and re-ar- ticulated according to new goals and priorities. Higher education should offer students the opportunity to develop aptitudes and acquire advanced but flexible competencies and skills that go beyond mere professionalism and instead focus on the development of a sound professionality. Keywords: professionality, professional epistemology, work-related learning. * Maura Striano, PhD, Professor of General and Social Pedagogy, Department of Humanistic Studies, University of Naples Federico II, Italy. Email: maura.striano@ unina.it. SUPPORTING INFORMAL LEARNING IN HIGHER EDUCATION INTERNSHIPS 63 Walker A.R. 2001, Proquest Dissertations and Theses 2001, Columbia University Teachers College, New York. Walker A.R. 2001, Proquest Dissertations and Theses 2001, Columbia University Teachers College, New York. g Walton G. and Matthews G. 2017, Exploring Informal Learning Space in the University: A Collaborative Approach, Routledge, London. Watkins K. and Marsick V.J. 1992, Building the learning organization: a new role for human resource developers, «Studies in Further education», 14(2), 115-29. for human resource developers, «Studies in Further education», 14(2), 115-29. Wenger E. 1998, Communities of practice: Learning, meaning and identity, Cambridge University Press, Cambridge. Wenger E. 1998, Communities of practice: Learning, meaning and identity, Cambridge University Press, Cambridge. g y g York L., Marsick V. 2000, Organizational learning and transformation, in Mezirow J. & Associates (eds.), Learning as Transformation: Critical perspectives on a theory in progress, Jossey-Bass, San Francisco. HIGHER EDUCATION AND WORK-RELATED LEARNING FROM PROFESSIONALISM TO PROFESSIONALITY Maura Striano (University of Naples Federico II)* 1. Introduction The European Commission has recently pointed out that higher education plays a unique role in the economic, political, and social development of member states, in view of the dramatically changing scenario, within which demand for highly-skilled and at the same time socially-engaged people is both increasing and changing. As stated in the Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee and the Committee of the Regions on a renewed EU agenda for higher educa- tion, in the immediate future, half of all jobs are projected to require high-level qualifications; additionally, jobs are becoming more chal- lenging, complex, and flexible. g g pl Within this scenario, Europe needs more high achievers who can be entrepreneurial, manage complex information, think autono- mously and creatively, use resources, including digital ones smartly, communicate effectively, and be resilient. In parallel, countering the growing polarization of European soci- eties and a distrust of democratic institutions calls on everyone – in- cluding higher education staff and students – to engage more actively with the communities around them and promote social inclusion and mobility (EU 2017). y This requires an overall reframing of higher education curricula according to specific educational objectives, considering either the necessity to sustain students in the development of aptitudes, compe- 66 MAURA STRIANO tencies and skills that qualify them for any kind of job, or to reduce the increasing gap between high and low achievers. Every student should be acknowledged and valorized on the ba- sis of their talent and not of their background, and actively engaged in their learning project which should include the implementation of basic skills (literacy, numeracy, digital competence) as well as the development of transverse skills (communication, learning-to-learn, entrepreneurship, problem-solving…). Moreover, students should be equipped with tools useful for a full and effective engagement in economic and social life, to cope with the challenges and requests of a fluid and flexible occupational sce- nario and of a highly complex political and social situation at both local and global levels. HIGHER EDUCATION AND WORK-RELATED LEARNING 67 students are progressively engaged in professional practices within a protected setting where they are progressively accompanied towards the profession they have chosen through a guided training in pro- fessional tasks and performances, according to a common frame of reference. Professional expertise is learned through observation, and the imitation of professional practice and supervision by an expert performing a regulative task, and not an epistemic one. When engaged in experiences of professional practice, students are therefore trained to acquire specific professional behaviour, but are not accompanied in the development of a specific professional epis- temology characterized by a special way of using and implementing the funds of knowledge they have had access to during their stud- ies, and of constructing new empirical forms of knowledge within the practices, deeply influenced by ideologies, attitudes, and ways of thinking and understanding. In point of fact, the reference to ‘professionalism’ in higher edu- cational curricula does not make room for a crucial element in the development and interpretation of a professional identity, which is what Evans, referring to Hoyle (1975), defines as ‘professionality’: an ideologically-, attitudinally-, intellectually-, and epistemologically- based stance on the part of an individual, in relation to the practice of the profession to which she/he belongs, and which influences her/ his professional practice» (Evans 2002: 6-7). The focus is on proce- dures, tasks, and performance, and not on perceptions, reflections, and understanding, which are crucial in the development of a well- expressed and well-grounded professionality. Learning is not therefore understood as a circular process but as a linear one, and the focus is not on the epistemological dimension that grounds professional practice, but on the performative and pro- cedural dimension of the practice. Students are not asked to elaborate and incorporate their knowl- edge into a specific form of agency sustained by a definite epistemol- ogy, but to apply and transfer their knowledge to specific situations through performances guided by defined protocols and routines, and this is extremely risky in long-term professional outcomes. If students are not accompanied and sustained in the construction of a ‘professional’ profile, or in the development of ‘professionali- ty’, they are not likely to be fully engaged in a professional role that reaches apical levels, nor be satisfied with it. 2. Higher Education from professionalism to professionality If we analyse and compare higher education curricula for specific professions, we can see that they are designed according to a clear- ly identified professional profile, characterized by specific funds of knowledge and competencies that are to be acquired and implement- ed on both theoretical and practical levels. Degree Courses such as Medicine, Psychology, Pharmacy, and Ed- ucational Sciences, offer students both classroom courses, seminars, and workshops, as well as an internship experience in specific pro- fessional contexts or a practicum, which is mainly designed to relate knowledge and practice through the observation of an expert pro- fessional and a progressive engagement in professional practices with the supervision of a tutor.i p This approach is oriented by what Evans (2008: 7) defined as ‘pro- fessionalism’, a reference concept that delineates «the content of the work carried on by the profession, as reflected in accepted roles and responsibilities, key functions and remits, range of requisite skills and knowledge and the general nature of work-related tasks» and incorpo- rates shared elements of professional culture and practical knowledge. Within this framework, professionalism provides us with an idea of professions that is mainly oriented by reference to a continuous procedural pattern becoming a professional essentially implies the acquisition of advanced and specialized forms of knowledge that re- quire to be operationalized in specific tasks and roles, and practised and internalized in order to build up professional expertise that can be observed and evaluated according to a performative approach. g p pp Accordingly, the learning experiences that make up the curricula are organized following a theory-practice linear sequence, and the HIGHER EDUCATION AND WORK-RELATED LEARNING HIGHER EDUCATION AND WORK-RELATED LEARNING The development of sound professionality requires a deep analy- sis and exploration of the cultural and ideological grounds that have sustained the choice of a determined profession; reflection on indi- vidual attitudes, idiosyncrasies, and preferences, but also on the ‘social representations’ that individuals and groups have about that profes- 68 MAURA STRIANO sion, over and above an active engagement in forms of thinking and understanding that are strictly connected to it.i sion, over and above an active engagement in forms of thinking and understanding that are strictly connected to it.i One thing to consider is that there is a significant division in higher educational curricula between those oriented towards the acquisition of knowledge, competencies and skills useful within a specific professional field of practice (clearly defined and delimit- ed) and those that are mainly oriented towards the acquisition of knowledge, competencies and skills that are not tailored to a spe- cific professional role or practice, but provide students with tools that can be used in various professional contexts, other than the ones previously referred to. p y Within this framework, Degree Courses such as Literature, Phi- losophy, and History are not organized according to this framework, but offer students the opportunity to become engaged in various cultural experiences and to reflect on them in depth at seminars or workshops, but without being directly engaged in any kind of pro- fessional practice. p Consequently, students may have the opportunity to develop vari- ous kinds of competencies, but the curricula do not specifically focus on the acquisition or empowerment of what have been identified as the ‘key competencies’ for lifelong learning. Moreover, students are not accompanied and sustained in making use of their knowledge or in building up more advanced and sophis- ticated forms of knowledge within possible working situations, and are therefore not sustained in the construction of any specific kind of professional epistemology. Finally, they rarely confront experiences that support the develop- ment of aptitudes, competencies and capabilities which are the basic fabric for an active, participatory, and reflective engagement in any professional role or in public life. p p This calls for a profound reframing of higher education curricula, which should offer students the opportunity to explore their ‘calling’ and vocational orientation, but also their own cultural and personal resources, not only through dedicated services but throughout their curricular activities and experiences. HIGHER EDUCATION AND WORK-RELATED LEARNING HIGHER EDUCATION AND WORK-RELATED LEARNING 69 aid of mediating instruments such as workshops, seminars, and on- line platforms that let students engage in the development of either a professional epistemology or a professional identity, both oriented towards the development of sound professionality.i The main challenge to face is to build up a significant and useful interconnection between knowledge and practice within the learning processes of higher education curricula, and this requires the refram- ing of curricular contents, learning materials, learning approaches and instruments, as well as learning settings, through a meta-curric- ular approach. HIGHER EDUCATION AND WORK-RELATED LEARNING Moreover, to help them construct a professional epistemology, students need to be accompanied to learn through confrontation and reflection on practical issues and problems emerging from the field, with the contribution of experts who do not provide procedural models to observe and imitate, but offer a professional point of view highlighting how professionals normally frame and view situations. The whole higher education curriculum should therefore be re- organized from within, focusing on the interconnection between theory and practice, curricular disciplines, and practicum, with the 3. Thinking and learning to connect knowledge and practice According to a traditional approach, within higher education cur- ricula, the relationship between knowledge and practice is a linear one: knowledge anticipates and prepares practice, and practice is the field of application of ideas, models, theories previously acquired and elaborated within a classroom setting, and individual or collective study experiences. In professionalizing courses, the practicum experience arrives late, and is mainly conceived as a field experience, somehow disconnected from the academic courses, even when it is sustained by a learning project supervised by both an academic tutor and an external one, which often offers students the possibility to collect data and materi- als to be elaborated for their final dissertation. The practicum experience is rarely anticipated and prepared with- in those academic courses that engage students in forms of academic thinking and learning and not of professional thinking and learning. g g p g g A first challenge is therefore to engage students in forms of pro- fessional thinking and learning (which require a specific epistemic positioning) within academic courses in order to build up a signifi- cant learning continuity within the curriculum according to a cir- cular and nonlinear pattern. f A second challenge is to offer curricular and disciplinary contents highlighting their practical and professional implications with a spe- cific focus on contextual and situational references, which implies a reconstruction and reorganization of learning materials in different forms, using distinct languages and supporting tools. A third challenge is to make use of elements and materials emerging from professional practice to build up significant learning situations which also implies the engagement of professionals in the construc- tion, elaboration, and offering of learning materials to be used within academic contexts. 70 MAURA STRIANO Each curricular discipline, even the most theoretical, has a mean- ingful relationship with life and professional practice and this must be explored in depth and emphasized to offer students meaningful learning experiences. In fact, each discipline is the organized and systematic by-product of processes of inquiry developed to achieve a better understanding of specific phenomena and situations, as well as to solve problems and to act on the surrounding environment to bring about signifi- cant changes, and this root is the key for an epistemic re-positioning of the students towards the disciplines they encounter on their aca- demic pathway. 3. Thinking and learning to connect knowledge and practice p y An inquiry-based approach to academic disciplines is therefore par- ticularly effective in helping students in constructing, reconstructing, and reorganizing their cultural background, which works as a plat- form that sustains further academic and professional learning. Moreover, through interiorization of the pattern of inquiry, stu- dents learn to master increasingly articulate and sophisticated cul- tural and practical challenges, since this pattern frames either their approach to and use of new forms of knowledge, or their professional commitment. By engaging the students in problems emerging from genuine practical and professional situations presented by senior students or by professional testimonials in the form of case studies, it would be possible to prepare the practicum experience in advance, and create a circular connection between academic and professional learning. i p g This approach to academic disciplines is particularly significant with- in liberal study curricula since it highlights the possibility of engaging the students in the development of transverse abilities and competencies (such as problem-finding and problem-solving competencies…) which is the basis for future experiences in a lifelong lifewide learning perspective, and plays a key role in any kind of professional occupation. The traditional dichotomy between vocational and liberal disci- plines and fields of study as well as between higher education curri- cula and programmes is no longer effective within current educational and political scenarios, which require students to be equipped with a set of transverse work-related competencies, not specifically con- nected to a pre-determined professional profile, but essential to help the students in finding their way in the future. f i g y Work-related competencies are effective in identifying and detail- ing goals, in defining consistent sustainable projects, in testing and working out hypotheses within real-life contexts and situations and are sustained by a reflexive epistemology, which is the grounding of a sound professional engagement and a constructive professional de- velopment in any field. 71 HIGHER EDUCATION AND WORK-RELATED LEARNING Magnell (2016) highlights several approaches to include work-relat- ed learning in academic curricula: added on by someone else; added on by the profession; integration of teaching and learning activities and integration with added value. Each approach is connected to a specific but diverse understanding of the function of education for academia and of education for work outside academia, which include various perceptions of roles, types of work-related learning activities, and their integration in the standard curriculum. 3. Thinking and learning to connect knowledge and practice Undoubtedly, the integration of teaching and learning activities and the integration with added value are particularly challenging, since they require a thorough reorganization of both the curriculum and the institutional organization. 4. Work-related learning and the construction of professional epistemology In not providing meaningful work-related learning experienc- es, higher education does not offer students the possibility to build a specific professional epistemology (i.e. the way professionals use, reframe, and build knowledge) which is the core of any professional role. Moreover, there is the risk that professionalizing higher education might lead the students towards a positivistic epistemology of practice. As Schön has highlighted in an analysis that can still be applied to current higher education scenarios, professional education is based on a positivist framework, which privileges the technical, the test- able, and the objective, and separates ends from means while failing to train for the real problems of practice (Schön 1992). Consequently, the epistemology of professional practice built in- to the very structure of professional schools and research institutions has been set forth as an exercise of technical rationality, that is, as an application of research-based knowledge to the solution of problems of instrumental choice. Schön suggests reconsidering the question of professional knowl- edge referring to an alternative epistemology of practice, which takes full account of the competence practitioners display in situations of uncertainty, complexity, and uniqueness, and is mainly based on the capacity to practice reflection on and in action (Schön 1992). Drawing on Schön, Kinsella (2010) highlighted the need for an epistemology of reflective practice based on a series of constitutive ele- ments: a broad critique of technical rationality; a vision of professional practice knowledge as a form of artistry; a constructivist understand- ing of the processes of knowledge construction; acknowledgment of the significance of tacit knowledge for professional practice knowl- 72 MAURA STRIANO edge, and, finally, the overcoming of body-mind dualism to recog- nize the knowledge revealed in intelligent action. According to this framework, higher education should therefore be strongly learner-centred and focused on the necessity to engage students with real problems emerging from the practice contexts, making use of methodologies such as problem-based learning, inqui- ry-based learning, and role playing, in order to sustain them in the process of constructing a reflective professional epistemology, useful to build sound professionality. HIGHER EDUCATION AND WORK-RELATED LEARNING HIGHER EDUCATION AND WORK-RELATED LEARNING 73 getting to know the language, narratives, and funds of knowledge that underlie communities of practices within different professional tradi- tions. This would lead students to approach a professional engagement based on a sound awareness of the conditions under which they come to actively participate in productive life. There is, indeed, especially in Italy, a lack of preparation of gradu- ates in relation to their professional identity, since this demands con- stant confrontation with professional experiences and professional contexts. Moreover, the construction and development of a professional identity requires acknowledgment of the existential potential of the job as well as the acquisition of languages, categories, narratives, and ‘war stories’ that are the inner fabric of any professional adventure. This calls for a strong and steady relationship between higher edu- cation contexts and professional organizational ones in addition to a constant engagement of experienced professionals as mentors and tutors for future graduates to sustain their legitimate and progressive participation in professional activities and practices. 6. Conclusions The main challenge for higher education today is to sustain students in both the acquisition of professional expertise, and in the develop- ment of a critical and flexible professional epistemology that cannot be separated from a professional identity, in order to sustain an effec- tive transition from the academic to the working life. Lave J. and Wenger E. 1991, Situated Learning. Legitimate Peripheral Participation, Cambridge University Press, New York. 5. Developing professional identity through legitimate peripheral participation Professional epistemology is one key element of a clearly defined and acknowledged professional identity, which, as Pratt, Rockmann and Kaufmann (2006) point out, can be understood according to three different perspectives considering career and role transitions, socialization, and identity work. y Professional identities are therefore constructed and developed through the process of career building based on the distinct roles played within career advancement: this is not the sum of the roles, but an integration of the experiences and expertise connected to these roles. As highlighted by Greil and Rudy (1983), the organizations with- in which a professional operates play a crucial role in the develop- ment and shaping of professional identities, bringing about significant changes over time to different degrees. Lave & Wenger (1991) explored how, within organizations, ‘com- munities of practice’ progressively sustain individuals in the construc- tion of a definite professional identity through processes of ‘legitimate peripheral participation’ that acknowledge and legitimate the entry of novice professionals into a specific field of practice. Individuals struggle to actively construct their professional iden- tity through an interactive and problematic process which engag- es them in highly demanding ‘identity work’ (Pratt, Rockmann, Kaufmann, 2006).f To be effective, this professional ‘identity work’ needs to begin and be carried out very early in the course of academic studies, which should therefore be arranged in forms and ways that sustain the stu- dents in focusing on and designing their own career path, prefiguring advancements and developments but also moments of crisis, change, and transformation. f Moreover, higher education should offer students the opportunity to develop a sound understanding of organizational processes as well as References EC 2017, Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee and the Committee of the Regions on a renewed EU agenda for higher education, EC, Brussels. g g f g Evans L. 2008, Professionalism, professionality and the development of education professionals, «British Journal of Educational Studies», LVI (1), 20-38. Greil and Rudy 1984, Social cocoons. Encapsulation and Identity Transformation Organizations, «Sociological Inquiry», LIV (3), 260-278. Hoyle E. 1975, Professionality, professionalism and control in teaching, Houghton V. et al. (eds.) Management in Education: The Management of Organizations and Individuals, Ward Lock Educational in association with Open University Press, London. Lave J. and Wenger E. 1991, Situated Learning. Legitimate Peripheral Participation, Cambridge University Press, New York. 74 MAURA STRIANO Kinsella A. 2010, Professional knowledge and the epistemology of reflective practice, «Nursing Philosophy», XI (1), 3-14. Magnell M. 2017, Employability and work-related learning activities in higher education; how strategies differ across academic environments, «Tertiary Education and Management», XXIII (2), 103-114. g ( ) Moreland N. 2005, Work-related learning in higher education, The Higher Education Academy, York. y Moore T. and Morton J. 2017, The myth of job readiness? Written communication, employability and the ‘skills gap’ in higher education, «Studies in Higher Education», XLII (3), 591-609. Pratt, Rockmann and Kaufmann 2006, Constructing Professional Identity; The Role of Work and Identity Learning Circles in the Customization of Identity among Medical Residents, «Academy of Management Journal», XLIX (2), 235-262. Schön D.A. 1992, The crisis of professional knowledge and the pursuit of an epistemology of practice, «Journal of Interprofessional Care», VI (1), 49-63. Speight S, Lackovic N. and Cooker L. 2013, The Contested Curriculum: Academic learning and employability in higher education, «Tertiary Education and Management», XIX (2), 112-126. Francesco Lo Presti (University of Naples Parthenope)* Abstract: Starting from both an Italian phenomenological perspective and some international works that develop the guidance theme in the direction of ‘educational guidance’, this chapter will briefly present the aims, instruments, and results of the Parthenope University Research Unit of the PRIN EMP&Co study, which focused on the specific segment of university guidance. The reflec- tion generated by the study provides information and data to identify guidance methods through a pedagogical approach. Keywords: pedagogy, educational guidance, higher education, life-project. 1. Introduction The contemporary era is characterized by a substantial diversifica- tion of professions. The rapid changes in workplaces and the uncertain nature of employment situations are forcing individuals to constantly come to terms with continuous changes in their lives, which implies the need to learn to constantly rebuild a sense of Self. Flexibility and mobility in the professional field therefore require education and training that provide people with support that goes beyond the mere transfer of contents and knowledge. They need to equip individuals with the ability to cope with the experience of change. All those ac- tivities aimed at allowing individuals to recognize and develop their own aptitudes, skills, and competencies therefore become central in the educational experience, in order to consciously build and man- age their life project; all such activities correspond to the term edu- cational guidance. * Francesco Lo Presti, PhD, Assistant Professor of General and Social Pedagogy, University of Naples “Parthenope” Italy, Email: francesco.lopresti@uniparthenope.it. GUIDING AT UNIVERSITY. THE PROTAGONISTS’ POINT OF VIEW Francesco Lo Presti (University of Naples Parthenope)* 2. Theoretical premises, subjects, and methodology Educational guidance appropriate to the current socio-cultural background should therefore be defined as a continuous process that accompanies all the phases of existence, aiming to put the individual in a condition to make conscious choices in order to exert control over their own life and to be positive and effective at personal, so- cial and professional levels. However, the current state of education- 78 FRANCESCO LO PRESTI al guidance has not yet seen the existence of a guidance system that concretely places itself as an educational and training support system developed within people’s overall lifespan of. Most of the guidance activities currently being developed in the field of formal education refer to some training paths for individuals, identified as particularly critical, such as moments linked to the choice of their educational and professional pathway. In particular, the selection and entrance phase of the University represents one of the moments that sees the widest production of activities, aimed at supporting individuals in coping with this delicate phase. Therefore, while emphasizing the need for a guidance system to define the horizon of its action to take in people’s whole existence, education, and choices, the research presented in these pages concen- trated on University guidance practices in Italy, understood at present as the most critical and sensitive crossroads of the training path. Most of all, the research concerned the approaches and guidance practices of University entrance in Italy. The observed perspective chosen is of a pedagogical matrix; i.e., it interprets guidance as a process of an intrinsically educational na- ture, identifying education in general as a fundamental process which itself expresses guidance characteristics. In other words, the idea is assumed that an education which fully responds to its prerogatives must necessarily fulfil the fundamental goal of making individuals in training capable of coping in the context of complex living expe- riences and to guide themselves in their ability to choose and train. g y Discussing guidance should therefore, in our opinion, be based on the key theme of human formation (Cambi 2003; Massa, Deme- trio 1991) both in terms of institutional formative pathways and the mechanisms linked to them, and in the field of scientific research. This horizon acquires the existential concreteness of educational phenom- ena (Massa and Demetrio 1991) as a guiding principle for designing effective guidance and, therefore, for an ‘up-to-date education’. 2. Theoretical premises, subjects, and methodology In this sense, in recent years, the role and specificity of pedagogy have adopted a much clearer stance (Cunti 2008, 2015; Domenici 2003; Loiodice 2009; Lo Presti 2010, 2015; Pombeni 1996), through the design of methods and training instruments intended to integrate critical capabilities on their own, and on cognitive and identity pro- cesses that guide choices in relation to the opportunities in educa- tional contexts (Bateson 1972; Bertolini 1988; Bruner 1990; Dewey 1946; Gardner 1991; Massa, Demetrio 1991). ) In this direction, the overall aim of the research was to investigate both the most widespread practices in the Italian University system and the related models, with the specific intention of verifying the presence of educational dimensions, i.e., theories and methods focusing GUIDING AT UNIVERSITY. THE PROTAGONISTS’ POINT OF VIEW 79 on the goal of the overall maturation of an individual in projecting capability of choice. p y From a methodological point of view, the research favoured a phe- nomenological-hermeneutical perspective (Bertolini 1988; Bertin, Contini 1983; Cavana, Casadei 2016; Husserl 1954; Merleau-Ponty 1945). The assumption of this theoretical horizon therefore, led the interest not towards mere facts, but in the direction of the impact they have on individuals through the meaning they assume in their lives: «phenomenology is seeking realities, not pursuing truth» (Mor- tari, Tarozzi 2010: 18). In this way, in the background, the research acquired the assumption that deeper understanding needs to pass via an individual’s own standpoints. Similarly, the research strategy was of a qualitative nature (Clandinin, Connelly 2000). q y Starting from these assumptions, the research developed in two phases. The first featured representatives of governance and guid- ance operators (rectors’ delegates, coordinators and/or branch office operators); instead, the second involved groups of students attending their first year of University.ii i y y The specific purpose of the first phase was to identify the guidance models and practices in use at the Italian Universities involved, taking a closer look at their educational and training dimensions. The sec- ond, on the other hand, sought to identify students’ guidance needs and the deepest individual aspects at the basis of their choices and life planning and, secondly, to try the same line of inquiry with students as a possible self-reflective guidance value strategy in the field of in- coming University guidance. 3. The first research phase: aims, instruments, results This phase involved, as anticipated, privileged interlocutors of the academic world and institutional representatives engaged in guidance actions. Specifically, 10 subjects (pro-rector, guidance delegate, op- erators, teachers) were involved, placed in key positions in the pro- gramming and managing of the above-mentioned proceedings at the Universities of Padua, Florence, Siena, Naples “Parthenope” and Na- ples “Federico II”. The instrument chosen to collect research data, in qualitative terms, was the semi-structured interview designed and built to obtain profound, critical, and objective knowledge of the research subject from subjective points of view.i j j p The interview was organized on the basis of specific thematic ar- eas related to a description of the services offered, the mission and the approach pursued, the organization and management of activities, the methodology and instruments used, the professional skills and competencies 80 FRANCESCO LO PRESTI involved, the evaluation of practices, the system actions implemented, and the interplay between guidance and University teaching. The interviews were carried out with guaranteed anonymity at the various univer- sities and were audio-recorded with the consent of the participants and subsequently screened and transcribed in full. involved, the evaluation of practices, the system actions implemented, and the interplay between guidance and University teaching. The interviews were carried out with guaranteed anonymity at the various univer- sities and were audio-recorded with the consent of the participants and subsequently screened and transcribed in full. The aim of this phase was, therefore, to return to the knowledge, representation, and beliefs, including implicit ones, which are at the ba- sis of guidance actions in order to obtain a background model. In other words, from an individual point of view, it set out to identify and examine implicit and explicit patterns at the origin of the university guidance system, in a search for the meaning, logic, and educational dimensions that lie behind the practices, to define an image of the approaches through which it is organized and implemented in the institutional contexts in question. Certainly, the results outlined do not constitute extensible or gen- eralizable data, since it is not a quantitative statistical survey; more exactly, according to the phenomenological-hermeneutic approach, they represent a key to reading the object which, through attention to the singularity of the cases, provides levels of knowledge and depth that contribute to clarifying its nature. 3. The first research phase: aims, instruments, results y g The interviews were analysed categorically with Atlas-ti 6.0 soft- ware (Muhr 2000), based on a network structure consisting of a cor- pus of hermeneutical units and an intuitive and creative approach to data usage. The generative research question that motivated the categorical analysis was created based on the famous formula of Glaser (1998) “What’s going on here?” and was expressed in “How does guidance happen in Italian university contexts; which approach do the prac- tices imply?” 3.1 Data analysis 3.1 Data analysis The analysis of the interviews was divided into three phases: ini- tial or open encoding, which consisted of line-by-line text analyses, ac- cording to the presupposition that ‘All is data’ to identify the minimal meaning units (text pieces) and the thematic recurrences in the form of precisely defined ‘labelled phenomena’; focused encoding, which al- lowed identification of conceptually wider macro-categories through a redefinition of the initial labels and their transformation into more mature concepts; theoretical encoding, which allowed development of a conceptual model that supplemented the main categories.i p pp g In the initial coding phase, 85 codes were identified (Code map) and these referred to all the sensitizing concepts that emerged from the first analysis of the corpus of interviews, regarding the initial search GUIDING AT UNIVERSITY. THE PROTAGONISTS’ POINT OF VIEW 81 inquiry. Subsequently, the codes considered redundant were grouped into a smaller number (32), then further reduced to 21. In the focal- ized encoding phase, the codes became 4 broader macro-categories, which through conceptual work were appropriately renamed and as- sociated through semantic links. From these reports, in the last phase (theoretical encoding), the central category was identified, induc- tively identified by following the hierarchical work on the categories emerging from the data, from which a general theory on the Univer- sity Guidance Process was integrated and delineated. This theory ex- presses all the points of view, facts, beliefs, and implicit knowledge of those interviewed, sorted into a unique configuration that generalizes the contents on the basis of categorical comparisons and combinations. g p From this general theory comes a guidance approach denominat- ed matching, in which choice is represented as the best result of the relationship between the individual and the university’s educational offering. In this sense, the guidance is interpreted by the individuals involved as an instrument that links what the student likes doing to the corresponding Degree Course. This guidance approach would thus seem to refer to theories on personal-environment adaptation, which are defined as matching theories (Parsons 1909), and which de- scribe choice as the most ‘economic’ result in the relationship between individual characteristics and environmental ones. In addition, a generalized approach to guidance is mainly inter- preted as a marketing strategy aimed at suggesting and attracting large, anonymous crowds of students in which every individuality is de- stroyed (Demarie, Molina 2007). Political Model This category essentially represents a series of points of view and acquisitions that follow a hierarchical mode in determining the plan- ning of guidance strategies; a mode in which the rules, visions, and educational values that the guidance determines seem more compat- ible with the requirements of the University system, rather than be- ing tailor-made to the individual student. The process outlined by the categorical analysis of the corpus highlights how guidance is un- derstood as coherently based on a vision of a linear pathway in which choice is interpreted as already clearly present in an individual, being directly linked to a specific training course and hence a presumed aim of the working world that corresponds to it. Literature Model This category expresses points of view that generally place indi- viduals and their characteristics at the centre of guidance processes; in fact, it would outline an alternative to the political model in con- ceiving the paths that lead individuals to build their own identity, educational paths, and career. However, for reasons likely to be traced both to logistical and infrastructural crises, and in a common difficul- ty in clearly identifying a widely recognized and shared theoretical- methodological background, this kind of model fails to be coherently and concretely placed to guide planning. An awareness regarding the importance of referring to a scientific-theoretical model of guidance emerges, supporting and helping the political model, which, how- ever, remains unchained to the practices, staying confined to the background of a merely declarative dimension of the principle lines. Students as painters of themselves This category expresses awareness of the complex dynamics that interact in individual decision processes, in which the significant in- dividual narrative is supremely relevant. As a result, the subjects in- terviewed showed that they recognized the complex aims of effective guidance, clearly identifying the subjective and variable nature of the dynamics that make up the student learning experience; however, even in this case, such awareness did not match the relevant practices. 3.1 Data analysis The emerging theory is indicated as being of a predominantly informative nature, and is appreciated for its usefulness in the immediate future; in other words, it is assumed that information may possess the enlightening power to provide students with an instrument to recognize themselves in their own future. It emerges that the individual and the institutional dimension blend in favour of the needs of the latter to the detriment of recognition of the former, where, on the contrary, an individual’s needs to project, construct, and give meaning to their existence, should in our opin- ion, become the primary aim of university guidance. In short, even if, according to recent theoretical models, guidance interventions should provide a response to individuals’ need to re- flect on themselves and know themselves to construct a life project that fully represents their subjectivity, it is clear how such models do not actually find a precise correspondence between theoretical/de- clarative aspects and practical/applicative developments in the actual perpetuation of an informative-divulgating approach. From the general theoretical project that emerged, four macro- categories of synthesis were obtained, connected by semantic links, 82 FRANCESCO LO PRESTI which explain the University Guidance Practices in the view of the interviewers and offer the opportunity to reflect critically on them. The four macro-categories that emerged are as follows. GUIDING AT UNIVERSITY. THE PROTAGONISTS’ POINT OF VIEW 83 ing as if the latter did not already incorporate a guiding prerogative. In other words, the planning of guidance and the management of ‘ordinary’ didactics appear to be out of step: teaching does not usu- ally cover guidance and does not benefit from the actions carried out by guidance services, except where it is believed to benefit from the presence of previously well-trained students. Teaching and guidance are therefore represented as two fields disengaged from one another, although the teaching experience never plays a neutral role in subject guidance processes, whether intentionally or unintentionally. The theoretical system derived from qualitative analyses of da- ta therefore leads to the conclusion that, for the subjects involved, the prevailing concept of University guidance, even if tendentially structured according to an approach and a mission based on more or less clear theoretical guidelines, still has some gaps. We would like to emphasize the disagreement with the strong need of individuals to be accompanied in the task of giving meaning to their existential projects and projecting themselves towards the future (Benasayag, Schmit 2004). ) To conclude, analysis of the set of results linked to this phase very quickly highlighted, the following critical points: • Substantial dissolution between emerging models and the quality of practices actually developed • Failure to refer to a shared system of projecting and guidance man- agement (absence of a recognized and unambiguous legacy) g g g g y • Unmodified direction of guidance on the educational side • Failure to recognize individualization and centring elements on the subject. Guidance-teaching dichotomy The fourth category groups the opinions that describe the lack of recognition of a meaningful relationship between guidance and teach- GUIDING AT UNIVERSITY. THE PROTAGONISTS’ POINT OF VIEW 4. The second research phase: aims, instruments, results The second phase, as anticipated, directly involved the students, aiming to identify their guidance needs, the deepest individual as- pects at the basis of their choices and life planning, and possible guid- ance strategies to respond to these. In this case, the instrument chosen and considered the most suitable to achieve the objectives was the focus group (Merton, Kendal 1946), which, with its interactive mode, represented a useful instrument both to recover deep-seated infor- mation about the points of view of the students involved, and to ac- tivate a reflective interactive posture that could guide the subjects themselves to become aware of their active and constructive role in the processes of choice. This second aim, in particular, was intended to correspond to the intention, which we have already referred to, to experience a short ‘educational path’ of a reflective nature, dur- 84 FRANCESCO LO PRESTI ing which participants could talk and swap opinions on the theme of guidance by drawing on the elements of reflection useful to activate internal self-revaluation processes, concerning their own knowledge and competencies. The use of the focus group was therefore linked to the choice of using narrative to generate dynamic reflection on complex themes that affect the individual’s guidance within a context of group interac- tion, the latter being seen as a resource useful to collect information, points of view, personal problems, stances, but also to collectively pro- duce alternative ideas and viable solutions (Duggleby 2005; Krueger, Casey 2000). y ) The planning that preceded the focus groups was split into a phase of conceptualization and development of the themes and of the re- lated questions and techniques; a phase of choice and recruitment of the potential participants, and finally, a stage of logistics and project- ing organization regarding the manner of conducting the discussion and the moderators to be involved. The participating students were recruited from membership lists through the guidance information desk by a personal request for par- ticipation by email. Starting from the consensus obtained, heteroge- neous groups were created by age and scope of training in order to promote greater variety and exchange point of views and to promote a certain dynamism in the discussion. GUIDING AT UNIVERSITY. THE PROTAGONISTS’ POINT OF VIEW 85 research, was arranged and adapted to the educational story which be- comes a life project. In the work described, the focus group was used as a qualitative instrument of the narrative revelation, but also as a training moment for the students involved and a chance for the re- search group to build a pedagogical university guidance proposal that dealt with educational issues. 4. The second research phase: aims, instruments, results As regards the implementation phases, groups were created of 12 members and a moderator team, consisting of a moderator, respon- sible for presenting questions, stimulating materials and facilitating discussion, promoting and encouraging the students to participate; and an assistant moderator, as suggested by Krueger (1994), with the task of video-recording the sessions and writing any memos. The chosen mode of conduct was mixed, and, broadly speaking, it had the moderator playing a marginal role during group discussions and an active role in launching themes and supporting discussions with encouragement and meaning feedback. The setting for the discus- sions − warm, friendly, permissive, and non-threatening (Krueger 1994) was a neutral space at the University’s facilities that the students regularly use, and was therefore familiar to them and non-inhibitory. g y y Considering the complexity and multidimensionality of the ob- ject of the focus group, it was decided to create 3 sessions per group one week apart, to give the necessary time to reflect on the personal aspects that emerged during the discussion and to produce narrative material useful for the next session. The structure of the focus group sessions started from the way in which Savickas and Hartung (2012) organized career history production according to the famous formu- la Telling-Hearing-Enacting My Story, which, in the case of the present GUIDING AT UNIVERSITY. THE PROTAGONISTS’ POINT OF VIEW 4.1 Data analysis The qualitative data collected through the 3 focus group sessions was subjected to categorical and content analysis with the support of Atlas.ti software. The analysis of the corpus was divided into three phases: initial or open coding, which identified the meaningful text parts containing the sensitizing concepts (60 codes); focused encod- ing, which redefined the labelling work as well as 20 conceptualized macro-categories; theoretical coding, which identified and elabo- rated the most mature concepts in the form of a conceptual model (9 codes). The analysis therefore allowed the formulation of a theory that explained the process leading the participants to fathom and/or change their meaning in life.i g g The core of the conceptual system has been defined as Fragility of Choices and it represents the widest and most branched category emerging from textual analysis. The theme of choices is redundant in the participants’ narratives and is described as an element character- izing their life paths in a pervasive way, since, although it represents a growing and formative opportunity for the person, it does seem to have been lived in negative terms, as a weight on the shoulders and a time of difficulty to cope with. In fact, the description of the choices that emerged from the analysis refers to dimensions such as those of obligation, conditioning by others, renunciation, and, above all, an absence of those fundamental aspects – knowledge, passions, interests – starting from which decisions are made that consider both ratio- nal and emotional-affective aspects. In other words, the core category highlights that the choices acquire deep meanings which transcend a mere selection from among different alternatives, in order to deci- sively affect the future. Not only does this create the fear of failing to make sufficiently weighty and profitable choices, it does not tolerate the possibility of making mistakes either. p y g Around the central core just described revolve another 8 macro- categories, held together by semantic links, which explain how the participants come to build their meaning in life: • Recursive transitions • Ambivalence about institutional education; • Personal experience; 86 FRANCESCO LO PRESTI • Significant adults; • Guidance disconnected from needs; • Self-guidance; • Vocation comes at work; • Sense of existence between work and knowledge. The imminence and obligation of choices intensifies due to, and at the same time as, recursive transitions which mark the participants’ life paths. 4.1 Data analysis The transitions referred to, are, for example, the canoni- cal ones that affect the community of teenagers and young adults in the transition between different orders and degrees of formal educa- tion. In fact, although change and transitions represent a structural datum of people’s whole life-cycle, those described in the narratives seem to have a potential for criticality precisely because of the need to make some choices that clearly also include a degree of resolution with oneself and the surrounding environment. Thus, what appeared to have, at least in the previous decades, an evolutionary potential in terms of emancipatory change (transition as an exclusively positive event) is characterized today by its involutional character, to procras- tinate and/or stagnate. What emerges, therefore, is worry, and the need to identify educational and professional paths that are satisfac- tory and congruent with one’s personal expectations and aspirations. Moreover, the ambivalence of institutional education emerges, which, despite undertaking for individuals one of the privileged con- texts for assuming a ‘form’, in the sense of acquiring a specific way of being and living, seem to be ineffective in supporting and educat- ing choices. In this regard, it emerges, above all, that high schools are described as a pathway which, instead of welcoming and ‘keeping’, ‘alienate’ the subject as a place where teenagers cannot perceive the possibility of recognizing themselves. Instead, the side of personal experience is interpreted as the most significant in relation to identity-building and self-knowledge; ele- ments that prelude the choice and, therefore, help clarify the exis- tential projects. The process leading to Self-determination is clearly seen by others, in particular by significant adults, parents, and teachers, who, in a few cases, were described as fully able to offer the instruments to make the self- construction of choice paths concrete, for example, by offering them- selves as an example or a guide, since they are more often considered as figures predominantly engaged in providing prescriptive indications and advice that channel their idea of others into pre-established paths. What emerges from the quality of the teachers’ educational work reflects in a substantial way the meaning that guidance assumes in institutional contexts. According to the participants, guidance is un- GUIDING AT UNIVERSITY. THE PROTAGONISTS’ POINT OF VIEW 87 dervalued, tackled superficially, not supported by specially trained educational figures, following an informed approach, considered virtually ineffective in responding to their needs. 4.1 Data analysis However, the par- ticipants did identify an alternative guidance instrument that they categorize with their work experience. In fact, this represents one of the most pregnant occasions in understanding what they intend to be, since it offers an opportunity to practically experience profes- sional roles through the introduction of knowledge, skills, and ap- titudes that only in this way they discover they have. It follows that vocation comes at work through discovery of self-related aspects of the professional sphere that were previously latent. Consequently, guidance is described by the participants not as a habitus born and cultivated in formal educational contexts, but as a form of self-guidance that takes the traits of an adaptive response to the lack of educational actions in this direction. In other words, the participants looked independently outside institutional contexts for the instruments they needed to make choices that were not hasty and superficial.i pi All this interrelated system of contexts, processes, reference fig- ures, and divisions that affect the personal and professional sphere con- tribute to determining what represents their meaning in life. What guides and motivates the sense of existence is also in this case em- bodied in elements such as personal fulfilment and satisfaction con- cerning work and, in a more general perspective, the need to know and discover oneself and the surrounding world to find constructive forms of self-expression. p There seems to be a certain degree of autonomy in the exercis- ing of choices and, therefore, in the ability to guide oneself, but this is generated mostly by cases of absolute necessity, which turn to a natural guidance predisposition, rather than a sense of responsibility and self-awareness. In other words, self-directed choices and paths are accompanied by a continual latent sense of restlessness, as a re- sult of a ‘leap into the dark’ rather than the result of a project that combines self-knowledge and opportunities. This seems to indicate a tendential ineffectiveness on the side of educational responsibilities, which do not emerge as significant in the perception and interpreta- tion of the subjects in relation to their guidance needs. In this direc- tion, what appears relevant is the reference to work and the category of direct experience as the main sources of guidance; sources linked, however, to chance, and above all not filtered by a systematic critical exercise of the subjective value it could have. 4.1 Data analysis In other words, in the individual interpretations, ‘personal experience teaches’ more than educational institutions, although this does not directly imply that experience itself is the subject of critical analysis. 88 FRANCESCO LO PRESTI 5. Conclusion GUIDING AT UNIVERSITY. THE PROTAGONISTS’ POINT OF VIEW 89 tinuous evolution of the skills expressed by the educational system towards the ability to meet, interpret, and support the singularity and complexity of the most current needs of subjects in training. References 5. Conclusion Thus, the path ultimately highlighted some elements of knowl- edge that are useful in defining the pathways of educational guid- ance to be tested, validated, and improved on successive occasions within further contexts comparable to more typical characteristics. These elements are:f • the effectiveness of educational work carried out within group dis- cussions as a privileged place for the expression of a narrative-dia- logic perspective;l • the use of narrative as a reflective and viatical instrument funda- mental for the development of knowledge, awareness, training, and self-education;i • acknowledgment of an educational benefit perceived by students in terms of self-awareness, of reflective and guidance competencies that would also indicate the need to plan guidance routes according to systematic, structured, accompanying forms, which are repeated at good stages marking the academic year;f g g g y • the fact that, despite focus groups having been conducted in differ- ent contexts, universities, and territories (which also led to diversi- ty in the field of objective opportunities), there was a strong overlap in the aspects that characterize student experiences with respect to themes of choice and the future in general, as well as of the generally expressed guidance needs. The theme of the competency of choice, as the central guidance cat- egory, appeared transversely, in the light of the outcomes, deeply bound up with constant exercise in self-questioning and reality. The option to direct guidance practices to the concreteness and density of the subjective and intersubjective dimensions of making meaning, rather than merely providing information and knowledge, or mea- suring abilities and aptitudes, appears effective with respect to the aim of highlighting the link with personal experience as a subjective experience of reworking meaning that acts as a theory guiding action. The dialogic-relational dimension, too, proved to be a structured and effective reflective space (governed by models, methods, and instru- ments) for constructing meaning and, therefore, ‘directions’. Consequently, all of this reinforces the idea of an adequate peda- gogical approach to guidance, and points towards further commitment to concrete experimentation with consistent practices and education- al-reflective instruments that make them operational within hetero- geneous and pluralistic contexts in a sharing and systemic view. g p g y Our work has been moving along this road and the information that has emerged seems to confirm the need for a further and con- GUIDING AT UNIVERSITY. THE PROTAGONISTS’ POINT OF VIEW References Benasayag M. and Schmit G. 2004, L’epoca delle passioni tristi, Feltrinelli, Milano (orig. ed. 2003). g Bertin G.M. and Contini M.G. 1983, Costruire l’esistenza, Armando, Roma. Bertolini P. 1988, L’esistere pedagogico, La Nuova Italia, Firenze. Bruner J. 1990, Act of Meaning, Harvard University Press, Cambridge. Cambi F. 2003, L’autobiografia come metodo formativo, Laterza, Roma-Bari Cavana L., Casadei R. 2016, Pedagogia come direzione. Ricerca di senso tra dinamiche esistenziali ed esigenze professionali, Aracne, Roma. Cunti A. 2008, Aiutami a scegliere. Percorsi di orientamento per progettare e progettarsi, FrancoAngeli, Milano. g Cunti A. 2015, Orientarsi da giovani adulti. Educare al desiderio tra formazione e lavoro, «Pedagogia Oggi», 1. g g gg Duggleby W. 2005, What About Focus Group Interaction Data?, «Qualitative Health Research», 15, 832-840. Glaser B.G. 1998, Doing Grounded Theory. Issues and Discussions, Sociology Press, Mill Valley. Husserl E. 1954, La crisi delle scienze europee e la fenomenologia trascendentale, il Saggiatore, Milano (orig. ed. 1954). Krueger R.A. and Casey M.A. 2000, Focus groups: A Practical Guide for Applied Researchers (3rd ed.), Sage, Thousand Oaks. ( ) g Krueger R.A. 1994, Focus Groups: A Practical Guide for Applied Research (2nd ed.), Sage, Thousand Oaks.l Lo Presti F. 2005, Il senso del sé. Percorsi autoriflessivi nella formazione, Pensa Multimedia, Lecce. Lo Presti F. 2010, Educare alle scelte. L’orientamento formativo per la costruzione di identità critiche, Carocci, Milano. Massa R. and Demetrio D. 1991, Le vite normali. Una ricerca sulle storie di formazione dei giovani, Unicopli, Milano. f g p Merleau-Ponty M. 1945, Phénoménologie de la perception, Gallimard, Paris. Merton R.K. and Kendal P.L. 1946, The Focused Interview, «American Journal of Sociology», 51. 541-557.f Morin E. 2001, I sette saperi necessari all’educazione del futuro, Raffaello Cortina, Milano (orig. ed. 2000).f Morin E. 2015, Insegnare a vivere. Manifesto per cambiare l’educazione, Raffaello Cortina, Milano (orig. ed. 2014). Mortari L. and Tarozzi M. 2010, Phenomenology as Philosophy of Research: An Introductory Essay, in Mortari L. and Tarozzi M. (eds.), Phenomenology and Human Sciences Research today, Zeta Books, Bucarest, 9-56. * Bruno Rossi, Honorary Professor of General and Social Pedagogy, University of Siena: Email: bruno.rossi@unisi.it. FRANCESCO LO PRESTI 90 FRANCESCO LO PRESTI Muhr T. 2000, Atlas.ti Short User’s Guide, Scientific Software Development, Berlin. Savickas M.L. and Hartung P.J. 2012. My Career Story. An Autobiographical Work Book for Life-Career Success, <http://www.vocopher.com> (01/18). Bruno Rossi (University of Siena)* Bruno Rossi (University of Siena)* abstract: The guidance experience, pedagogically considered, is above all the process of identification, recognition and consolidation of individual resources of which the person is the bearer (attitudes, capabilities, inclinations, expectations, desires, projects), is the conquest of one’s own identity, the research of constitu- ent elements of personality, the awareness of ‘being-different-from-everyone- else’. Within this theoretical assumption, the narration is a suitable placement as estimable and relevant device for self-guidance and transformation of the self. In the guidance actions the use of narrative, reflective and autobiographical para- digms can represent an effective aid in the direction of the redefinition of iden- tity, empowerment and self-empowerment. Narration is the search for the truth about the self, or even more the process of re-thinking and re-design the self. It means to discover and achieve a deep knowledge about the self, to reconstruct one’s own life plan, is to de-form to re-emerge, to re-form it. In a broad sense, it is in some way to ‘die’ to be reborn regenerated. Keywords: narration, identity, project, skills assessment. Keywords: narration, identity, project, skills assessment. If the watchword of the culture of orientation has generally been ‘aptitude’, it is now possible to affirm that the real keyword is ‘identity’. Every valid guidance experience allows people to meet their inimitable typicality and define the ideal of a Self that is generative of the intent to circumscribe its role within the social fabric accompanied by a readiness to bring meaning to subjective existence. Conquering self-oriented competence is an educational goal of no small account, thinking particularly of the fact that the health of any personality is identified by an image of itself that is both complete and unified, including not only the concept of what it is, but also of what it wishes to be and should be. Self-awareness is equivalent to reconstitut- ing the identity of a subject seeking the achievement of essential goals, through constructions and reconstructions, structuring and destructur- ing, learning and unlearning. Being conscious of one’s own worth and need, perceiving oneself as different, contributes in no small measure to eliminating any sense of in- significance and existential banality, and brings the necessary strength to respond responsibly to the appeals of life. Suffice to recall the concept of psychological education to refer to the fundamental educational task of fos- tering the ‘I’. This is a task which is very different from nurturing escap- ism and solipsism, individualism, and selfishness. What is at stake is the 92 BRUNO ROSSI emancipative undertaking to make people master their psychic life and thus help to defend them against prejudices and conditioning, mortifica- tion, and manipulation, and to conquer a force of expansion and a gener- ation of humanity that nurtures their personal, social, and cultural lives. The concept of the development task may serve to indicate program- ming indispensable for psychosocial education, for a positive and reward- ing growth, and for the conferral of direction, unity, and consistency in one’s evolutionary journey. In this sense, the various development tasks are arranged as educational commitments, which, as such, demand the possession of principles and rules, and ideals and values, that can bring order to the many decisions a human being is required to make. Keywords: narration, identity, project, skills assessment. f This concept leads us to focus on the commitment and effort, cour- age and creativity, the availability of renewable energy and the drive towards perfection that a subject must express in taking shape, thereby emphasizing the fact that the various evolutionary achievements have nothing to do with the occasional and contingent nor axiological neu- trality and intimism. An authentic personal development, while it bears the beauty of adventure and constant discovery, can also be appreciated for its strong passion for regulative principles that are beneficial for the human being to grow and improve. SELF-TELLING TO ORIENTATE ONESELF 93 (what they actually are, are worth, possess, what they know, can and want to create), they valorize themselves, measuring their value against external reality, produce their own existential project, exercise a capac- ity of personal initiative, inventiveness and creativeness capable of pro- ducing high rewards for themselves and the community. Orientation is essentially a way to manage their own life according to vocationally significant decisions from an independent and free being engaged in the choices of planning life in which and with which they foster the full development of their personality. For this reason, orienta- tion cannot mean one technique or some improvised advice that reduc- es it to a point of view or opinion in a process prompted by specialized staff or an operation carried out from the outside by experts, nor in an event of the aptitudinal-measuring-diagnostic-informative type, but in a process that finds its beginning and evolution in a person’s ontological substantiality and ethics, while necessarily noting the importance of so- cial factors, so that, not infrequently, the dynamics of decision-making are the product of a compromise. p p In this sense, self-orientation follows on directly from introspection and self-assessment, i.e. from a critical and objective analysis of one’s de- sires and aspirations, capacities, and interests, both hidden and obvious from the precise assumption of ‘being-in-the-world’, in other words, from the consideration of one’s own self interrelated with the experien- tial reality that has significance for the self. In short, the roots of self-knowledge lie in self-exploration and self- objectification, thanks to which human beings can precisely trace their destiny, establish their own existential project, and take the decisions for their own life independently, developing appropriate forms of origi- nal assertiveness to defend themselves from pseudo-vocations and exter- nal pressures, while taking note of adaptations and pseudo-participative dynamics. y Within this theoretical framework, narrative can find a suitable loca- tion as an estimable relevant pedagogical instrument of self-orientation and self-transformation. In guiding actions, the use of reflexive autobio- graphical narrative paradigms can be the guarantor of an effective aid in redefining the identity of the existential design of empowerment and self- empowerment, i.e. gaining a set of facts, skills, and relational modalities that can facilitate independent research strategies to achieve the goals of innovation, constructiveness, and social and professional protagonism. 1. Self-knowledge and the self-project These are considerations which help to underline that orientation, even sooner than being scholastic and professional, is something personal. We are in the presence of a dynamic maturation process that requires the use and operation of all the dimensions and forces of the person understood in a realistic way, and that, in its realization, activates the affective, cog- nitive, volitional, moral, and relational spheres. In this way, orientation is equivalent to the integral formation of the personality since achieving full possession of their aptitudes and exploitation of their potential allow people to implement and practise subjective, free, and intelligent tasks that are therefore truly creative, in virtue of a continuous transcendence of their existential situation and an intentional projection towards oth- ers and reality itself. In saying this, we are sustained by the conviction that the guiding experiences are the detection, recognition and consolidation of the re- sources people possess (aptitudes, capacity, enthusiasm, inclinations, de- sires, plans), is the conquest of their own identity, is the search for the constituent elements of their personality and awareness of their own ‘being-different-from-everyone-else’. gf y Through a continuous analysis of their motivations, aspirations, inter- ests, and expertise, people come to build their own image of themselves SELF-TELLING TO ORIENTATE ONESELF SELF-TELLING TO ORIENTATE ONESELF p p g There is a close bond between an ideographic conception of orienta- tion and narrative methodology: orientation cannot be substantiated by the narration itself. Life stories told in the first person bolster the guid- ance process, and ultimately establish identity processes. The legacy of stories possessed by people is the source of their stability and transfor- mations, their compass, an instrument to interpret their own world and 94 BRUNO ROSSI contribute to building it. In this way, the orientation shifts its centre of gravity from ‘orienter’ to orienting, acknowledging that these compe- tencies and powers, abilities, and resources, have a potential for self-con- duction and self-direction. contribute to building it. In this way, the orientation shifts its centre of gravity from ‘orienter’ to orienting, acknowledging that these compe- tencies and powers, abilities, and resources, have a potential for self-con- duction and self-direction. SELF-TELLING TO ORIENTATE ONESELF Being able to tell of oneself means making discoveries within ourselves and about ourselves, means being able to reconstruct our own life plan, means de-forming in order to re-form, to gain a new form, in some ways means a ‘dying’, to be reborn regenerated. Self-telling does not mean making a mere chronicle of one’s own past, since the affective, ethical, aesthetic, and cognitive dimensions are all involved. Self-telling is to give meaning to experience, renaming it, re-evoking it, commenting on it, with the conviction that this is not to be understood merely as a practice in itself, but also as an understanding of the meaning of what was and is being experienced, as a process of questioning the past and the present.l g p p q g p p Self-telling becomes precious reflective practice for the processing of one’s own biography, the interpretation of one’s own formative biog- raphy, for the reconstruction of the meaning of personal existence and personal life choices, for the meaning of events, for conferring meaning on expectations and projects, desires and duties, as well as for the under- standing and connection of happenings and events intended otherwise to separation, fragmentation and desemantization, as well as for (new) awareness-raising of one’s power and sense of self-efficacy. f g pfi y Commemorating oneself is offered to people not so much as a me- chanical repetition of the past but as an itinerary in and for itself in order to give shape, as an activation of existentially significant experiences to draw on, inter alia, in order to gain awareness, assume responsibility, and clarify, deepen and bring meaning to one’s life. The past can be consid- ered a burden that slows and hinders change, or a container of events and experiences that can be used to tackle the problems of the present and to give meaning to life, to stem the suffocating arrogance of the present, to eliminate constraints and obstacles that hinder the process of self-emancipation and self-idolization, to gain the tools and resources to make subsequent experience formatively more solid and productive. q p y p Historical memory can help people become aware of hopes, dreams and utopias, projects which, if interrupted or reabsorbed over time, still maintain the concrete potential of germinating the new and never-before- seen. Being human also means remembering what we were. 2. The story, hermeneutics, rewriting Self-telling is the search for a truth about oneself, taking care of, re- thinking, replanning oneself, being reborn. The history of a life possessed may become a valid navigation chart, thanks to which we can orient and guide ourselves, undertake a journey towards and tend to ourselves, expand and gain mastery over ourselves. Self-telling is a chance to re- write what can no longer be relived, to interpret the past in the light of the present in the prospect of a future which is a promise of regenerat- ing possibilities, of new investments and new forms of action, re-moti- vation, and learning. g Self-telling is rekindling the memory. This is relevant to estimate the contribution it can offer regarding the processes of self-description and self-reflection, investigation and discovery of re-evocation and introspec- tion, of self-seeking and biographical awareness, self-acknowledgement, and self-assessment, of a reconstruction that becomes interpretation and edification. i When people re-read and re-interpret themselves, reconsidering their existential journey and understanding themselves over again, it becomes possible to rewrite their own ‘individual novel’, the re-construction of identity and therefore a change and a growth, managing to experience things with greater awareness and therefore discovering that they are more a protagonist of their own story. Through the hermeneutics of the autobiographical road and an under- standing of the motivations and dynamics of things that have happened as well as their many meanings, people can identify their own roads and evolutionary and transformational seasons through which they developed. Through this hermeneutics, it is possible to give an explanation to one’s own past by giving meaning to the various experiences, decisions, and choices, and to impart a sense of one’s own future, to become aware of one’s own affective, social, cognitive, and moral history, and to exert a planning impetus, to explain and critically review learning and cog- nitive processes occurring during actions that produce particular epis- temic postures, to orient motivations, targets, and formative itineraries, to discover unexplored values and interests to be transformed into ob- jectives that can allow us to make best use of our subjective potentiali- ties, to gain an inner coherence and continuity in space and time, and to formulate realistic assumptions about our future. 95 SELF-TELLING TO ORIENTATE ONESELF SELF-TELLING TO ORIENTATE ONESELF In virtue of a recall, of a selective, critical, and interpretative recall, sympathetic rather than explanatory, processes of self-knowledge and self-awareness are triggered, dynamics of re-evocation and self-probing are activated, investigations of self and biographical awarenesses are prompted, learn- ing and relearning occur, a self-identity profile is gained that is simulta- neously diachronic and synchronic. y y At stake is the progressive development of a dynamic self-portrait through the multiplicity of identity that dwells within the subject, through the different identities that have oriented and continue to ori- ent one’s actions, the choices imposed or desired, personal representa- tions and projections. 96 BRUNO ROSSI Thus, starting from oneself permits the emergence of that subjectivity that is so strongly demanded by current scenarios. Starting from oneself also permits hermeneutics, self-recognition, re-appropriation and redefi- nition of one’s self, actions and one’s own context, re-interpreting and re-weaving one’s own story, self-consciousness as a subject that learns, (re)discovery of one’s own learning style, refinements of one’s skills as an aware learner, the achievement of new learning mode. Using the narrative experience, it is possible to help learners gain an idea of themselves as a subjectivity that can decide, choose, and act. For this reason, a valid activity of empowerment is outlined, favouring the gaining of agentivity and self-directionality. Thinking in particular of the work experience, what is in question is the ability to promote in subjects the design of a professional self, starting from their life story, in order to emphasize the most significant elements, to help them recover and express the personal dimension of their forma- tive experiences, to prompt a critical dialogue with the motivational sit- uations and decision-making processes encountered, to think about the problems tackled on a daily basis, to display their theoretical and practi- cal knowledge, to learn, with the goal of self-transformation, from their personal biography, as well as facilitating the identification of a profes- sional act deemed promising and significant, and therefore the solicitor of professional guidelines and plans so as to provide a greater capacity for an intelligent and autonomous location within the working processes. g g p Of note is the close connection between professional development and the attribution of meaning to the formative or professional conduct and experiences, as well as the related maturation of the concept of the formative and professional selves. SELF-TELLING TO ORIENTATE ONESELF 97 terests, beliefs of effectiveness, professional styles, motivation to work, professional values), without failing to observe that knowledge of em- ployment possibilities, professionalizing activities, the gaining of skills and technical-specialist capacities alone cannot bring completeness to professional identity. SELF-TELLING TO ORIENTATE ONESELF It is important to accompany subjects to define not only their professional interests but also their goals (remu- neration, autonomy, responsibility, relational quality, recognition, pride, self-esteem and self-confidence, learning, and professional development) that they seek to achieve by means of their work, and the importance that this plays in their life. p y In question is the opportunity to help them gain guidance skills (self- planning, awareness of their own resources, decision-making practices, self-assessment) capable of sustaining the quality and efficacy of personal actions geared to check and direct noteworthy events characterizing the development of their careers. There may be a feeling of impossibility both to understand human conduct ignoring their intentions, and to understand these, ignoring the contexts in which they have meaning. We find it essential to emphasize the need for the formative experience to help people reflect on the sin- gularity of their character and knowledge, to develop a realistic idea of themselves, an appropriate vision of personal potential, inclinations and basic skills, whether technical-professional or transverse (professional in- SELF-TELLING TO ORIENTATE ONESELF 3. Skills assessment, guidance technology By means of a re-enactment of the experiential, subjects can be made aware of their potential, can acquire a reflexive gaze at themselves and the way in which they have organized and structured their personal experi- ences, and can think in terms of skills and therefore think of themselves as a subject-in-a-situation, can re-describe themselves placing themselves 98 BRUNO ROSSI within specific binding places, can reflect on what they are able to do, even in terms of applicability in the world of work, on what should be improved in relation to the demands of the world of work, as well as on what they could do to more easily enter or re-enter the job market through greater responsibility with respect to their future work, and thus catch a glimpse of the brand new, the not-given, the not-yet, resources and personal riches that have remained unknown, unexplored, and unused. f within specific binding places, can reflect on what they are able to do, even in terms of applicability in the world of work, on what should be improved in relation to the demands of the world of work, as well as on what they could do to more easily enter or re-enter the job market through greater responsibility with respect to their future work, and thus catch a glimpse of the brand new, the not-given, the not-yet, resources and personal riches that have remained unknown, unexplored, and unused. f At stake is the aid offered for the reconstruction and analysis of person- al, educational, and vocational history, of recognition, of the ownership and integration of the abilities and skills acquired, identifying strengths and areas of increase in personal skills, evaluation of the applicability and transferability of the skills possessed to other working environments, other geographical locations, and professional roles. In this sense, it may be considered formatively significant to accompany subjects in gaining the ability to update, enhance, and modify their skills, boosting basic ones as well as expressing their critical capacity to operatively translate them, both in specific learning environments and in an instrumental way. 3. Skills assessment, guidance technology In this regard, we should learn to appreciate skills assessment, think- ing especially of its contribution not so much in identifying the degree of mastery of one’s skills based on abstract taxonomies, nor so much in terms of roles, but as recognition of real work and therefore of located knowledge, knowledge in action, local and contingent knowhow, con- crete action abilities, personal capacity expressed and acted out ‘locally’, in contexts of organized action, of the possibility of responding success- fully to a typical question, therefore far from idealized and generic mod- els of theory and practice in the workplace. y p p The application of skills assessment seems particularly proper and ef- fective in those professional places characterized by dynamic variables and subject to multiple and continuous changes that determine a de- mand for new goals and new skills, different tasks and roles, different professionalism and responsibilities, of intuitive interiorized and refined capacities in virtue of which to deal creatively and effectively with prob- lematic situations. f Through recourse to skills assessment, at stake is the aid offered to organizational subjects to become (re)builders of their own employment history (choice, initial work, training, vocational development) and in- terpreters of a professional assessment, and therefore to examine and valorize their potential and personal psychosocial resources (representa- tions, aptitudes, values, interests and motivations related to work) and professional ones; to recognize and verify the knowledge and knowhow acquired, their experiences and unspoken aspirations, the intelligence and skills built inside the workplace (in different working situations), the learning-by-doing gained, and to facilitate the identification of the most suitable training for personal needs, to draw up a project for professional development and manage a repositioning as regards work, employment transition, mobility and re-insertion into the job market, along with any ongoing adjustment and occupational redesign. SELF-TELLING TO ORIENTATE ONESELF 99 3. Skills assessment, guidance technology pi g y Favouring the methodological path of self-exploration and self-assess- ment rather than that of diagnosis and self-assessment, skills assessment is not to be seen solely as a repertoire of technical skills acquired by sub- jects during their educational and professional life, but also as an instru- ment to allow the investigation, destructuring and rebuilding of some of the components of their subjective identity, together with the recon- struction of that set of skills and resources of different kinds which en- able them to manage their own tasks at a given moment of professional development and organizational life. Far from identifying it as a diagnostic or evaluative instrument, or as a mode for selecting personnel, we can look at skills assessment as a for- mative experience that allows people to redefine their own patterns of thought and action, and therefore as an experience that can bring substan- tial lifeblood to the planning self, to facilitate a disposition to reflective planning, and therefore to contribute to the maturation of self-reliance and an ability to take charge of their own future by measuring themselves against the world of work and educational opportunities. 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Antonia Cunti (University of Naples Parthenope)* Antonia Cunti (University of Naples Parthenope)* Abstract: The purpose of this paper is to discuss and reflect on the Neapolitan section of the PRIN project from a critical perspective. The research underlines the relevance of combining the dimensions of meaning, continuity, and change, giving individuals the chance to try out promising educational itineraries where guidance is understood as a pedagogical tool of higher education contexts. Keywords: higher education, personal and professional identity, self-realization, guidance, counseling. 1. Introduction Guidance is undoubtedly a complex theme, primarily because it represents the development of educational processes. Indeed, it is from its close relationship with education that the necessity arises to link it to, amongst others, the processes to develop the Self, the various forms of conditioning and educational care, and the quality of inter- personal and social relationships. It represents a critical perspective coherent with the fields explored by the PRIN project, also consid- ering the trajectory it followed before becoming a part of education- al research. Traditionally, guidance has been a field of psychological and sociological research: the former, throughout its complex evolu- tion, focusing predominantly on the possibility of harmonizing stu- dents’ motivations (not only cognitive, of course) with those of the workplace. These studies, while at various times giving precedence to one or the other, have aimed at producing a match that would satisfy both, and therefore benefit people’s wellbeing as well as the system development. The latter adopts a mainly analytical point of view of the processes of change within social systems in respect to individual working existence. Both approaches describe and link objects and/or phenomena, however, such a perspective falls short when the chal- lenge facing guidance becomes that of creating, both educationally and structurally, contexts, relationships, and organizations capable of stimulating and growing needs, desires and strategies, as well as 104 ANTONIA CUNTI guidance skills, to evolve developmental paths of thinking, feeling and acting; in other words, to develop the Self. GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH Young adulthood today is a particularly problematic moment of anyone’s lifespan, investigated in depth by many research and disciplin- ary perspectives. The transition to a new condition can be identified when, at the end of high school, young adults are not only asked to simply wonder about their formal education and professional future, and ideas of the latter begin to steady in their minds and within what we might call a ‘speaking community’, as more than just a concept that will soon concern them; a multitude of opportunities, points of view, information, and individual and social criticisms, but, above all, the necessity to make choices that will directly impact their fu- ture, decisions for which they are responsible. One of the educational roles that universities perform is student job placement, not only in terms of supporting the learning of knowledge/ competencies coherent with specific cultural and professional profiles, but also of contributing to the development of one’s personal, educa- tional, and professional Self. The learning process is focused on a ‘learn- ing-as-adults’, i.e., their own knowledge and knowhow are prioritized to allow them to structure their identity, while the major difference be- tween university and school puts young adults in an adult relationship with knowledge, their education, the sense of this for their lives, for the realization of their Self as individuals, men/women, and citizens. The educational quality of these systems must therefore be di- rected primarily at education itself, teaching students to take care of their own education, to create and sustain an aptitude to learn dur- ing their lifetime (Alberici 2002), and to identify, define and enhance their own individual, dynamic and emancipatory desires. This form of support must therefore regard methodological and innate guidance (Cunti, Priore 2014). Supporting such educational processes requires the dismissal of a concept of learning that corresponds to something well defined, therefore with the ability to adapt to those dimensions recognized as a personal and social success (OECD 2017). Desired learning, rather than being intended as what someone wants to become, consistent with own experiences and the ability to elaborate these in the pursuit of transient, emancipatory, and trans- formational goals, is often flattened by being already decided, where formal education urges individuals to do everything on their own. 2. Guidance as an educational emergency in Higher Education It is increasingly widely acknowledged that guidance has become not only that of creating upon what we have, or of developing our potential, but also of making sense out of all of this by means of re- flection on individuals’ development within their own contexts. If this is to be one of the main pedagogical goals in guidance process- es, the educational task in this area – and therefore also the social and political tasks, can be none other than to design, verify, and en- hance theoretical and practical structures to define, detail and clarify the role of pedagogy. It was from this perspective that the Neapoli- tan research unit worked, focusing on guidance as a fundamental educational tool, and at the same time a complex subject bordering many others, and requiring both interdisciplinary and transdisci- plinary approaches. p y pp Although guidance is a lifelong issue, particularly due to the weak- ening of social and community support in transitional life-events, and because of the precarity of self-determination during adulthood, it is especially important for young adults, and therefore also the main educational systems that take care of them: school and university. Postmodern characteristics (Bauman 2005, 2000; Lasch 1984) create a widespread crisis which is felt profoundly by young adults in higher education, since society requires them to overcome several developmental tasks crucial to their social recognition as adults. If the choices have multiplied vertiginously, the youth crisis is nevertheless connected as much to the unavoidability of making choices regard- ing their own future, as to the unpredictability of the consequences that each choice might lead to (Cunti 2008; Cunti and Priore 2014; Cunti, Priore, Bellantonio 2015). Postmodernist society, deeply marked by risk (Beck 1986), uncer- tainty, and precarity (Bauman 2005), inspires a view of the future as a threat (Benasayag, Schmit 2004) rather than an opportunity or perhaps a promise (Cunti et al. 2015). This inability to trust in the future prompts many young adults to remain firmly in the present, so that a consequence of disenchantment (Cambi 2006) is the very underwhelming realization of an imagined future that should allow young adults to think of themselves in a guise different from the pres- ent, nurturing desires and expectations for the future. All the above calls for an educational undertaking to deal with the unexpected (Morin 2001: 14). 105 GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH The almost excluded value of learning is inherent to the ‘freedom- from’, at best anticipating often systematic and improvised attempts to pursue the ‘freedom-to’ (Berlin 1969), in this case welcomed as an educational task to remove obstacles that might surpass the conquest of the identified goals; accordingly, schools and universities cannot avoid educating to desire, to realize oneself as a person and a worker. In light of the above, guidance opens up to multi-faceted inter- ventions primarily regarding the institutional processes of designing, 106 ANTONIA CUNTI structuring and putting into practice systems whose heart is the de- velopment of students’ competencies, and the identification and re- alization of their desire and/or need for educational emancipation. In this sense, this part of the research, through contact with both uni- versity representatives and students to collect real and perceived data, encouraged reflections on the theme and offered an important study from which arise criticisms, and potential and significant areas for improvement which universities can work on in synergy. structuring and putting into practice systems whose heart is the de- velopment of students’ competencies, and the identification and re- alization of their desire and/or need for educational emancipation. In this sense, this part of the research, through contact with both uni- versity representatives and students to collect real and perceived data, encouraged reflections on the theme and offered an important study from which arise criticisms, and potential and significant areas for improvement which universities can work on in synergy. p y gy It is well known that educational systems, universities included, are attributing an increasingly key role to guidance; in recent decades, ever since it became a strategic theme for the organizational and ed- ucational development of these institutions, the need for research to improve the productivity of systems, as well as students’ emancipative wellbeing, has become more important. Consequently, pedagogical research on guidance is called to offer its contribution, to contrib- ute and enhance knowledge, interventions, and improvements; from this point of view, the ability of institutions to make decisions based on the need for evolution represents the most important indicator of quality in guidance. GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH 107 incorporate this relationship. The challenge is to connect them cre- atively, in order to explore living and working environments, iden- tifying elements corresponding to the characteristics of individuals, and basing these processes on self-reflection with a view to discov- ering one’s authentic desires. From this comes a need to bring in- dividuals to the forefront, giving them the choices of responsibility and honour for systems and their own personality, given that educa- tion today cannot be separated from the concept of each individual as an integral part of their living and learning environments. Hence, the issue of an ‘ecological individual’ (Bateson 1972) represents both a priority and a gamble for guidance. We might ask ourselves, then, considering the principle aspects covered by the PRIN research, how the aforementioned aspects were explored while adopting the key to reading educational guidance. One key dimension regards the relationship between general and local, which gives rise to a question of order and structure that is in- herent to the research. As we have already underlined, guidance rep- resents a priority for educational systems, the aim being to advance and streamline social and economic systems, both for the satisfaction of individuals and the development of communities. From such a per- spective, the correspondence between European guidelines (ELGPN 2017) and their implementation in each country is irrefutably criti- cal. At a structural level, the particular conditions to be created are not envisaged, thus there is always the risk of stopping at the stage of good intentions, without eliciting processes based on best practices capable of creating, testing, and implementing the identified options. The PRIN project shows that guidance is often an add-on, rather than an integral part of education and other systems, where its inte- gration would require changing the organizational culture, especially regarding the promotion and optimization of generative and leading systematic interactions. With regard to educational research, which is essential to improving practices, it seems that the key to developing rigorous methods is transferability rather than generalization; using the former we can appreciate the diversity of contexts and document research processes and results in order to increase our knowledge and our ability to develop guidance methods. GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH The possibility of contributing to the growth of the system by one of its most important components, i.e., students, is not discounted, but must be accompanied by educational processes that press students to reflect on their own living environments and personal evolution; the PRIN research in question has assumed this fundamental value through the action of focus groups to learn from oneself in comparison with others, intercepting views and multiple experiences, as well as differentiated growth prospects, fundamental to activating closed forms of mind without which it is much more difficult to imagine, and imagine oneself, in unknown ways. fi g , g , y Referring to the perspective adopted by this research, and its meth- odological and procedural choices, there are two things to consider: firstly, the fact that guidance has somehow induced educational re- search to embrace and share with psychological and sociological dis- ciplines, rigorous methodologies based on a methodological pluralism with both quantitative and qualitative aspects. It has also pushed such research to adopt an empirical base and confer a transformative di- mension on the scientific field, with the intention of creating a use- ful, collaborative strategy for the development of society. Secondly, the challenge of maintaining the link between individuals and their contexts, without which guidance cannot be properly examined and transformed; this complexity calls for targeted actions that consis- tently refer to the web of elements which in turn depend on interdis- ciplinary and transdisciplinary aspects. Nowadays, the link between individual and environment arguably necessitates the educational construction and implementation of new adaptation processes which GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH With reference to university guidance in particular, the circularity between knowledge and the practices of various contributors could include students as consultants in the process of action-research and its implementation, in which a qualitative and quantitative exploratory dimension is combined with an aspect of planning to improve institutional guidance. The inten- tion is not to enforce a rigid barrier between the people benefitting from a system or action programme and those dedicated to obtaining satisfactory results, but instead to allow everyone involved to become 108 ANTONIA CUNTI a researcher in their own right, and to act for both their own good and that of the system they are participating in. Indeed, rather than changing behaviour according to external advice, it is more produc- tive to focus on self-reflection, both individually and in groups within institutions, to explore methods of triggering a virtuous circularity involving all relevant parties and the quality of their interactions. a researcher in their own right, and to act for both their own good and that of the system they are participating in. Indeed, rather than changing behaviour according to external advice, it is more produc- tive to focus on self-reflection, both individually and in groups within institutions, to explore methods of triggering a virtuous circularity involving all relevant parties and the quality of their interactions. A further aspect is the transverse nature of the methods in an at- tempt to cope with the complexity of guidance itself, as well as that of university systems. In this regard, one of the most important aspects of guidance relates to didactics, given that the subject of university guidance is so tightly interwoven with that of didactic processes (Fab- bri, Melacarne 2015; Fedeli, Grion, Frison 2016). The realization of a varied and interconnected approach to guidance necessitates using didactics as a guidance oriented to one’s whole education. Knowl- edge processes must be supported and enhanced since the ways in which they are determined can lead to the building of a subjectiv- ity capable of knowing and improving for the rest of people’s lives; this requires the examination, primarily, of the methodological pro- cesses necessary for the construction of knowledge (Domenici 2009). Transversality also refers to the necessity for a kind of education that keeps levels of thinking, feeling, and acting together. GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH In this sense, best guidance practices regard the implementation of a laboratorial methodological instrument to guide students through self-reflection on their own guidance as individuals and workers, a process that can be implemented, enacted, and improved. GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH 109 become aware of their own capabilities and the way in which they can emphasize and grow them, as well as using them in conjunction with other resources, settings and people, and seeing change as evo- lution: this is decidedly one of the greatest and most important edu- cational challenges. The need highlighted is that of accompanying students’ educa- tional courses, which does not mean simply transmitting contents, but rather, concentrating on how these contents aid students in de- veloping their own individual identity. University contexts should, therefore, provide systematic accom- paniment and support for students during their career, not only in cases requiring specific and specialized intervention. A second aspect is to enact a guidance-based didactic method, since what is being im- parted is not merely the quality of the content but the way in which it becomes learning. The ultimate challenge for university contexts is to spread the culture of the ‘helping requirement’ in tricky situa- tions, meaning that the possibility of counseling should be considered by students as an opportunity to be accompanied throughout their career in order to cope with critical situations. Entering, albeit in a synthetic way, the merits of the divisions identified, they should ac- company and support a full expression of the abilities of choice, for an empowerment aimed at the enhancement of personal and envi- ronmental resources. The accompaniment of knowledge processes is geared to a guidance that enables individuals to build an ‘Educational Ego’ (Cunti 2008: 23), in other words a Self-building process as an individual capable of learning and enhancing for a lifetime. This first subdivision opens up to education and didactics, to the guidance aspects of knowledge, provided that the methodological level in the process of accompany- ing knowledge-building is emphasized, also in the sense of acting in relationships in all those contexts where education is carried out in collective environments, in contrast to transmissive models. The intrinsic guidance value of formal-learning knowledge re- quires an appropriate methodological itinerary so that it can express itself. In other words, scientific disciplines have a guidance value in themselves, recognizing this presupposes certain educational and di- dactic qualities. Learning and teaching processes are primarily pro- cesses of guidance to knowledge and culture; in this sense, guidance can be act as a strategy for educating thinking also to build oneself and the future. 3. Guidance aspects in Higher Education As mentioned previously, educational settings are called to the fore of the struggle against this general sense of crisis, in particular, higher education contexts. For this reason, guidance has become a central theme in educational policies both in Italy and internation- ally (ISTAT 2016). The crisis revolves around the vision of future, in other words, the ability to imagine ourselves in the prefigurative dimension created by human beings, or to project ourselves into a guise different from the present. The difficulty in visualizing a future in which we could realize our desires prompts us to amplify and em- phasize the present, a phenomenon which, alongside the consequen- tial procrastination rather than action, particularly characterizes the young world (Benasayag, Schmit 2004). Hence it would appear to be an educational role to promote the development of capabilities (Sen 1999), i.e., the collection of resources available to a person and their ability to enact most of them. Only then can they concretely act and GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH Thus, learning to be part of the process of creating knowledge can make one become an artisan of one’s own; knowing how to grasp the dynamics at the basis of the construction of scien- tific knowledge cannot mean being vigilant about those that stim- ulate one’s cultural education; knowing how to draw from time to 110 ANTONIA CUNTI time on the knowledge that is useful may mean learning not to de- pend on what someone knows, wanting to express oneself as an in- dividual even in the presence of culture and towards education. In general, difficulties in educational contexts are challenging to inter- pose between teaching and guidance, as if didactic activity did not incorporate an orientative prerogative and the didactic experience was anything but neutral in the individual guidance process, regard- less of whether this happens intentionally.i pp y As far as the definition and relationship between work and educa- tion is concerned with individual existences, this second subdivision refers to quite specific approaches, to a kind of informative, predic- tive, and diagnostic work/education guidance (see skills assessment) or verging more on the promotion of resources. The innovative per- spective could be of Self-realization, where the work dimension has a very important definition value, oriented to enhance the theme of desire and what is important to oneself. Moreover, recent interna- tional documents have shown that often what is good for the individ- ual is already defined and corresponds to the quality of competencies and achievements by which an individual can best be sustained. In this sense, also some of our research data (Cunti, Priore, Bellantonio 2015) underline a perspective of adaptation to the environment by coinciding with what can be done, and this also means that flexibil- ity is understood as adaptation to the environment. Priority should, however, consist of giving the floor to the individual, in a historical spirit in which the fragmentation of the working context is not indif- ferent to the fragmentation of the Self − even Richard Sennett with The Flexible Man (1999) warned that flexibility could lead to corro- sion of the Ego. GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH 111 ficulties are found, ways of seeing inadequate ones that constrain the achievement of objectives, and, at the same time, a ‘clinical’ recov- ery of one’s own history, experiences and past, and then of research and the accomplishment of desires. In the foreground, from a meth- odological point of view, the narrative approach and reflexivity are considered exquisitely pedagogical dimensions. Guidance is under- stood here as educational counseling, i.e., as one form of counsel- ing (Simeone 2002) and methodological accompaniment that creates the conditions for activation and a full expression of the abilities of choice. Also to the fore are the ways in which individuals live their relational universes to allow for an exploration of what, for example, characterizes or hinders the realization of personal expressive and ex- istential incentives, and the descent into the realm of desire, anchor- ing them to their own story, to their own identity manifestations. This perspective of counseling represents a significant ramification of the guidance theme in the international literature (Savickas 2005, 2002; Guichard 2010, 2005), and is a useful key to methodological and educational perspectives on guidance. p p g In the international context, the dimensions of caring and be- ing cared for have been designed specifically with reference to work choices, with a view to making them synchronic to individual pro- pensities and transformations of those individuals’ existential paths; in general, it is no longer the environment that guides occupational and professional choices favouring the expression and realization of individual specificities, but individuals who are helped to become aware of themselves and their desires (Savickas 2011, 2001). The ac- companiment of people to reflect on their subjectivity which mani- fests in the different aspects of existence, fully reflects the work of the Self according to evolutionary circularity and self-emancipation; life-designing counseling therefore looks at individuals in all their complexity and tension to impress coherence and design vitality on the various roles and functions, supporting them above all in the most critical moments of transformation (Guichard, Di Fabio 2010, Savickas et al. 2009). GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH g If the work dimension has a very significant definition of Self and the educational task can be considered above all to teach individuals how to cultivate their own desires, it is important to note not only the presence of so many professional biographies characterized by dis- continuities in choices, providing that the responsibility for these is almost entirely on the individual; therefore, retrieving the pedagogi- cal dimension of guidance can mean educationally working on those that are two key concepts of navigation in the society of complexity, as well as two pedagogical words: narratives and project. The last dimension, but not for its importance, is that of coun- seling, which is a methodological and educational interpretation of guidance that contributes to changing visions, re-activating resources, and imagining new paths. Counseling is understood here as a meth- odological agent for the re-definition of design in demanding situa- tions. Working with others can be seen as a critical approach in the sense of an obstacle analysis, an examination of reality in which dif- GUIDANCE SPECIFICITY IN EDUCATIONAL RESEARCH 4. Conclusion The Neapolitan section of the PRIN research looked at the role of guidance processes in creating the education of students in higher education, highlighting, specifically, the importance of combining the dimensions of meaning, continuity, and change, in other words, the development, structuring and permanence of the Self and, above all, experimenting with possible educational itineraries. If it is the 112 ANTONIA CUNTI quality of educational experiences that are promoted to make a strong impact on people’s guidance processes, it is essential to accompany educational processes in all the contexts where they occur, also by identifying emerging pedagogical questions on guidance that can also become research questions beyond education, to then try to create and test possible pedagogical and guidance responses. References References Alberici A. 2002, Imparare sempre nella società della conoscenza, Bruno Mondadori, Milano. Bateson G. 1976, Verso un’ecologia della mente, Adelphi, Milano (orig. ed. 1972). Bauman Z. 2002, Modernità liquida, Laterza, Roma-Bari (orig. ed. 2000). Bauman Z. 2006, Vita Liquida, Laterza, Roma-Bari (orig. ed. 2005). Beck U. 2000, La società del rischio. Verso una seconda modernità, Carocci, Roma (orig. ed. 1986). 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La mentalità della sopravvivenza in un’epoca di turbamenti, Feltrinelli, Milano (orig. ed. 1984).f Morin E. 2001, I sette saperi necessari all’educazione del futuro, Raffaello Cortina, Milano (orig. ed. 2000).i OCSE/OECD 2017, Definition and Selection of Key Competencies Executive Summary, <http//www.oecd.org/dataoecd/47/61/35070367.pdf> (01/18). Savickas M.L. 2001, Toward a Comprehensive Theory of Career Development: Disposition, Concerns and Narratives, in Leong F.T.L. and Barak A. (eds.), Contemporary Models in Vocational Psychology: A Volume in Honor of Samuel H. Osipow, Lawrence Erlbaum, Mahwah, 295-320. Savickas M.L. 2002, Career Construction. A Developmental Theory of Vocational Behavior, in Brown D. and associated (ed.), Career Choice and Development, Jossey-Bass, San Francisco, 149-205. Savickas M.L. 2005, The Theory and Practice of Career Construction, in Brown S.D. and Lent R.W. (eds.), Career Development and Counseling: Putting Theory and Research to Work, John Wiley & Sons, Hoboken, 42-70. Savickas M.L. 2011, Career Counseling, American Psychological Association, Washington. Savickas M.L. 2015, Designing Projects for Career Construction, in Young R.A., Domene J. and Valach L. (eds.), Counseling and Action, Springer, New York, 16-17. Savickas M.L. et al. 2009, Life Designing: A Paradigm for Career Construction in the 21st Century, «Journal of Vocational Behavior», 75, 239-250. J Sen A.K. 2000, Lo sviluppo è libertà, Mondadori, Milano (orig. ed. 1999).l Sennet R. 2010, L’uomo flessibile. Le conseguenze del nuovo capitalismo sulla vita delle persone, Feltrinelli, Milano (orig. ed. 1999). Simeone D. 2002, La consulenza educativa. Dimensione pedagogica della relazione d’aiuto, Vita e Pensiero, Milano. * Vanna Boffo, PhD, Associate Professor of General Pedagogy, Department of Education and Psychology, University of Florence, Italy. Email: vanna.boffo@unifi.it. Vanna Boffo (University of Florence)* Abstract: Starting from questions inspired by the tremendous changes taking place in the worlds of education and employment, this research focuses on the role of employability in the building of new skills in higher education contexts. This longitudinal survey conducted on a sample of 52 graduates from the Educational Science sector found certain key characteristics in the transitions to the world of work, allowing reflection on those educational activities whose impact is more favourable to develop job placement. Keywords: employability, adult education, higher education, competences, work pedagogy. EMPLOYABILITY AND TRANSITIONS: FOSTERING THE FUTURE OF YOUNG ADULT GRADUATES Vanna Boffo (University of Florence)* 1. Introduction: from research to the existing state of affairs In a context of constant and rapid changes worldwide, the quest for meaning that involves universities, and especially Italian universities, puzzles teachers, researchers, and citizens alike. And this quest has be- come transformed, for the most part, into a question of what knowledge, skills and capabilities will be needed to enable all the students who reach the final levels of higher education to enter the world of work fully in- formed and accountable for their own future. Some medium- and long-term drivers are shaping a world that is as much in flux as it is different from the past; suffice to think of demo- graphic changes such as the ageing of the population, but also of glo- balization or the digitization expressed by the advancement of robotics, Big Data, the Internet of Things, Artificial Intelligence, and the digital platforms that interconnect new forms of cohabitation within the «Smart City» (Brynjolfsson, McAfee 2014; OECD 2017). In the face of these ground-breaking changes that are not so easy to understand, higher ed- ucation must reflect on its ability to achieve satisfactory educational ob- jectives that will allow its graduates to keep pace. This is especially true in Italy where a growth in access to third-level education, the university, together with an improvement in the quality and relevance of graduates’ skills is considered one of the most vital challenges by the OECD, along with the MIUR and ANVUR (OECD Skills Strategy: Italy 2017: 11). In fact, what we know is that, in addition to having a small number of graduates and graduate workers compared to the average in European 118 VANNA BOFFO countries, Italy also has a limited flow of graduates arriving in the world of work. Only 20% of Italians graduate between 25 and 34 years of age, compared to 30% of the OECD average (Skills Strategy: Italy 2017: 11). Italian graduates have a lower level of skills (26th place out of 29), in their levels of both literacy and numeracy, i.e. our graduates are at the same level as graduates in countries such as Sweden, Germany, and Finland. It is also necessary to improve the relevance of university studies with respect to the skills required by the job market, the skills gap being par- ticularly pronounced where several companies are struggling to find the required profiles for their business development. EMPLOYABILITY AND TRANSITIONS process. The principal studies on employability were conducted between the mid-1990s and the end of the first decade of the 2000s (Harvey 2000, 2001, 2003; Knight, Yorke 2002, 2003; Yorke, Knight 2006) and attest the commitment that higher education should take to train/educate for work, for a profession, but above all to build the capabilities to remain/ persevere in view of the transitions of a world in constant evolution and change (Morin 1999, 2001, 2015; Nussbaum 2010, 2011). 1. Introduction: from research to the existing state of affairs il q pi p As the OECD report indicates, this urges significant reflection on what are the links between university courses, outgoing competencies, transi- tion abilities, and the capacity to enter the world of work. Too often, the outgoing qualification does not reflect the skills learned and possessed, creating doubts over the possibility of proper alignment and especially the chance of befittingly meeting the supply and demand requisites. On top of this, often the employers themselves have long since left universi- ty, if they ever attended it, or are not adequately familiar with the trans- formation in higher education courses. There is reassuring data coming in when graduates have learned high-level technical, professional, and cultural skills, when their profile is clear, when their preparation is im- mediately understandable; in these cases, the graduates’ applicability and employability become motivating factors, effectively supporting a safe and certain transition (OECD 2017: 12). The theme of work placement for graduates is consistently linked with that of Italian University courses in the 2000s, alongside the problems of employment for young adults with higher education qualifications, and an Italian job market marred by structural contradictions. If on the one hand, it seems that there is a lack of work, on the other, there are vacan- cies and sectors struggling to find the best candidates. gg gi As part of the PRIN project on Employability and Competences, the University of Florence research unit took its cue from an analysis of the transition of graduates towards their first work placement (Ecclestone 2007; Furlong, Cartmel 2007; Teichler 2007, 2013; Eight, Atzmüller, Berthet, Bifulco, Bonvi et al. 2015) with the aim of studying the access methods, the desire for professional advancement, the awareness of the skills acquired and needed for work and daily life, and the tools/tech- niques/roads useful in defining the professional goal. qi g p g It is assumed that, subtended to the theme of placement, the dimension of employability developed during their education can be found, being a process category related to the educational path more than a product cat- egory for the job search. The transition theme is explained, interpreted, and reasoned through that of employability. What we might say is that, in higher education, the transition to work is an embedded employability 119 EMPLOYABILITY AND TRANSITIONS 2. From employability to transitions Here, there is a strong link with education of a pedagogical pattern, and with learn- ing processes that are self-directed, organizational, and subject-based. Studies on employability were developed in the Anglo-Saxon area starting from the 1960s, but only at the end of the ‘90s did substantial thinking begin to theorize on the centrality of employability in under- standing the role of educational institutions for the job market: «In essence, the debate is about what employers want and what higher education insti- tutions can do to enhance the employability of students» (Harvey 2003: 3). The question arose from observation of the demands of the world of work that did not find in UK graduates the skills required to advance production. The problem was not the economic growth of a country, but the education of the students. About 15 years ago, at the beginning of the 2000s, Harvey, the then director of the Center for Research and Evaluation at Sheffield Hallam University wrote: There is a growing awareness in the UK of the importance of higher ed- ucation in providing the innovation and creativity for the development of a knowledge-based economy in an increasingly competitive global market. Three major policy initiatives have contributed to this over the last decade: 1. widening participation and improving retention; 2. en- hancing employability. 3. lifelong learning. Both higher education and the graduate labour market are changing rapidly. The student intake is becoming more diverse, in age, background, previous educational ex- perience and interests and ambitions, although the government’s efforts to broaden the social base of the undergraduate population has recently been characterised as a limited success (Harvey 2002: 4). A definite sign that a gap of approximately 20 years separates us from other countries when it comes to reflection on employability. A reflec- tion that has grown in various disciplines from economics, to quality as- surance, sociology, statistics, and education engineering. The result has been a study of different forms and models using both qualitative and quantitative research. In general, even in countries where the debate on the presence, mon- itoring, and evaluation of employability has been more intense, there has been reluctance to include the concept in reflections on the future of universities. To speak of the future is never easy, but even more so in this case. What should the role of the university be in a country? 2. From employability to transitions The main question of the investigation looked at the problem of un- derstanding the process of transition of young graduates from the tak- ing of their Degree, to their search for a job, and their placement. What deep dynamics underlie the initial transitions to the world of work of young adults (22-29 years) leaving higher education? More specifically: how do graduates look for work, how do they prepare for the transition step, and, above all, how do they go about building employability dur- ing their years of university study? g y y y The objectives that emerged from the analysis of the research ques- tion concerned: 1) Understanding the attitudes of the young to work; 2) Interpreting life prospects; 3) Mapping implicit strategies for transi- tions; 4) Observing the range of choices of young people; 5) Acquiring elements to improve university teaching; 6) Looking for the presence of employability in the practices of university curricula. In effect, if the theme of the research work focused on understanding the transition pro- cess, it appears that this process has been understood as an indicator of the building of employability. What we can say is that the main objective was to include a central category for the construction of professionalism. If the PRIN research as a whole looked at higher education, includ- ing calling, orientation, teaching, internships, and transition to work, we could say that the category of employability underlies a process that has been identified to justify the term ‘Higher Education’. This reflection has been maturing in a historical, cultural, social, and political period in an Italy squeezed between the world economic crisis of 2007 and its inability as a country-system to recover the lost pre-crisis positions, emerging from an economic mire that mainly affected the most heavily disadvantaged and among these, undoubtedly, young adults. In this state of affairs, al- though the graduates themselves have reacted (AlmaLaurea 2017), it is the structure of the university institution that has shown the principal shortcomings. At present, ministerial recommendations strongly steer study courses towards constructing professional skills, but without these being backed up by cultural reflection to support the transformations that higher education is being called upon to make by law. Employability is 120 VANNA BOFFO the category that justifies work orientation, conceptualizing the need to acquire skills for lifelong learning, which makes it possible to model traditional teaching on work-related or digital learning. EMPLOYABILITY AND TRANSITIONS 121 the dichotomy «Learning as Education» or «Learning as Training» (Har- vey 2003); speaking of employability means dealing with Learning and Training in the same breath. Not in opposition but in conjunction. The writings of Harvey, Yorke & Knight (2006) confirm this, and empty the field of erstwhile rhetoric. i Therefore, the models that have followed in recent years, for the most part ‒ since the debate is young ‒ can be summarized in three different visions. One is integrated with and broadens the previous one, provid- ing us with an exhaustive picture of the sense and meaning with which we, today, look at the process of employability as being embedded with- in every curriculum of studies that steers the educational path towards a natural development from the place of learning to the world of work. l p p g One interesting reflection on the theme of employability in univer- sity contexts was conducted by Harvey (2002, 2003, 2006), and at the same time by Yorke & Knight (2002, 2004, 2006) in the early 2000s, even if the whole debate had been extensively prepared by reflection on higher education in the English-speaking world over the previous two decades of the 1990s. Both the prospect of Harvey and that of Yorke & Knight link the concept of employability to higher education and raise the problem of the use and presence of an ability ‘to live’, so that fitting- ly solid bridges can be built with the world of work. In a social context that demands more and targeted skills for a greater number of graduates to create better growth for countries with a fairer distribution of eco- nomic and social resources, reflecting deeply on the category of employ- ability becomes key to understanding in which direction efforts need to be made to improve university teaching, create work placements and internships, and construct specific links with businesses, associations, and the public and private production sectors. Harvey’s definitions, on the one hand, and Yorke & Knight’s on the other, introduced didactic- pedagogical elements and broadened the concept until it founded an in- novative way of considering higher education. In 1999, Harvey offered this definition: «The employability of a graduate is the propensity of the graduate to exhibit attributes that employers anticipate will be necessary for the future effective functioning of their organization» (Harvey 1999: 4). 2. From employability to transitions High- er education should, indeed, question the meaning of the transforma- tions it needs to make in a world that is changing, is being transformed, shifting direction. The educational factor is always present even if we are talking about learning. Thus, we must not be afraid to veer towards EMPLOYABILITY AND TRANSITIONS EMPLOYABILITY AND TRANSITIONS In fact, the real problem would seem to be the attributes that a grad- uate should possess, which should match those that businesses require. Instead, Harvey stresses that the fundamental problem is the consider- ation that employability is a process and it is this that creates the real prob- lem, in the final analysis: employability raises fundamental questions about the purpose and struc- ture of higher education. Employability is not about training or provid- ing add-on skills to gain employment. On the contrary, employability is about how higher education develops critical, reflective, empowered 122 VANNA BOFFO learners. Despite appearances to the contrary, the real challenge is not how to accommodate employability but how to shift the traditional bal- ance of power from the education provider to those participating in the learning experience (Harvey 1999: 13). learners. Despite appearances to the contrary, the real challenge is not how to accommodate employability but how to shift the traditional bal- ance of power from the education provider to those participating in the learning experience (Harvey 1999: 13). Equally inviting of reflection is Yorke and Knight’s definition, which is by far the most sweeping and decisive: «a set of achievements – skills, understandings, and personal attributes – that make graduates more likely to gain employment and be successful in their chosen oc- cupations, which benefits themselves, the workforce, the community, and the economy» (Yorke, Knight 2005: 3). The work of the Learning and Teaching Support Network (LTSN) and the Enhancing Student Employability Co-ordination Team (ESECT) coordinated by Mantz Yorke from the early 2000s, has been a point of reference, unique of its kind, to understand the passage from considering employability as a concept of a hazily economic nature, to a category with a clear com- mitment to learning, i.e. to the pedagogic-didactic spheres of higher education. Starting from a series of texts, research, and published vol- umes, processed using evidence-based research methods, the opportu- nity arises to reflect on the educational paths, teaching practices, and the pedagogical content of the category. EMPLOYABILITY AND TRANSITIONS 123 taken in a single solution, that of the Focus Group for prior knowledge, and in-depth interview to get to know the individuals. The Focus Group form provided some fixed fields for a mutual understanding of the re- search subjects. The semi-structured interview was then conducted us- ing a second form. The protocol was rigorously developed at each step, in the sense that the contacts, the Focus Group, the period between the first meeting and the subsequent meetings, were steps scheduled tem- porally and materially in an identical manner over the years of research. p y y y The expected results focused on four fields of reflection: 1) Volition; 2) Skills; 3) Channels; 4) Expectations. The subjects of the investigation included 52 graduates (39 from the University of Florence, 2 from the University of Padua, and 9 from the University of Wuerzburg in Ger- many). The data collected from the 10 samples of graduates who took part, from June 2014 to July 2016, covered 110 in-depth interviews. The samples were selected from the LM57&85 MA course in Adult Educa- tional Sciences, Continuing Education, and Pedagogical Sciences at the University of Florence; the LM50&57 course in Educational Services Management and Continuing Education at the University of Padua; the MA in Educational Sciences at the University of Wuerzburg. A com- parative interpretation of the interviews was carried out jointly by the group of researchers. Some series of metadata or indicators were defined after comparison of the graduates’ studies. p g Particular care was taken over the role of the interviewer/researcher. Three researchers covered this function flanked by three junior research- ers who recorded and photographed the successive phases of the group meetings and interviews. The aim was to obtain constant feedback on the attitude of the researcher when listening, plus willingness to change practices or behaviour where they appeared inappropriate to the research work. Researchers also assumed the role of careful facilitators of the path of ‘self-care’; the interview became a means for the graduates to reflect on their past learning and studies, while, in some cases, a series of two or three interviews carried out at a distance of six months acted as ap- pointments for job orientation and the improvement of transversal com- petencies of the personal kind. 3. The survey method for reflection on transition pathways The research conducted at the University of Florence followed a quali- tative survey method, and used a comparison with two samples of gradu- ates, one interviewed at the University of Padua, during the LM50&57 study course, the other at the University of Würzburg, during the MA course in Educational Sciences. The survey method allowed us to grasp the phenomena to be studied according to a map that was not predeter- mined, but constantly being modified, as happens when the prospect of work fits a context of ecological research of a pedagogical-educational type, with socio-anthropological characteristics (Glaser, Strauss 1967; Gla- ser 1992; Guba 1990). Consequently, application of the Grounded Theory approach proved to be a research practice in the field that allowed us to first draw out the analysis categories and then the interpretation, starting from the subjects, or better, starting from the interviewees’ experiences. One important trait of the method was the longitudinal data collection. The research strategy was based on Case Studies, which provided pre- cise indications on the procedures to be followed to conduct the survey itself (Mortari 2007). The Case Study proved useful in a line of inquiry that thoroughly analysed the sample of young adults, all MAs. The sur- vey technique, i.e. the data collection mechanism, was twofold, even if EMPLOYABILITY AND TRANSITIONS 4. The results of the longitudinal survey The analysis of the data from the Focus Group constituted the refer- ence background from which the working group began to highlight the collocation of each graduate. The Focus Group constituted the first level of knowledge on the theme of the transition to work, and was used as a moment for mutual reflection in constructing the relationship between the researchers and those investigated. 124 VANNA BOFFO The interviews were transcribed in full and constitute the main corpus of the research data. They were analysed using a metadata template relat- ing to educational characteristics of the subject, the educational actions lived/undertaken, the type of employment taken pre/post-graduation (Training, Apprenticeships, Mobility, Theses, Laboratories, Employment Before Graduating, Coherent/Non-Coherent Employment, Adverse/ Supportive/Indifferent Educational Actions, Work-Place Learning Po- tential – WPLP, Employee Value Proposition – EVP). , p y p ) The data analysed from the interviews showed that: 1) the transition from university to the world of work involves the way of being young adults in higher education, while the transition itself is a way to call the transformation a modification of the sense of one’s own self, one’s own interiority, one’s vision of the world; 2) the transition does not begin with the degree, but is prepared by previous studies, both in terms of hetero- managed and self-managed educational terms while, within university and secondary school curricula, it has a determinant family matrix; 3) the skills to operate in professions are the result of expertly constructed educational pathways, even if sometimes not fully consciously, devel- oped through specific courses that allow the gaining of experience and make the knowledge already learned applicable; 4) work placements are a constant (jobs, coherent casual jobs, return to education); 5) the exis- tence of a post-graduate degree period in which the young adult gradu- ally becomes aware of their new condition, a path that sometimes lasts 10 months and more; 6) the transition period is lived with the determi- nation to reach a goal coherent with the studies; 7) the gap between the pre-graduation and post-graduation volition is manifest; 8) awareness of the difference between the skills learned and those required by the job market is detectable; 9) informal networks for research work rather than the use of social or formal networks are identifiable; 10) workplaces are considered venues with a high potential for training and orientation. EMPLOYABILITY AND TRANSITIONS and development of skills (reports, communication, problem-solving, context analysis, data usage, self-efficacy) can be defined in higher edu- cation pathways. 4. The results of the longitudinal survey g p g Research results to be considered: 1) employability is a summary cate- gory that holds the highest rate of pedagogical knowledge on educational action to be undertaken in the pre-degree and educational process with- in university curricula, translating a process of change that may occur before graduating, and potentially supporting the process of transition, preparation and delivery; 2) placement is the end result of an education- al process, we need not worry about how much nor how our students have learned, we need to worry about where they will apply what they have learned; 3) the relationship between education during study cours- es, university education (mobility, traineeships, apprenticeships, work- related and work-based learning, E-learning and M-learning), and the world of work, needs to be strengthened and developed synergistically; 4) business culture, the culture of professions and knowledge of the job market need to be implemented in university curricula; 5) the location 125 EMPLOYABILITY AND TRANSITIONS 4. Final remarks: interpreting the data At the end of the research, we might return to reflect on the initial request and be aware that it could be divided into three parts depend- ing on whether the data analysis is going in a micro, meso, or macro direc- tion. At the micro level, the question could be reformulated thus: how do young adults face the transition to work? At the meso level, we could ask the question: how does the curriculum of the university’s educational offering support the transition to work, in terms of skills? At the mac- ro level, we could ask the research what the role of the university in the development of employability is. The answers to these questions can be considered in these terms: 1) Employability represents a process of change which prepares and ac- companies the transition process. It represents a category umbrella, retaining pedagogical awareness of educational actions that need to be developed to obtain a Degree; 2) Placement is an educational process that starts far back, it is not nour- ished by knowledge, but by skills of a technical and transversal na- ture, acquired in institutional and formal situations, but also in those of non-formal and informal learning; 3) The relationships between the study course, university education and the world of work are decisive for the prospects of transitions oriented to faster placements; 4) Work culture understood as the preparation for a profession through the acquisition of transversal skills is a discriminating factor in the tran- sition after graduation. 5) Drawing up an inventory of the skills required by the job market for Professional Profiles in the Educational Sector and the Social Econ- omy: new professionals/entrepreneurs/experts.i 6) The transition to the first job takes approximately 6-10 months after graduation, and represents only the first of several successive steps. In no case had stable jobs been found, but only fixed-term contracts in the best of cases. 7) A close synergy with the world of work is necessary for the creation of competence. The most obvious problem lies precisely in this last point, which we could call the ‘competence supply problem’ or how to train good minds for first-class professionalism. 126 VANNA BOFFO In the past, the issue of competence supply was resolved through the training of good managers. This was matched by business models where busi- ness schools took care of people’s growth. Brynjolfsson E. and McAfee A. 2014, The Second Machine Age. Work, Progress, and Propserity in a Time of Brilliant Technologies. W.W. Norton & Company, New York. Ecclestone K. 2007, Keynote Presentation to Researching Transitions in Lifelong Learning Conference, University of Stirling, 22-24 June 2007, <http://www. leeds.ac.uk/educol/documents/175854.doc> (01/2018). y p y g Glaser B. 1992, Basics of Grounded Theory Analysis, Sociology Press, Mill Valley. Glaser B. and Strauss A. 1967, The Discovery of Grounded Theory. Stategies for Qualitative Research, Aldine, Chicago. ( ) Furlong A. and Cartmel F. 2007, Young People and Social Change: New Perspectives, (fully revised 2nd Edition), Open University Press, Buckingham. 4. Final remarks: interpreting the data Today most companies theo- rize and practise more for lean training models and brain mobility.f It takes staff with seniority – competent professionals – and short- to medium-term projects. This has consequences on the function and the university of mismatch problems faced by graduates who are not first-rate professionals. Rare cases of real unemployment: students are activated. But in all cases, a significant phenomenon which we attribute a symbol- ic value that forces students to extend by one year the period of training for entry into the labour market: the lengthy period for entering the job market. The year after graduation has a crucial function for the encoun- ter with the job market, with ‘coherent’ work and investment in train- ing. This is a year in which young people: re-define their professional identity (false expectations) seek alternatives to training, especially when they have begun to work (knowledge of MDL segments), build and ex- ploit networking, use dynamic learning. In the end, three questions arise from the research in support of grad- uates’ future transitions to working life: 1. Can we bring work culture into the construction of study course curricula (without limiting this to apprenticeships, certain events, or services)? How can we support the construction of a coherent future for professional identity through university teaching? 2. Since a professional future depends less and less on headhunters and more on the social networks (personal, professional, entrepreneurial, institutional) that young people belong to, can the University take this into account? 3. The happiest cases of transition to work found were those in which there was a close relationship between teaching and applied research. How can we encourage this practice in a public university? How can we encourage this practice in a public university? References Brynjolfsson E. and McAfee A. 2014, The Second Machine Age. Work, Progress, and Propserity in a Time of Brilliant Technologies. W.W. Norton & Company, New York. Ecclestone K. 2007, Keynote Presentation to Researching Transitions in Lifelong Learning Conference, University of Stirling, 22-24 June 2007, <http://www. leeds.ac.uk/educol/documents/175854.doc> (01/2018). ( ) Furlong A. and Cartmel F. 2007, Young People and Social Change: New Perspectives, (fully revised 2nd Edition), Open University Press, Buckingham. 127 EMPLOYABILITY AND TRANSITIONS EMPLOYABILITY AND TRANSITIONS Guba E. 1990, The Paradigm Dialog, Sage, Newbury Park. Harvey L. 1999, New Realities: The Relationship Between Higher Education and Employment, Keynote presentation at the European Association of Institutional Research, Lund, Sweden, <https://goo.gl/5JnchK> (01/2018). Harvey L. 2000, New Realities: The Relationship between Higher Education and Employment, «Tertiary Education and Management», VI (1), 3-17. i Harvey L. 2001, Defining and measuring employability, «Quality in Higher Education», VII (2), 97-109.i Harvey L. (ed.) 2003, Transitions from higher education to work, A briefing paper, <http://www.qualityresearchinternational.com/esecttools/esectpubs/ harveytransitions.pdf> (01/2018). Knight P.T. and Yorke M. 2002, Employability through the curriculum, «Tertiary Education and Management», <http://www.leeds.ac.uk/educol/ documents/175854.doc> (01/2018). Knight P.T. and Yorke M. 2003, Employability and Good Learning in Higher Education, «Teaching in Higher Education», VIII (1), 3-16, <http://dx.doi. org/10.1080/1356251032000052294> (01/2018). g ( ) Morin E. 1999, La testa ben fatta. Riforma dell’insegnamento e riforma del pensiero, Raffaello Cortina, Milano.f Morin E. 2001, I sette saperi necessari all’educazione del futuro, Raffaello Cortina, Milano.f Morin E. 2015, Insegnare a vivere. Manifesto per cambiare l’educazione, Raffaello Cortina, Milano. Mortari L. 2007, Cultura della ricerca e pedagogia. Prospettive epistemologiche, Carocci, Roma.i Nussbaum M.C. 2010, Not for profit. Why Democracies Needs the Humanities, Princeton University Press, Princeton. y Nussbaum M.C. 2011, Creating capabilities: the human development approach, The Belknap Press of Harvard University Press Cambridge Massachusetts y Nussbaum M.C. 2011, Creating capabilities: the human development approach, The Nussbaum M.C. 2011, Creating capabilities: the human development approach, The Belknap Press of Harvard University Press, Cambridge, Massachusetts. Otto H.-U., Atzmüller R., Berthet T., Bifulco L., Bonvin J.M., Chiappero- Martinetti E., Egdell V., Halleröd B., Kjeldsen C.C., Kwiek M., Schröer R., Vero J., Zielenska M. (eds.) 2015, Facing Trajectories from School to Work Towards a Capability-Friendly Youth Policy in Europe, Springer, London. OECD 2017, Getting Skills Right: Italy, OECD Publishing, Paris, <http:// dx.doi.org/10.1787/9789264278639-en> (01/2018). g ( ) OECD 2017, OECD Skills Strategy Diagnostic Report: Italy, OECD Publishing, Paris. OECD 2017, Strategia per le competenze dell’OCSE – Sintesi del rapporto: Italia, OECD Publishing, Paris. Teichler U. 2007, Does Higher Education Matter? Lessons from a Comparative Graduate Survey, «European Journal of Education», 42, 11-34. Teichler U. 2013, Universities Between the Expectations to Generate Professionally Competencies and Academic Freedom: Experiences from Europe, «Procedia – Social and Behavioral Sciences», 77, 421-428. Yorke, M. and Knight P.T. 2006, Embedding employability into the curriculum, The Higher Education Academy, York. Paolo Federighi (University of Florence)* abstract: Entering a study course and the subsequent transition to the labour market is no longer the rule. In the past, routes were linear, today transitions are non-linear. The research undertook to contribute to studying the factors that promote university students’ transition to work. Some of the research activities have been devoted to a longitudinal study where the ways Educational Science graduates manage their own professionalization strategies have been investigated. It is a cluster for which in several countries the lowest return has been estimated in relation to higher education investment. If we manage to clarify the terms of non-linear transitions paths we can succeed in understanding the types of meas- ures to be introduced, how to relate them to processes that will professionalize students, when to activate them, and how to evaluate their impact. keywords: higher education democratisation, non-linear transitions, profession- alization measures in higher education. keywords: higher education democratisation, non-linear transitions, profession- alization measures in higher education. NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET Paolo Federighi (University of Florence)* 1. Transitions, efficiency and fairness Since the Seventies, the idea that the production system requires peo- ple with good levels of higher education has gradually gained ground in public policies. Over this same period, the higher education systems of many countries have expanded rapidly. This has had significant and pro- found impacts on labour markets and in the way in which employers use highly educated labour. «These expansions have, for the most part, been predicated on the assumption that more education is good for individu- als and for society as a whole, not only in terms of economic outcomes like wages or employment, but also for a wide range of social outcomes like improved health, reduced crime and higher well being» (Machin, McNally 2007: 5). y ) In more recent years, the OECD has focused government strate- gies further by shifting the emphasis from the quantity of accesses, to the quality of the learning outcomes of investment in higher education: «Labour market relevant knowledge and skills drive and support eco- nomic growth, innovation, and the evolving needs of the labour mar- ket» (OECD 2017: 16). The number of years of study or the certification acquired are still considered relevant, but only on condition that these correspond to a satisfactory level of professional skill that meets the needs of the labour market. This approach has inspired strategies to reform higher education sys- tems and has strengthened the request for more coherence «between the 130 PAOLO FEDERIGHI level of (graduate’s) skills and the skills required at work» (OECD 2017: 16) as for closing the gap between university and industry. The traditional education model based on transmitting knowledge in the various fields of study has started to change in favour of a model that advocates the development of competences. The introduction of a competence-based approach in the defining and preparation of the learning outcomes is probably the most distinctive feature of the current university reform and is closely linked to the en- hancement of graduate employability, given that it gives a higher pro- file to and involves their professional ability and its applicability in the workplace (Coromina Saurina, Villar 2010: 24). Consequently, the entire process of educating students has become an object of study and modifications: from guidance in upper secondary schools, education and training during the period of study, to the tran- sition to the labour market. NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET Nonetheless, the automatisms have not been replaced by an equal- ly linear transition process that ensures a place in the labour mar- ket after the studies are over. The linear segmentation into three synchronized phases (study, job matching between demand and supply, employment) recurs infrequently. The transitions are non- linear. There is no longer a proportionality between the actions put in place and their effects, i.e. between entering a study course and the subsequent passage to employment. According to some studies (Burke, Threadgold, Bunn 2017), the absence of linearity is related in a unique way to the paths of university students from historically under-represented backgrounds when they transition as graduates into an insecure labour market. Therefore, deepening knowledge of the paths that characterize the process of transition from higher education to work helps to under- stand what mechanisms to put in place to reduce the negative impact of a graduate’s socioeconomic status, and to restrict higher education’s social reproduction systems (Harrison 2017). Clarifying the terms of the non-linearity of transition paths lets us understand the types of measures (activities and services) to introduce, how to relate these to processes that will professionalize students, when to activate them (at the end of the university career or during the study course), and how to evaluate their impact. The Emp&Co research undertook to contribute to studying the factors that promote university students’ transition to work. A part of the research activity has been devoted to a longitudinal study of the ways in which Educational Science graduates manage their own pro- fessionalization strategies in the first two years after the end of their university studies. The choice of this segment of the population aimed to collect new facts on the numerous ways in which the processes of professionalization and transition to the labour market operate in dif- ferent academic sectors. This type of study is known to be rare. At the same time, this is a sector that belongs to the field of ‘education’ and therefore to one which, according to all the studies, ensures a wage premium that is considerably lower than other sectors, both in the short term and considering cumulative earnings over 20 years (OECD 2017: 55). 1. Transitions, efficiency and fairness The success of the transitions to the labour market is no longer left to individual destinies. The mechanisms and dy- namics that regulate them have been made visible, their logic studied, and then refocussed through corrective actions.i The outcomes of higher education are not identified simply with the skills possessed by graduates, but by the quantity and quality of «graduates who are part of the labour force. They include employment status, earnings, and the match between the level of their skills and the skills required at work, etc. Outcomes also take account of graduates who are not enrolled in further schooling, and are not part of the la- bour force» (OECD 2017: 16). ( ) Possession of the skills requested is not enough to justify person- al and social investment. What counts is the employment status, the wage premium, and the timeframe within which these results are achieved. The devices that accompany the transitions should there- fore be corrected by reducing the impact of the invisible factors that favour those with an exclusive social and family background, and promote integration into the labour market irrespective of merit. If not, the mechanisms accompanying the transition of graduates to- wards the world of work will only strengthen and aggravate the uni- versity’s social reproduction. Expansion of the access to higher education has dealt a blow to the very automatisms of industrial society, the direct relationship between school and factory. Companies too have abandoned the old business school models and their function of induction. In the past, the mech- anisms to be activated were relatively simple. The routes were linear, as were the automatisms that ushered people towards a relatively pre- dictable and stable professional future. NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET 131 2. Professional growth paths 2. Professional growth paths The interpretation of the set of professional growth paths for Educa- tional Science graduates shows the presence of three driving forces that guide individual activities: g 1. Collection of information on the potential labour market and on how to establish a coherent professional profilei 1. Collection of information on the potential labour market and on how to establish a coherent professional profilei 2. The gaining of professional certification (with the function of signal- ling) and the enrichment and visibility of professional potential 3. Gradual building of independence in life and work. We are using the term ‘driving forces’ and not ‘steps’, since there is not a temporal succession between these three components. If anything, it is a matter of objectives undergoing constant evolution, which those concerned work on synergistically, albeit with differentiated strategies and levels of effectiveness (Boffo 2017; Torlone 2017). To achieve these objectives, while carrying out their university stud- ies, the students follow paths characterized by: • a commitment of various kinds to work activities (in addition to internships) • a commitment of various kinds to work activities (in addition to internships) p ) • participation in vocational training activities • building of «dynamic learning networks» (Reich, 1991). At the end of university studies, the paths are divided further with: • breaks from every type of formative and professional commitment At the end of university studies, the paths are divided further with: • breaks from every type of formative and professional commitment • further developments in the building of «dynamic learning networks» • participation in structured activities targeted directly at job placement • the beginning of the path to develop a professional career with transitions from the first, to the second, and the third employ- ment contract (again within the 18-month period considered by the research). • further developments in the building of «dynamic learning networks» • participation in structured activities targeted directly at job placement participation in structured activities targeted directly at job placement • the beginning of the path to develop a professional career with transitions from the first, to the second, and the third employ- ment contract (again within the 18-month period considered by the research). The individual paths differ in the combination of the main types of activity (study-work-networking-idleness), beyond the rhythms of their duration and succession. NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET Most of all, this is a cluster for which in countries such as Sweden, «the lowest return has been estimated (the others include Religious Studies, Psychology and Mathematics/Natural Sciences)», but in others, such as Spain «a negative return is estimated for graduates with degrees classified as ‘other social sciences and humanities’» (Machin, McNally 2007: 23). Therefore, a study of the processes that affect graduates in extreme situations should enable us to better understand the factors of exclusion and inequality which could affect graduates in all the pro- 132 PAOLO FEDERIGHI fessional sectors most exposed to the risk of a mismatch, not due to a lack of competence, but to their socioeconomic status. fessional sectors most exposed to the risk of a mismatch, not due to a lack of competence, but to their socioeconomic status. NONLINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET Figure 1 – Two interpretive models. Linear Model Higher education Job Placement Labour Market Life and work experience Prior learning Life and work Life and work Job Current Job Interpreting professional growth paths using a non-linear model means attributing a value to each type of life and work experience had by the stu- dents; it means that the development of a professional career is the result of a set of learning outcomes (prior learning). From an educational perspec- tive, the achievement of a fi rst employment contract and then subsequent ones is interpreted as the result of what was learned in formal education, through individual dynamic learning networks, the educational and cul- tural infrastructures accessed, and experiences of work (whether paid or unpaid). The Emp&Co research allowed us to observe in the graduates’ professional growth paths, the following four types of educational activity: p g p g yp y • Formal education comprising the customary academic path enriched by internships, study abroad, and participation in research. Both during stud- ies and after graduation we can observe the tendency to return to further training of a professional kind. This may be directed to strengthening areas of competence not covered by the university off ering, to acquire knowledge of new professions, or to build new networking relationships. g p g p • The building of networking relationships and dynamic learning networks occurs in all professional growth paths with a positive outcome. Accessing networking and strengthening it seems to be related to graduates’ family backgrounds and a capacity for autonomous initiative in the creation of new social and professional networks (associations and business). As well as constituting a hub of opportunity, networking represents a context in which it is possible to achieve mutual learning and swap knowledge. p g p g • Access to and use of educational and cultural infrastructure and services appears to be a further component present in numerous growth paths of graduates. This is a factor that has been investigated only marginally. Access to services and programmes provided by youth policies proved of use in the cases found (e.g. Youth Guarantee Programmes). 2. Professional growth paths Entry into the labour market and professional growth are not the result of a linear process followed with discipline, in which each step is based on proportionate effects produced by the previous one. It is instead the result of a set of learning outcomes ac- quired through diverse types of activity (including those carried out in higher education) on which the new job is founded. 133 NONLINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET NONLINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET What remains to be investigated further is the recurrence and importance of the use of digital infrastructures as well as generic cultural ones (from the library, to the museum, music, and so on), both for their function of direct access to information on the labour market, and their medi- ated function of skills development and the creation of networking. 134 PAOLO FEDERIGHI • Lastly, work proves to be part of the opportunities that work culture had already generated before university studies ended. Work is simul- taneously the expected result and the means through which the stu- dents develop their ability to learn and produce new knowledge. The opportunity to move from one job to a different and better one could be interpreted as the fruit of prior learning accumulated through the different educational activities participated in. Therefore, the detectable differences in individual paths which empha- size the non-linearity of the trajectories can be attributed to the quality of the opportunities enjoyed. To explain the stasis or change found in the individual paths observed, we could use a descriptive model that combines the various positions covered (idleness or non regular work, unpaid em- ployment, standard contract), with varying levels of educational quality potentially expressed by the activities the graduates participate in (certifi- cation; vocational training, services, and networks; continuous improve- ment processes through joining soft or hard innovation projects). Figure 2 shows the existing relationship for new graduates between educational activities, position, prospects for growth in the quality of work, and the expectation of moving to a new job. The weights assigned to the indi- vidual boxes have a purely indicative value and correspond to the prob- abilities found to progress towards a better position. Position number 9 is considered the most advantageous, since it is open to new positions. Figure 2 – Learning activities and new graduates’ positions. Figure 2 – Learning activities and new graduates’ positions. Figure 2 – Learning activities and new graduates’ positions. 3. Professional growth and development at work 3. Professional growth and development at work 3. Professional growth and development at work In the context of the Emp&Co research, work, as a means for person- al education and an end for its realization, is treated as a priority since it is a resource for personal growth. Other aspects, although relevant (e.g. the ROI from university education) were not investigated. NONLINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET NONLINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET NONLINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET 135 From this perspective, the most signifi cant factor to consider is the workplace learning potential, in other words, what can be learned by a worker covering a specifi c productive position at a given location. We are referring to the learning processes that are part of the productive ac- tivities of everyday life. y y The research showed how the characterizing data (the mode) con- sists of: • possessing regular work experience coherent with the type of stud- ies carried out, • possessing regular work experience coherent with the type of stud- ies carried out, • being employed when the interview was carried out, • having had more than one job experience. All the graduates had passed through small casual jobs and then moved towards positions related to identifi ed and defi ned professional profi les. Vertical and horizontal mobility, also towards economic sectors (ag- riculture, tourism, commerce) not coherent with the training received united all the subjects examined. The cases of vertical mobility involved a passage towards more qualifi ed professional roles, or towards forms of contracts with greater guarantees. The voluntary termination of an employment contract is an experi- ence common to all employees, and is connected to the choice of: as- suming an entrepreneurial role, changing organization, or returning to education. In Fig. 3 we describe the fi ve possible directions the profes- sional mobility of new graduates can take. Figure 3 – Career Lattice. [Adapted from: Young, B., Ladder vs. Lateral Career Paths, in Career Development Toolkit, 2011, <http://www: hrmcareerdevelopment.blogspot.it>] Figure 3 – Career Lattice. [Adapted from: Young, B., Ladder vs. Lateral Career Paths, in Career Development Toolkit, 2011, <http://www: hrmcareerdevelopment.blogspot.it>] Figure 3 – Career Lattice. [Adapted from: Young, B., Ladder vs. NONLINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET Lateral Career Paths, in Career Development Toolkit, 2011, <http://www: hrmcareerdevelopment.blogspot.it>] Development Toolkit, 2011, <http://www: hrmcareerdevelopment.blogspot.it>] Enrichment Growing within existing job Vertical Increasing responsibility within organisation Horizontal Change job within organization without changes in status/pay/level Realignement taking a step back to pursue different career options Lateral Change job outside organization without changes in status/pay/ level Enrichment Growing within existing job Vertical Increasing responsibility within organisation Horizontal Change job within organization without changes in status/pay/level Realignement taking a step back to pursue different career options Lateral Change job outside organization without changes in status/pay/ level Enrichment Growing within existing job Vertical Increasing responsibility within organisation Horizontal Change job within organization without changes in status/pay/level Realignement taking a step back to pursue different career options Lateral Change job outside organization without changes in status/pay/ level Vertical Increasing responsibility within organisation Lateral Horizontal Enrichment Growing within existin job Realignement taking a step back to pursue different career options 136 PAOLO FEDERIGHI The dynamism that appears after the end of university is particularly intense and expresses itself in an active search for new career prospects with a willingness to change professional contexts and roles. From an educational standpoint, this behaviour can be attributed to two factors: the process of building professional identity, and an increased ability to read the positive or negative signs of the workplace learning potential.i The process of building professional identity intensifies with the posses- sion of additional information on the potential labour market and the maturing of new expectations with respect to previous work experi- ences. The comparison between past and future potential is probably an accelerator of the beginning of the process that leads new graduates to engage in the building of their own professional identity. Since these are professions exposed to constant evolution, and persons simultaneously engaged in distinct roles and tasks, the task of building «meanings that individuals attach to themselves in the context of work» becomes even more complex. NONLINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET In fact, this commitment – starting from their person- al attributes, membership of social groups and professional roles – leads individuals to give «meaning to who they are and what they do in the workplace» (Caza, Creary 2016: 4).i p y Whether we are talking of professional identification (as a state of be- ing that explains how individuals view themselves vis-a-vis their profes- sion – Caza, Creary 2016: 7), or of the process of building professional identity (the way in which individuals have the agentic role of creating their own professional self-image – Caza, Creary 2016: 7), in both cases, graduates are prompted to redefine their professional identity both be- cause of prior learning, and fresh job opportunities. p g j pp Furthermore, two further elements must be considered, due to the particularity of the professional group of reference for Educational Sci- ence graduates. This, more and better than other professional profiles, helps to understand the phenomenon of transforming the boundaries of traditional professions, of their hybridization, and of another two phenomena that affect young people: the high probability of not only changing jobs more than once during their career, but also of changing profession, plus the gradual spread of the phenomenon of multiple pro- fessional identities linked to the simultaneous employment of persons in two or more jobs. This is a phenomenon that is not limited to those who take the entrepreneurial road or choose to be freelance, but also those who are self-employed. The idea of a single-profession career is becom- ing outdated. Many professional workers today are not simply acquiring one set of specialized skills and knowledge, but instead are accumulat- ing multiple sets of skills, and applying these in quite distinct ways (Ca- za, Creary 2016: 13). y The workplace learning potential is a factor towards which the attention of skilled workers is increasingly turning, and which is also used to ex- NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET 137 plain the attraction for low-wage premium study paths. This is the same dynamic to which individual professions and careers/professional iden- tities are exposed, making the workplace the setting to build their own professional growth, and not only in terms of career development. The Emp&Co research shows that it is not only the expectation of a wage premium that determines the decisions of students and graduates. NONLINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET The Employee Value Proposition (Minchington 2010) is an element that bet- ter explains present and expected job satisfaction, and that motivates the high propensity to occupational mobility found in the research. Within this frame is the evaluation of learning potential of a workplace, defin- able as “the power of a work setting to integrate learning at work with the result of behavioural changes and the generation of new knowledge. Such a workplace offers accessible information, opportunities to learn, and real support by peers and managers” (Nijhof, Nieuwenhuis 2008). On this basis, it makes sense to assume that students and graduates with some work experience develop perception and knowledge of the learn- ing mechanisms present in a workplace and are driven to assess their relevance in relation to expectations of professional growth. Thus, their choices can be guided by an assessment of each of the factors that are in- cluded in the workplace learning potential: • The strategic dimension, i.e. the development prospects of an organi- zation, objectives for growth in the medium and long term. • The strategic dimension, i.e. the development prospects of an organi- zation, objectives for growth in the medium and long term. • The organizational dimension, i.e. the richness of cultural containers, the knowledge accumulated by the organization, its level of struc- turing, its definition of management processes and innovation (Vi- cari 2008). • The existing distributive rules which regulate access to knowledge of the organization and to their creation: timeframes, ways of cog- nition, and the types of process people are engaged in (Eraut 2007), the possibility of creating endogenous know-how producing new knowledge in relation to the product, customer, market, and process that the organization needs. 4. Employability and restriction of higher education institutions’ reproductive functions 4. Employability and restriction of higher education institutions’ reproductive functions The traditional educational model based on the transmission of knowl- edge in the different fields of study does not have as its benchmark an image of the student and graduate corresponding to the professional identity and prospects found. Enhancing the relationship between students and their labour market is a challenge that requires universities to unavoidably adapt and modern- ize their educational offering: «One of the main objectives of higher edu- 138 PAOLO FEDERIGHI cation is to provide its graduates with the skills needed to succeed in the labour market» (OECD 2017: 9). ( ) It is well known that access to the labour market is determined as much by economic factors (the state of the economy, labour market regulation, the existing stock of skills), as by the graduates’ charcteristics. Studies on the impact of factors such as ethnicity, gender (for the earnings gap compared to men), socioeconomic status (family background and quality of networking), age, or disability, show how each play a role in the labour market and can affect the market outcomes for certain graduates. Research similar to that of Emp&Co carried out in Australia (Burke, Threadgold, Bunn 2017) shows “how social and cultural differences (e.g. socioeconomic status, gender and ethnicity) and inequalities (e.g. of access to and participation in HE and paid work) «are not only interrelated, but are bound together and influenced by the intersectional systems of society (Collins 2000: 42). […] This lens helps to uncover how socioeconomic status is bound together with social and cultural differences in the educational and work structures». Other research shows that the social background often produces a du- rability of disposition that is not easily dismantled or modified in and through university contexts. This is what emerged from a study carried out on the functions of higher education in Jordan and Egypt (Assaad, Krafft, Salehi-Isfahani 2014): Family background, gender, and geography, and to some extent sec- ondary school performance, appear to play a much larger role in labour market outcomes, even in this select group of graduates, than the type or quality of their higher education institutions. This suggests that the labour markets do not in fact reward skills, or do so to a very limited extent, so that neither higher education institutions nor students have any reason to seek out the type of higher education that builds produc- tive skills (Assaad, Krafft, Salehi-Isfahani 2014: 16). 5. Measures to support employability The gulf between the university and the world of work makes the definition of students’ investment choices and career more problemat- ic and open to risk, especially for those who are inequitably located in navigating their aspirational journeys (Harrison 2017). The success of the transitions to the labour market is not attributable solely to the quality of individual self-improvement projects. «Those without access to supportive social, economic and cultural capital and the readiness to negotiate transitions effectively are typically those who require these capacities the most» (Billett, Thomas, Sim, Johnson, Hay, Ryan 2010: 484). In these cases, but not only, the success of the tran- sitions also depends on the willingness and ability of higher education system to adopt practices and behaviours that help develop labour mar- ket-relevant knowledge and skills. But to be able to train students, it is necessary that individual universities engage in paths of institution- al learning that will enable the same university to learn how to trans- form themselves and create their own new social identity and their new positioning with respect to the labour market. The construction of a process of capacity building at university involves governance interven- tions based on policy levers to enhance labour market relevance. At the system level, these results are obtained with governmental policies that act: on the criteria for funding universities; on regulations and on the rules that govern the everyday life of businesses, organizations and stu- dents; on information strategies to influence the behaviour of students and higher education institutions; on activation of the organizational resources of the various public agencies (from ANVUR to the various ministe- rial bodies) to help achieve their goals by steering or influencing higher education systems. y There are however practices and behaviours that do help develop la- bour market-relevant knowledge and skills, which universities can adopt as a learning process and organizational change that are relatively auton- omous with respect to the action of governmental policy levers. Below, we examine a selection of some of the measures consistent with the Emp&Co research results, whose effectiveness is confirmed by studies carried out at an international scale (OECD 2017: 64-74) relat- ing to the following final considerations: NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET 139 4. Employability and restriction of higher education institutions’ reproductive functions Changing the function of the university’s social reproduction does not depend on measures taken at the level of organizational and teach- ing models. Yet it can be restricted through strategies that mitigate the effects and enhance efficiency, by acting, for example, on the period of adjustment, the time it takes for a graduate to become effective in the workplace. This means putting the university in a condition to train «transformative employees», «people who can use higher-level skills, such as analysis, critique, synthesis, and multi-layered communication to facili- tate innovative teamwork» (Harvey 2003: 11). The task of the university is not then limited to facilitating the meeting between demand and offer of employment, «in essence, the emphasis is on developing critical, re- flective abilities, with a view to empowering and enhancing the learner. Employment is a by-product of this enabling process» (Harvey 2003: 3). 3. Student admission policies and practices, and academic support for students The procedures by which students are admitted and supported in their studies can also make a difference in the relevance and quality of the skills developed and this ultimately affects labour market outcomes. Stu- dents who are selected by higher education institutions based on previous strong academic results and completion of relevant pre-requisite subjects (at secondary school or through other tertiary education) are more likely to succeed in higher education (Hiss, Doria 2014). 4. Curriculum design and delivery «The curriculum is at the core of higher education learning: in any field of study, a well-designed curriculum is an important step towards ensuring that students develop good skills that position them for labour market success» (OECD 2017: 67). The construction of curricula is ex- posed to the challenge of the dynamics of transformation of professional groups, of the hybridizations of various profiles, their constant changes, and of various forms depending on organizational contexts. The answer to this problem can only partially be entrusted to continuing education provided after the end of university studies and in workplaces. To this must be added the fact that the idea of a single-profession career is becom- ing outdated. Part of the disciplinary knowledge provided will not only become quickly obsolete, but also irrelevant with respect to the sundry figures and professional roles that students will be required to interpret. i g p q p The interdisciplinary response, not based on a mere juxtaposition of curricula in different disciplines, could constitute an effective launchpad for curriculum design. However, it clashes with both the prevalence of organizational models based on the separation between disciplines, and with the lack of information on the future of the professions. In a national economic structure dominated by small- to medium-sized enterprises, forecasts of the skills demand for the medium to long term are scarce. 2. Informed student choice 2. Informed student choice The students’ choice of study must be able to rely on existing infor- mation. Guidance activities must focus on providing information on genuine employment prospects. The students’ choices must not be influ- enced by the need of individual universities to increase their number of enrolments. Information on employment prospects is a service that must be ensured throughout their career, also to help reshaping their choices. 1. The capacity of higher education institutions to respond to labour market demand f This depends above all on the ability to offer relevant study pro- grammes. The availability of human resources (i.e. academic and support staff), and physical infrastructure have a significant impact, and depend on local choices (selection of personnel, investments). 140 PAOLO FEDERIGHI 5. Learning and teaching and delivery of curriculum «The ways in which the curriculum is delivered can also play an im- portant role in generating high quality skills that support good labour market outcomes for higher education graduates. The traditional ap- NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET 141 proach to higher education teaching has long been for an experienced academic to provide a lecture that “imparts knowledge” to students» (OECD 2017: 71). Teaching practices based on the lecture-based model are known to be ineffective. They do not take account of the fact that higher education institutions operate with adults, who normally work, and already have their own background knowledge and experience. In short: they do not consider how adults learn. The problem also concerns innovations in teaching, the use of technologies that are by now a part of people’s daily life. However, this limit can be overcome with the use of typical adult education methods (e.g. group activities, oral presen- tations, problem-solving scenarios) as they are developing professional and technical skills. The education of ‘transformative employees’ – i.e. people who are entrusted daily not only with tasks of mere execution, but the creation of new knowledge of products, organization, process- es, and markets – is mainly realized through the method of delivering the curriculum. 7. Internationalization The development of the international dimension of educational path- ways makes it possible to deal with labour markets other than local ones. The prospect of working abroad for a period of their lives, and thus being confronted with different national labour markets, is an opportunity that is gradually extending to all professional groups. The exclusive relation- ship between the university and the local economy covers an increasingly limited proportion of educational and research activities. «Undertaking part of a higher education programme in another country can enable students to develop important transversal skills and thus support good la- bour market outcomes» (OECD 2017: 72). Studying abroad helps students expand their knowledge of other societies, languages, cultures, and busi- ness methods, and develop cross-cultural competencies and sensitivities. These skills, in addition to the resiliency demonstrated by exiting one’s comfort zone to pursue education in a foreign setting can send a strong signal to employer about the employability of graduates (OECD 2013). 6. Work-based learning and workplace learning The Emp&Co research shows how the education of students also depends on the learning potential of their current job. The work-based learning provided by curricula must therefore also communicate with workplace learning processes in which the student participates. Participation in quality work-based learning (sometimes known as work- integrated learning) can improve labour market outcomes. There are sev- eral types of work-based learning processes in higher education. These include field experience, mandatory professional practice, co-operative education placements, internships, applied research, project learning and service learning. Graduate apprenticeships have recently re-emerged as another way of combining work and academic studies to enable people to ‘earn-as-they- learn’ (OECD 2017: 73). Participation in workplace learning processes puts the student in a position of dynamic interaction with the learning factors present at their own workplace. This may be a limiting factor, given that their image of work is determined by current experiences and their quality. At the same time, they constitute a wealth of theoretical, methodological, and practical knowledge, on which the student can exercise their critical- transformative skills, bringing innovation to this context. Moreover, the students are constantly exposed to a benchmarking exercise that puts them in the condition to give meaning and evaluate the theoretical and practi- cal experiences offered by the curriculum. For the university, this means learning to deal with the work experiences and cultures the students bring, and to manage and steer the learning processes that they generate. 142 PAOLO FEDERIGHI NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET 143 learning opportunities and post-graduation employment opportunities. Increasingly, they also provide counselling and advice related to new skill sets that are important both for getting a job and for succeeding in workplace and to develop entrepreneurial skills (OECD 2017: 74). The need for specialized support services exists primarily to perform the functions of guidance, coordination, quality assurance, and network- ing of the system. In fact, the set of activities described above constitutes a system integrated with all the functions of teaching, research, and uni- versities’ third mission, which requires its own body of strategic plan- ning and management. 6. Conclusion Transitions to the labour market are also dependent on the recruit- ment traditions, methods, and tools used by enterprises. The weakness of the systems currently in place is under debate. In 2016, France Stratégie estimated that the cost of inequalities in accessing qualified work is not far off €150 billion, due to wasted talent, unemployment, and idleness, a misallocation of human resources (Bon-Maury et al. 2016). fi ( y ) The research is making efforts to define new mechanisms that might reduce the technical inefficiencies and cultural weaknesses of the cur- rent ones. The aim is to go beyond the use of CVs, certificates, and the gut instinct of recruiters. To this end, the research has focused on recent applications of machine learning coupled with increased access to data, raising the possibility of improving hiring decisions with the help of al- gorithms (Danieli et al. 2016; Hoffman 2015). f Meanwhile, civil society is making efforts to promote non-gov- ernmental initiatives that can bring young people into contact with enterprises. University research should participate more directly in the study of technologies and organizational forms that could help higher education institutions rid themselves of outdated cultures and ideologies and solve the setbacks that prevent them from fulfilling their role. 8. Career Advice and support The results of the Emp&Co research show that the functions of career advice and support may not be concentrated on the moment of transi- tion between higher education and the labour market since these tran- sitions are non-linear, and because many of the students already have a job before completing their studies. The expected function is to foster the transition towards a new job, possibly of a better quality than the previous one. Career advice is emerging as a function relevant to all the measures listed above. It is a by-product of them. As Jääskelä & Nissilä argued (2015): «Pedagogy and guidance services (are) a systemic entity». It is however questionable whether this type of service can be limited to students, or if even universities should behave like other stakeholders of learning, and provide a ‘post-sales’ career advice service. As part of the Emp&Co research, the delivery of this type of service to graduates was tested. A final consideration is that this is an initiative that is sustainable and coherent with the ethics and values of a contemporary university. The service could play an even more significant role if all university teachers provided students with the contacts and information in their possession relating to the production world that they cooperate with. This function is also accompanied by career services. Many higher education institutions now provide career services or ca- reer centres to help students connect with prospective employers. These centres assist students by helping them apply for jobs, write their curric- ulum vitae or resumé, and prepare for job interviews. They also provide students with access to employers by organising job fairs and employer visits to campus, and by distributing employer postings for work-based NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET Assaad R., Krafft C. and Salehi-Isfahani D. 2014, Does the type of higher education affect labor market outcomes? A comparison of Egypt and Jordan, Working Paper 826, April 2014, Giza, Egypt, The Economic Research Forum (ERF). References Assaad R., Krafft C. and Salehi-Isfahani D. 2014, Does the type of higher education affect labor market outcomes? A comparison of Egypt and Jordan, Working Paper 826, April 2014, Giza, Egypt, The Economic Research Forum (ERF). 144 PAOLO FEDERIGHI Billett S., Thomas S., Sim C., Johnson G., Hay S. and Ryan, J. (2010), Constructing productive post-school transitions: An analysis of Australian schooling policies, «Journal of Education and Work», XXIII, 5 November 2010, 471-489. Boffo V. 2017, The Embedded Employability Process, in Boffo V., Fedeli M., Melacarne C., Lo Presti F. and Vianello M., Teaching and Learning for Employability: New Strategies in Higher Education, Milan-Turin, 161-165. Bon-Maury G., Bruneau C., Dherbécourt C., Diallo A., Flamand J., Gilles C., Trannoy A. 2016, Le coût économique des discriminations, France Stratégie, <http://www.strategie.gouv.fr/sites/strategie.gouv.fr/files/atoms/ files/19-09-2016_fs_rapport_cout_economique_des_discriminations_ final_web_0.pdf> (01/2018). Burke P., Threadgold S. and Bunn M. 201), Degrees of Class: Interrogating linear and non-linear transitions from higher education into the labour market, Paper presented at Higher Education Rising to the Challenge Balancing expectations of students, society and stakeholders, SRHE Annual Research Conference, 6-8 December 2017, Newport. p Caza B.B. and Creary S.J. 2016, The construction of professional identity, in Wilkinson A., Hislop D. and Coupland C. (eds.), Perspectives on contemporary professional work: Challenges and experiences, Cheltenham, 259-285. Edward Elgar Publishing. [Electronic version], from Cornell University, SHA School site: <http://scholarship.sha.cornell.edu/articles/878>. Danieli O., Hillis A. and Luca M. 2016, How to Hire with Algorithms, «Harvard Business Review», October 17, 2016. Collins P.H. 2000, Black feminist thought: Knowledge, consciousness, and the politics of empowerment, Routledge, New York.i Eraut M. and Hirsh W. 2007, The Significance of Workplace Learning for Individuals, Groups and Organisations, Oxford & Cardiff University, ESRC Centre on Skills, Knowledge and Organisational Performance, Oxford-Cardiff. Harrison N. 2017, Student choices under uncertainty: bounded rationality and behavioural economics, in Mountford-Zimdars A. and Harrison N. (Eds.), Access to Higher Education: Theoretical Perspectives and Contemporary Challenges, Routledge, London. g Harvey L. 2003, Transitions from higher education to work, Centre for Research and Evaluation, Sheffield Hallam University.f fi y Hoffman M., Kahn L.B. and Li D. 2015, Discretion in Hiring, Harvard Business School, Cambridge.i Hiss W., Doria K, 2011, Defining Promise: Optional Standardized Testing Policies in American College and University Admissions, Bates College, Lewiston, Maine. Jääskelä P. and Nissilä P. 2015, Identifying Themes for Research-Based Development of Pedagogy and Guidance in Higher Education, in «Scandinavian Journal of Educational Research», LIX (1), 24-41. References ( ) Machin S. and McNally S. 2007, Tertiary Education Systems and Labour Markets, «Thematic Review of Tertiary Education», OECD, Paris. Minchington B. 2010, The employer brand manager’s handbook, Collective Learning, Torrensville. NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET NON-LINEAR PATHS IN TRANSITIONS THROUGH THE LABOUR MARKET 145 Nijhof W.J. and Nieuwenhuis L.F.M. 2008, The Learning Potential of the Workplace, Sense Publishers, Rotterdam-Taipei. p p OECD 2013, Education Indicators in Focus: How is International Student Mobility Shaping Up, OECD Publishing, Paris. OECD 2017, In-Depth Analysis of the Labour Market Relevance and Outcomes of Higher Education Systems: Analytical Framework and Country Practices Report, Enhancing Higher Education System Performance, OECD, Paris. Reich Y. 1991, The Work of Nations: Preparing Ourselves for 21st Century Capitalism, New York, Knopf Publishing.i Torlone F. 2017, The Transitions of Young Adults to Define Individual Pathways of Professional Insertion, in Boffo, V. Fedeli M., Melacarne C., Lo Presti F. and Vianello M., Teaching and Learning for Employability: New Strategies in Higher Education, Milan-Turin, 175-190. g Vicari S. 2008, Conoscenza e impresa, «Sinergie», 76, 43-66. Young B. 2011, Ladder vs Lateral career paths, «Career Development Toolkit», hrmcareerdevelopment.blogspot.it (01/2018). PART II THE PERSPECTIVES OF THE PEDAGOGICAL COMMUNITY * Concetta Tino, PhD, Post-doc fellow in Education, Department of Philosophy, Sociology, Pedagogy and Applied Psychology - FISPPA, University of Padua, Italy. Email: concetta.tino@unipd.it. Concetta Tino (University of Padua)* Concetta Tino (University of Padua)* abstract: Data from Censis 2011 highlighted worrying aspects of school drop- outs (18%) and a NEET population increase (22.1%); the same elements were also highlighted by data from Cedefop (2014), where the issue of youth unemploy- ment (21.7%) was also mentioned. In addition to this are the disappointing results from OCSE-Pisa surveys demonstrating that Italian educational institutions fail to provide young people with the skills they need to effectively solve real-life prob- lems. In this scenario, at an Italian and European level, the importance of solv- ing these problems is repeatedly underlined, with the creation of instruments to interconnect the world of education and the world of work. Within this process, School-Work Alternance (SWA) programmes can find a place. This study focuses on the strategic action of their key actors in creating effective partnerships with external organizations. Based on this assumption, the research question asked was: what specific functions do teachers play within School-Work Alternance programmes? Methodology: a qualitative methodological approach was used; data were collected through semi-structured interviews addressed to 14 high school teachers, and subsequently analysed using Atlas.ti software in order to record the significant core categories that emerged. Results: the data collected showed that within the SWA system yet to be defined, SWA teacher/tutors and coordinators in school contexts have played a significant role within School-Work Alternance programmes to date. Final remarks: teachers involved in School-Work Alternance programmes have a strategic position. These results have some practical implica- tions at both educational/training and professional levels. Keywords: school-work alternance programmes, activity theory, boundary crossers, boundary objects, training. 1. Introduction The Italian and European debate on the importance of creating tools to integrate formal knowledge with informal learning developed in working environments is closely linked to the ongoing economic and social transformations that have been affecting Europe over the last ten years, and to the challenges they have generated. After the worrying results recorded in Italy by Censis (2011) regarding school dropouts (18%) and the increase in the NEET (Not in Education, Em- ployment or Training) population, with a percentage of 22.1% among young people aged between 15 and 29; subsequently, Cedefop (2014) showed how, as well as the dropout (17%) and NEET populations (18- 24 years: 29.3%), there was a considerable youth unemployment rate 152 CONCETTA TINO of 21.7%, compared to the average of 15.1% in the European Union countries. The results of the OCSE-Pisa (INVALSI 2012) surveys also contributed to reinforcing reflection on the importance of re- thinking the education-work relationship, developing methodologies and strategic learning practices that would be useful to integrate for- mal and informal learning, and theoretical and practical knowledge. The surveys demonstrated that Italian education institutions not only work differently in relation to the territory they belong to, and the type of school (high school, technical or vocational), they also fail to create those conditions that can help young people acquire skills that are useful for real life. This is a scenario that casts doubt on both the isolation and self-centrality of educational institutions and traditional teaching methods which have been dominated by a teacher-centred approach and the subject of study, thus denying students an active role in the process of building knowledge (Tacconi 2015a), as well as the opportunity to exercise power within participatory and active learn- ing contexts (Fedeli 2016). Therefore, an epochal change is needed to create synergic alliances both within the formal contexts of educa- tion, with the teacher as facilitator and the learner as responsible for and co-constructor of his own personalized teaching plan (Pastore 2016), and between public and private organizations that can facilitate the relationship between education and work, to create open systems and inter-organizational agreements to cope with the challenges that “liquid modernity” generates (Bauman 2000). of 21.7%, compared to the average of 15.1% in the European Union countries. 2. Theoretical framework School-Work is a system which, to be effective, needs to be built on a strong partnership between schools and workplaces, where the key actors must leave their comfort zones, cross the boundaries of their systems, and enter unfamiliar territory. In this assumption, the role performed by key teachers (tutors and SWA coordinators) dur- ing SWA paths is investigated in this study through the «boundary crossing» approach (Engeström, Engeström, Kärkkäinen 1995), whose roots lie within the cultural-historical activity theory of the «third generation» (CHAT) (Engeström 2001). According to this theory, in- teraction between systems can generate expansive learning through a cycle of collective transformation, when the systems partners act with awareness of the functioning of the organizations involved, considering them as goal-oriented activity systems; as multi-voiced communities inhabited by subjects with different stories, values and cultures, and where division of labour demands processes of explica- tion and negotiation; as organisms with their historicity made from tools, rules and artefacts; as places where differences and contradic- tions can create conflicts and lack of equilibrium, while at the same time these factors can become sources of innovation, transformation, and learning for the people and systems involved.f The relationship between different systems, required by today’s economic and social complexity, recalls the concept of boundaries, since the systems and the people who live within them, in order to overcome their specific specialization and the difficulty of dealing with problems, reach out through social and cultural practices, and through participation and collaboration within different contexts and organizations (Akkerman, Admiraal, Simons 2012; Daniels, Edwards, Engeström, Gallagher, Ludvigsen 2010), in an attempt to transcend the fragmentation (Hermans, Hermans-Konopka 2010), g p Within the school-work relationship, the boundary between the two systems of activity is represented by cultural difference and the potential difficulty of interacting. However, this same difficulty is also what Akkerman and Bakker (2011: 139) defined as «the poten- tial value of establishing communication and collaboration». This very process, during boundary crossing activities, involves two impor- tant components: the people involved or boundary crossers, and the ar- tefacts or boundary objects, built to realize the process itself. In SWA paths, teacher/tutors and SWA coordinators act as ‘crossers’ between systems, with the task of creating bridges between the two worlds as well as the contextual connections. 1. Introduction The results of the OCSE-Pisa (INVALSI 2012) surveys also contributed to reinforcing reflection on the importance of re- thinking the education-work relationship, developing methodologies and strategic learning practices that would be useful to integrate for- mal and informal learning, and theoretical and practical knowledge. h d d h l d l The education-work relationship «[…] questions the historical, and evident institutional difference between the tasks of schools (ed- ucation only) and the production world (work only), and strongly urges to redefine both the unitary identity profile of this segment of the education and training system (education and, or even through, work), and the human and educational value of production contexts (work and education)» (Tacconi 2015b: 9). From this perspective, a School-Work Alternance (SWA) programme, as a unitary synthesis of the two worlds kept separated for too long, can develop a fresh and more complete way of looking at work, not simply as a professional training tool, but also as personal development. Consequently, SWA probes the internal organizational of the education system, the teach- ing methods, the role of learners and teachers, but above all it leads to a questioning of the functions carried out by those key teachers who, over the years, and before the recent norm (L. 107/2015), have actively supported the development of SWA pathways and the build- ing of bridges between formal and informal learning contexts, try- ing to provide students with SWA experiences based as far as possible on the idea of situated learning practice and a strategic partnership between educational and production contexts (Tino, Fedeli 2015). SCHOOL-WORK ALTERNANCE PROGRAMMES 153 2. Theoretical framework This leads them to experience the natural ambiguity that the role implies: on the one hand they can appear as builders of bridges and alliances between the two worlds, 154 CONCETTA TINO capable of introducing new elements within the practices of the two different contexts (Wenger 1998); on the other, they may risk being seen as peripheral figures (Akkerman, Bakker 2011). Managing this ambiguity requires them to create dialogues with all the actors in the two contexts, to understand the different perspectives, and to develop boundary crosser leadership (Morse 2010). A boundary crossing process is not only determined by the ability of the people directly involved in moving to unfamiliar boundaries, but above all by the ability of the system to look at and venture beyond its boundaries, to negotiate within a third space characterized by a new culture as the result of a process of hybridity and dialogue (Gutier- rez, Rymes, Larson 1995) between separate organizational systems. y ) p g y Within a boundary crossing process, not only people are involved, but also important boundary objects as proof of a real process of ne- gotiation and sharing between schools and workplaces. On the one hand, they represent the strength of the synthesis of two different perspectives, on the other, they are the very image of flexibility, since they are concrete artefacts of a path built jointly by groups and different contexts, in the name of a process of collaboration, com- munication, reciprocal learning, and simultaneous participation in different domains (school and work), according to the principle of othering (Akkerman, Bakker 2011: 142) based on the ability of dia- logue and identification. 3. The study 3.2 Context and participants The context of the study was seven high schools in the north of It- aly. Specifically, it involved four technical institutions, one vocational institution, and two high schools. The teachers involved numbered 14, one teacher/tutor and one SWA coordinator for each education- al institution. Both had some SWA experience: this meant that the teacher/tutors had experience in supporting students on SWA paths within production contexts; while SWA coordinators were teachers with an overview of the design and practice of SWAs at the institu- tion they belonged to. 3.1 Objectives and methodology 3.1 Objectives and methodology This study wished to investigate the role of teachers involved in SWA programmes, to answer the following general question: «What are the functions of teacher/tutors and coordinators in SWA pro- grammes?» In order to identify useful guidelines to allow a more de- tailed data analysis, the general question was then dealt with in two specific questions: i) What are the real tasks of teacher/tutors and SWA coordinators? ii) How do SWA tutors and coordinators support school-work partnerships?i In this specific study, a qualitative approach was adopted whose pe- culiar aspects were: i) the ability to understand phenomena, through the frameworks by which people experience and interpret reality (Corbin, Strauss 2008); ii) the ability of the researcher to follow a flex- ible research design (Creswell 2013; Marshall, Rossman 2011); iii) the opportunity to look at the setting and the people as a whole, with- out reducing them to a mere set of variables (Tracy 2013; Yin 2011). 155 SCHOOL-WORK ALTERNANCE PROGRAMMES The survey technique used was the semi-structured interview thanks to its low level of directivity, due both to the presence of a draft on the topics to be treated and without a precise order, thus, only with the indication of a ‘perimeter’ to move within, and the role of the researcher in conducting the interview. This allowed the researcher to decide which topics to deal with, what sequence to fol- low, how to formulate the questions, but also to take a closer look at any issues emerging during the interview. In turn, interviewees were left free to express their opinions and ideas, indirectly influencing the communication flow and direction of the interview. In this sense, through the semi-structured interview, the interviewer and the in- terviewee had similar roles (Sala 2010). The interview addressed to the key SWA actors included seven dimensions (teachers’ functions, relationship with partners, students’ learning design, SWA programme implementation, learning, assess- ment, ideas for SWA improvement), each of them dealt with in ques- tions, with the aim of gathering the necessary information on the various aspects relating to the research goal, but without giving up the flexibility and openness required by the type of interview and the characteristics of conversation. 3.3 Data analysis The interviews were carried out between May and December 2015 at the various educational institutions involved, where the teachers worked, and after approval of their school principals. After receiv- ing the participants’ authorization, each one-and-half-hour inter- view was recorded. Subsequently, all interviews were transferred to an electronic format to allow the researcher a textual analysis using Atlas.ti.07 software. The analysis procedure mainly followed the top- down principle, a deductive approach where the content analysis was based on theoretical reference categories, but neither the bottom-up 156 CONCETTA TINO or inductive approach were excluded when emerging and useful cat- egories had been identified to better understand the object of study. gi j y The analyses conducted through Atlas.ti.07 generated 14 Primary Documents (PD) and 549 codes, and included: a) transcription of the interviews in digital format (PD); b) preparation of the Hermeneutic Unit (HU) as a setting for pre-codes and codes for groups-families and networks; c) identification of the quotations with the definition of labels by function: add coding and open coding; d) identification of the code families around which to group those codes that could bet- ter describe a macro-area or a core category (Tarozzi 2008); e) graphic representation of the most significant family networks. This last pro- cess proved useful as a summary map of the relevant categories iden- tified or emerged. 3.4 Findings Analysis of the interviews, carried out as described in the previous paragraph, produced significant findings on the object of study. The findings have been presented here as cross-themes on the two specific questions the general research question was split into.ii q g q p As regards the first specific question, «What are the real tasks of teacher/tutors and SWA coordinators?» five cross-themes emerged: i) the concrete development of the school-work partnership: SWA tutors and coordinators are the only teachers who constantly interface with representatives of production contexts. The possibility of main- taining or strengthening strategic and long-lasting partnerships with external contexts depends on their ability to emphasize the importance of the SWA projects, and to combine the needs of schools with those of external organizational contexts, develop- ing reliable relationships in the name of dialogue, transparency, and mutual respect; ii) the appropriate student-production context matching: in fact, this was considered a key factor by the participants; the effectiveness of the entire SWA experience had by the students depended on this. In order to avoid possible mistakes, it is necessary for SWA tutors and coordinators to know both the interests and potentiality of the students and the work contexts, in terms of professional and relationship practices;i p p iii) the organization of students’ SWA experiences; if as a first step, the SWA coordinators have the task of identifying a teacher/tutor for each class or group of students, providing them with the characteristics of external organizations that host students, in a second step, the teacher/tutors, after identifying the students’ interests, analysing and excluding logistical problems, have the task of contacting the 157 SCHOOL-WORK ALTERNANCE PROGRAMMES working contexts where they can place the students, negotiating the period and, if possible, the activities the students should do; iv) preparation of the documents that must support and provide proof of the stu- dent’s experience: there are many school contexts where the SWA tutors and coordinators are involved in preparing the necessary doc- uments: students’ training design, assessment and monitoring tools, and observation grids. 3.4 Findings These are boundary objects or mediating artefacts, which should provide proof of the process of sharing and dialogue between systems, the synthesis of different perspectives and not the fruit of activities carried out by individuals or at most by very few people without any form of internal or external sharing; v) the monitoring and assessment of experiences: tutors and coordinators are also in charge of this complex task. They monitor the expe- rience of every single student under their care, through periodic visits to the host organizations and informal conversations with workplace tutors, by collecting and analysing student logs, when the school requests them; by reading and assessing the student’s final SWA report; by preparing an assessment report of students’ SWA experience in order to present it to the class committee. This is an organism which, in most cases, can only take note of the assessment process, without playing an active role, because of its minimal participation within the whole SWA process. v) the monitoring and assessment of experiences: tutors and coordinators are also in charge of this complex task. They monitor the expe- rience of every single student under their care, through periodic visits to the host organizations and informal conversations with workplace tutors, by collecting and analysing student logs, when the school requests them; by reading and assessing the student’s final SWA report; by preparing an assessment report of students’ SWA experience in order to present it to the class committee. This is an organism which, in most cases, can only take note of the assessment process, without playing an active role, because of its minimal participation within the whole SWA process. The themes relating to the second specific question «How do SWA tutors and coordinators support school-work partnerships?» are two: i) the internal and external communication that SWA tutors and coordinators use as ‘glue’ between the two contexts. 4. Final remarks The findings showed how the relationship between school/work sys- tems requires recognition of both the boundaries between organizations and the activity of boundary crossers, whose role is to create links and bridges between different organizational systems (Aldrich, Herker 1977; Andersson, Andersson 2008), the willingness to inhabit that third space (Frenkel 2008; Gutierrez et al. 1995) creatively generating a synthesis of distinct cultures and perspectives. The core elements that emerged showed that SWA tutors and coordinators act as an important cross- system function between systems. In fact, they are those who, together with the students, carry the image of the school they belong to out- side; they connect students with local production contexts, and create links between school life and working contexts, attempting to reduce the distance between them, seeking to link content and methods to the needs of the outside environment. They are those teachers who have a clear awareness of the value of the experience and therefore declare themselves ready to sacrifice time and resources to accomplish it. They are the only teachers who are committed to navigating internal and external boundaries, involved in the communicative flows of negotia- tion and dialogue, influencing the effectiveness of internal and external partnerships, and student SWA experiences. They are the only teach- ers who work concretely and consistently in the construction of new crossing artefacts as evidence of a possible boundary-crossing process.i The findings highlighted another element on which the educa- tional and work contexts are presumed to reflect: only tutors and co- ordinators are the ‘protagonist-managers’ of an SWA system; in fact, they are supporters and promoters of partnerships, organizers and re- sponsible for students’ experiences, managers of internal and external communication flows, builders of boundary objects, and responsible for SWA experience assessments. All of this means that the two systems are still trapped within their boundaries, struggling to creatively live a third common space, as a common place of innovation, transfor- mation, and mutual learning, as a possibility for change that requires new relationships and new cultures (Akkermann, Bruining 2016). This scenario has some important implications in practice: SWA is an educational methodology that became mandatory for all study paths and for all students with the introduction of the Italian Law no. 3.4 Findings Participants stated how, within the institution they belong to, communication is useful not only to promote the sharing process, to motivate, and involve other col- leagues in taking part first in the building of students’ learning de- sign, then in the experience itself, but also as a tool for translating and filtering information; while, on the outside, communication becomes the lubricant of the partnership, since it generates trust and good relations between the parties, and facilitates the sharing of various perspectives; ff ii) the effort to combine different perspectives; it is precisely this theme that highlights the role of SWA tutors and coordinators as bound- ary crossers between systems. Participants showed a willingness to live within the boundary zone (Konkola, Tuomi-Gröhn, Lambert, Ludvigsen 2007), considered as a space free from predetermined rules and activities, and where a real dialogization process between school and work (Gutierrez et al. 1995: 446) could produce a syn- thesis of cultures and differences, generating effective learning ex- periences for the students and systems involved. 158 CONCETTA TINO 4. Final remarks 4. Final remarks 107 of 2015, so that tutors and coordinators, who have been SWA path managers and policy makers so far are required to reflect and gain awareness of the presence of boundary crossing processes in SWA pathways. It demands the full involvement of parties rather than in- dividuals. At the same time, it cannot be denied that tutors and co- ordinators play a key role within this process and that they should SCHOOL-WORK ALTERNANCE PROGRAMMES 159 be formally recognized a new professionalism that can be identified as the figure of an educational middle-management system (Fabbri, Melacarne, Allodola 2015). Additionally, the complexity of the func- tions given to them implies the recognition of the role of boundary crossers, and hence the need to think about appropriate educational and training paths capable of supporting these new professionals in the processes of integrating the objectives of different organizations, in the crossing of different boundaries that require them to know how to enact a dual professional identity (Richter, West, Van Dick, Dawson 2006) and how to enact their boundary crosser leadership in order to promote the overall involvement of systems. 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Email: donatella.cesareni@uniroma1. it; Ilaria Bortolotti, PhD Student, Department of Social and Developmental Psychology, Sapienza University of Rome, Italy. Email: ilaria.bortolotti@uniroma1.it. SCHOOL-WORK ALTERNANCE PROGRAMMES SCHOOL-WORK ALTERNANCE PROGRAMMES 161 Wenger E. 1998, Communities of practice: Learning, meaning, and identity, Cambridge University Press, Cambridge. Wenger E. 1998, Communities of practice: Learning, meaning, and identity, Cambridge University Press, Cambridge. Wenger E. 1998, Communities of practice: Learning, meaning, and identity, Cambridge University Press, Cambridge. Yin R.K. 2011, Applications of case study research, Sage, Thousands-Oaks. PROMOTING 21ST CENTURY SKILLS IN HIGHER EDUCATION THROUGH COLLABORATION AND ACTIVITIES Nadia Sansone (Sapienza University of Rome), Donatella Cesareni (Sapienza University of Rome), Ilaria Bortolotti (Sapienza University of Rome)* Nadia Sansone (Sapienza University of Rome), Donatella Cesareni (Sapienza University of Rome), Ilaria Bortolotti (Sapienza University of Rome)* abstract: The paper presents a model of university teaching where students are involved in concrete activities and collaborate while developing key compe- tences for the academic and working future. 52 students of the Degree Course in Psychology at the Sapienza University of Rome participated to the activity. The method of data collection and analysis combines qualitative and quantitative ap- proach. Results show a general perception of skills enhancement and a good ap- preciation of the course, especially in comparison with traditional learning modes. Keywords: skills, collaborative learning, project-based learning, technologies. 1. Theoretical introduction One of the main goals of secondary and higher education is to en- sure that students acquire useful skills to achieve success not only in their studies, but also in their future career and in daily life. A comprehensive list of 21st century skills has been provided by Binkley et al. (2012) who identified ten skills grouped into four clusters: ways of thinking (e.g., creativity and innovation, critical thinking, problem-solving, decision- making, learning to learn, and metacognition); ways of working (e.g., communication and collaboration – teamwork); tools for working (e.g., information literacy, ICT literacy), and living in the world (e.g., citizen- ship; life and career; personal & social responsibility). By mastering such skills, tomorrow’s citizens should be able to solve complex problems, in- novate work knowledge practices, create and support professional net- works, and cope with multiculturality and continuous change. To enhance meaningful and long-lasting learning that can promote knowledge acquisition as well as crucial skills development, more recent literature (Pozzi et al. 2012; Schellens et al. 2006) asks for educational ap- proaches in which to: fruitfully integrate individual and group activities, structure and guide focused learning discussions, think about specific tasks NADIA SANSONE, DONATELLA CESARENI, ILARIA BORTOLOTTI 164 around concrete activities, and fi nally, provide theoretically-based techno- logical mediation. Based on these concepts, the authors are presenting their proposal on how to innovate higher education accordingly, by designing a type of course in which students are involved in concrete activities, and collabo- rate to create knowledge and build signifi cant artefacts while developing key competences for their academic and working lives. y p g The course is based on the Trialogical Approach to Learning (TLA, Paavola, Engeström, Hakkarainen 2010), so-called because it integrates ‘monological’ and ‘dialogical’ approaches to learning with a third element: intentional processes involved in the collaborative creation of knowledge artefacts shared by and useful for the community. The acquisition and participation metaphors of learning (Sfard 1998) are, in this approach, em- bedded in the knowledge creation metaphor, which, by going beyond the many traditional dichotomies (Paavola, Lipponen, Hakkarainen 2004), focuses on the individual and social processes, conceptual knowledge and social practices needed to foster collaborative creativity (Fig. 1). The trial- ogical approach demonstrates its strong links with the Cultural Historical Activity Theory (CHAT) (Engeström 1987). PROMOTING XXI CENTURY SKILLS IN HIGHER EDUCATION 165 Table 1 – The six design principles. Design Principle Definition DP1 Organizing activities around shared ‘objects’ Formative action must converge on the realization of shared objects recognized as important and in- tended for actual use, beyond individual and social dimensions of learning. Shared objects are also rep- resented by shared practices and processes. DP2 Supporting interaction between personal and social levels It is necessary to combine individual work with that in a team, considering the diverse needs and “exploiting” inclinations and interests. DP3 Fostering long-term processes of knowledge advancement This principle emphasizes the importance of pro- viding enough time for iterative inquiry cycles and of supporting environments to let long-term pro- cesses take effect, including the creative re-use of previous practice and knowledge artefacts. DP4 Emphasizing development through transformation and reflec- tion between various forms of knowl- edge and practices Innovative ideas and practices can emerge more easily when learning involves various forms of knowledge and practices: declarative, procedural as well as tacit. DP5 Cross-fertilization of various knowledge practices across commu- nities and institutions Creating connections within other contexts inten- tionally promotes the acquisition of modes of in- teraction, ways of thinking and languages typical of contexts other than those of training. DP6 Providing f lexible tool mediation In line with 21st century society, the last principle affirms the importance of providing adequate and diversified technologies, selecting those most suit- ed to mediate collaborative activities and enhance aspects highlighted in the other design principles. Table 1 – The six design principles. Innovative ideas and practices can emerge more easily when learning involves various forms of knowledge and practices: declarative, procedural as well as tacit. Creating connections within other contexts inten- tionally promotes the acquisition of modes of in- teraction, ways of thinking and languages typical of contexts other than those of training. In line with 21st century society, the last principle affirms the importance of providing adequate and diversified technologies, selecting those most suit- ed to mediate collaborative activities and enhance aspects highlighted in the other design principles. The six principles synthesize the main pillars of the TLA: designing object-based learning activities in which both individual and collabora- tive work strategies are mobilized, creative processes are triggered, and educational technologies support each stage of the process. 1. Theoretical introduction Starting from Vygotskij’s thinking (1981), according to which our experience of the world is al- ways mediated by a tool, whether tangible or intangible, CHAT perceives knowledge as a collaborative construction mediated by cultural and social artefacts, and grounded in practical activities (Cole 1996). Figure 1 – The trialogical learning approach. [Sansone, Cesareni, Ligorio 2016] A trialogical approach is applied through six principles, the so-called ‘design principles’ (Tab. 1: Hakkarainen, Paavola 2009; Paavola, Hak- karainen 2014), which guide the planning of technology-based teaching and learning activities to facilitate shared eff orts of working with knowl- edge artefacts (Paavola, Lakkala, Muukkonen, Kosonen, Karlgren 2011). igure 1 – The trialogical learning approach. [Sansone, Cesareni, Ligorio 2016] Figure 1 – The trialogical learning approach. [Sansone, Cesareni, Ligorio 2016] A trialogical approach is applied through six principles, the so-called ‘design principles’ (Tab. 1: Hakkarainen, Paavola 2009; Paavola, Hak- karainen 2014), which guide the planning of technology-based teaching and learning activities to facilitate shared eff orts of working with knowl- edge artefacts (Paavola, Lakkala, Muukkonen, Kosonen, Karlgren 2011). A trialogical approach is applied through six principles, the so-called ‘design principles’ (Tab. 1: Hakkarainen, Paavola 2009; Paavola, Hak- karainen 2014), which guide the planning of technology-based teaching and learning activities to facilitate shared eff orts of working with knowl- edge artefacts (Paavola, Lakkala, Muukkonen, Kosonen, Karlgren 2011). A trialogical approach is applied through six principles, the so-called ‘design principles’ (Tab. 1: Hakkarainen, Paavola 2009; Paavola, Hak- karainen 2014), which guide the planning of technology-based teaching and learning activities to facilitate shared eff orts of working with knowl- edge artefacts (Paavola, Lakkala, Muukkonen, Kosonen, Karlgren 2011). PROMOTING XXI CENTURY SKILLS IN HIGHER EDUCATION In particular, trialogical activities are favoured by the use of environments and tools that allow participants to create and share, elaborate and transform, or- ganize different artefacts, making the related knowledge practices vis- ible, reflecting on them and evolving them. 2. Methodology DP2: Supporting in- tegration of personal and collective work DP1: Organizing ac- tivities around shared objects 2.1 Aims 2.1 Aims 2.1 Aims The study presented here aimed to observe students’ acquisition of knowledge and skills, and to gather useful information for improving learning design. Specifically, our research questions were: NADIA SANSONE, DONATELLA CESARENI, ILARIA BORTOLOTTI 166 1) Do students perceive an evolution in the level of skills they mastered at the beginning and at the end of the course? 2) How do students evaluate the course and its activities? 1) Do students perceive an evolution in the level of skills they mastered at the beginning and at the end of the course? g g 2) How do students evaluate the course and its activities? g g 2) How do students evaluate the course and its activities? 2.2 Participants 2.2 Participants Fifty-two 3rd year students (24 M, 28 F – avg age 21.8) from the Three- Year Degree Course in Psychology 2.3 Context 2.3 Context The course described here was on Experimental Pedagogy, and used blended learning by integrating face-to-face and online lessons hosted by the Sapienza Moodle (<http://elearning2.uniroma1.it>). The course lasted 10 weeks and was subdivided into three modules lasting approxi- mately three weeks each. Students were divided into six groups of about nine people each; the groups’ membership remained unchanged throughout the entire course. Students were divided into six groups of about nine people each; the groups’ membership remained unchanged throughout the entire course. The following table shows the learning design of the course, describ- g p p g g The following table shows the learning design of the course, describ- ing how the design principles were applied to the activities (Table 2). Table 2 – TLA principles in the course. Design principle Implementation in the case DP1: Organizing ac- tivities around shared objects The final object to be built by each group was a pedagogical sce- nario in which technology-mediated collaborative activities were to be designed. Learning topics and intermediate collaborative prod- ucts were the basis for the construction of the pedagogical scenario. In particular, the last artefact was a significant and useful ob- ject, connecting the group with the broader community. In fact, teachers from different Degree courses can all use these projects. DP2: Supporting in- tegration of personal and collective work Learning discussions were organized in which students had to bring their personal ideas as well as re-elaborations of scientific and pedagogical issues. Discussions and the following activities of product building were supported by allotting four specific roles: Social Tutor (task: promoting each group member’s par- ticipation), Synthesizer (task: writing a weekly summary of the group discussion), Sceptic (task: presenting alternative points of view to ‘warm’ the discussion), Head of the Collaborative Ar- tefact (task: arranging tools and materials for the collaborative classroom activity of building the artefact). Interaction between the groups was ensured by classroom pres- entations of the works and peer-review activities aimed at im- proving the group work. Reflection on students’ own participation and responsibility (agency) was solicited by compiling personal Learning Diaries, consisting of different stimuli to allow students to analyse their path more systematically and improve their own participation. Table 2 – TLA principles in the course. The final object to be built by each group was a pedagogical sce- nario in which technology-mediated collaborative activities were to be designed. D P4: F o s t e r i n g long-term process- es of knowledge advancement DP6: Providing flex- ible tools for devel- oping artefacts and practices DP5: Promot ing cross-fertilization of knowledge practices DP3: Emphasizing development and creativity PROMOTING XXI CENTURY SKILLS IN HIGHER EDUCATION 167 During the course, the students dealt with various kinds of knowl- edge, ranging from the theories studied and taught by the teach- er to concrete examples of didactic activities illustrated through films, charts, stories, and analyses of scientific articles, till the final phase of constructing their artefacts, which all of these dif- ferent formats had to become part of.l Personal and group reflection was repeatedly stressed in the group discussions around the teachers’ evaluations at the end of each module, also by compiling observation forms and reading these in groups, and finally, through the Learning Diaries. The learning activities lasted ten weeks and were organized in 3 different modules, thus including many moments of transition in which to reflect upon the previous phase. l p p p The course was designed so that theoretical knowledge on the teaching and learning themes was repeatedly taken up and re- flected on, in view of its practical application within the planned pedagogical scenario. p g g Moreover, the artefacts were reviewed and improved through ad- vice given: a) by the teacher in the plenary presentation, b) by the pairs through the peer feedback review – based on a specific tem- plate, c) by experts (primary, secondary and university teachers) during the final stage of constructing the pedagogical scenario. Students were able to experience the practices and methodologies of learning design in schools and higher education. Each group was supported and advised by an expert in the level of educa- tion chosen as the specific topic of their project. Thus, primary or secondary school teachers reviewed and provided feedback on their school-level projects, while a university physics pro- fessor provided feedback on a project to do with collaborative work between physics students, designing artefacts in that field. During the activities, several tools were used. Students interacted in small groups within Moodle courses. To perform the vari- ous activities, tools such as Padlet (brainstorming in the class- room), Google Drive Design Tool (to create shared conceptual maps), and Shared Google Documents (to collaboratively write the project) were used. To effectively apply the TLA prescriptions, each module had a spe- cific focus which the corresponding activities and products to be built came from, as shown below (Tab. 3). Table 3 – Discussions, activities, and artefacts in each module. 2.3 Context Learning topics and intermediate collaborative prod- ucts were the basis for the construction of the pedagogical scenario. In particular, the last artefact was a significant and useful ob- ject, connecting the group with the broader community. In fact, teachers from different Degree courses can all use these projects. Learning discussions were organized in which students had to bring their personal ideas as well as re-elaborations of scientific and pedagogical issues. Discussions and the following activities of product building were supported by allotting four specific roles: Social Tutor (task: promoting each group member’s par- ticipation), Synthesizer (task: writing a weekly summary of the group discussion), Sceptic (task: presenting alternative points of view to ‘warm’ the discussion), Head of the Collaborative Ar- tefact (task: arranging tools and materials for the collaborative classroom activity of building the artefact). y g ) Interaction between the groups was ensured by classroom pres- entations of the works and peer-review activities aimed at im- proving the group work. l p g g p Reflection on students’ own participation and responsibility (agency) was solicited by compiling personal Learning Diaries, consisting of different stimuli to allow students to analyse their path more systematically and improve their own participation. 1) First research question 1) First research question Forty-three anonymous pre-post questionnaires, namely, the Con- textual Knowledge Practices Questionnaire (Muukkonen et al. 2016). This was self-administered using Google Drive modules (<https://drive. google.com>) and consisted of: a) a pre-post section containing 21 items on perceptions relating to studies and abilities; b) a post-section consist- ing of 27 items examining the perception of any knowledge and skills acquired during the course and its activities. These items were organized in seven scales built around the TLA design principles. g p p Answers to the questionnaires were analysed using SPSS. 2) Second research question Th F G (FG ) PROMOTING XXI CENTURY SKILLS IN HIGHER EDUCATION Theoretical Contents Discussions/Activities Artefacts First Module Teaching/learning theories ‘The good teacher’: Discussion Conceptual map on ‘The good teacher’ Second Module School history, learning technologies Analysis of scientific articles Draft of the pedagogi- cal scenario stressing the theoretical principles Table 3 – Discussions, activities, and artefacts in each module. Table 3 – Discussions, activities, and artefacts in each module. 2.4 Tools and procedure The study presented here is based on the Design-Based Approach (DBA) (Brown, 1992; Collins, 1992; Design-Based Research Collective, 2003) which seeks to plan and implement innovative interventions, to then observe and analyse working elements as well as areas for improve- ment, and ultimately, re-design further interventions. The method of data collection and analysis combines qualitative and quantitative ap- proaches. Specifically, the following data were collected: 2) Second research question Three Focus Groups (FGs) were held at the end of the course. Stimuli aimed to detect students’ views on: their preferred activity (usefulness in terms of learning and skills); the role taken; pros and cons of group work; the assessment methods (ongoing, self-assessment, peer-observation); and the differences between the course just completed and traditional ones. To favour a critical and diversified discussion, the FGs consisted of stu- dents from each group. g p The transcripts were analysed using qualitative content analysis, aimed at defining categories to group the key concepts. 2.5 Results NADIA SANSONE, DONATELLA CESARENI, ILARIA BORTOLOTTI NADIA SANSONE, DONATELLA CESARENI, ILARIA BORTOLOTTI 168 Third Module Technologies and col- laborative learning Searching for best prac- tices on technology at school; school; collab- orative writing of the project Final pedagogical scenario 2.4 Tools and procedure 2.5 Results 1) Do students perceive an evolution in the level of skills they mas- tered at the beginning and end of the course? g g The results show a general perception of skills enhancement (Table 4), with statistically significant differences, both for the Work Compe- PROMOTING XXI CENTURY SKILLS IN HIGHER EDUCATION PROMOTING XXI CENTURY SKILLS IN HIGHER EDUCATION 169 tence scale (Wc), (t = –4.48 (42) p < .01), and for the Self-effi cacy scale (Se) (t = –2.955 (42) p < .01). At the end of the activity, the students felt that they had more collaborative work skills and felt greater confi dence in their skills. Table 4 – Diff erences in perception of incoming and outgoing skills. N = 43 (24F.; 19 M.) Entry Exit Work competence 3.60 3.95 Work engagement 3.53 3.53 Self effi cacy 3.86 4.03 Total 3.63 3.79 Table 4 – Diff erences in perception of incoming and outgoing skills. No signifi cant diff erences were found for the gender variable either in entry or in exit for Wc and Se. Instead, the Work Engagement scale, presented statistically signifi cant diff erences in exit (One-Way ANOVA f = 6.343 (42) p = .016). It seems that the course led the girls to feel more involved (M = 3.69) than the boys did (M = 3.23).i y Specifi cally, students believed that the course had allowed them to improve their skills in each of the areas investigated, the average score being above 3 in all seven scales (Fig. 2). In particular, they believed they had learnt to collaboratively build knowledge objects (scales 1 and 2, Average 4 and 3.91) and make these artefacts iteratively better (scale 4, 3.90), also thanks to the feedback received from their peers and the experts (scale 3, Average 4). Furthermore, they perceived that they had learnt new ways to use technologies (Average 3.88). However, they believed that they had only partially learned to collaborate and communicate in an interdisciplinary way (scale 6, Average 3.34). PROMOTING XXI CENTURY SKILLS IN HIGHER EDUCATION Figure 2 – Perception of skills development after the course (Likert scale 1-5) 3 3.2 I 3.4 3.6 3.8 4 4.2 1) Learn to collaborate on shared objects 2) Integrate personal and collaborative agency and work 3) Develop products through others’ feedback - - I I I I I I r - - 4) Continuous development knowledge objects 5) Understand different areas and practices of subject c t - 6) Communicate and collaborate in an interdisciplinary way 7) Learn how to exploit technology - I I 1 -- Figure 2 – Perception of skills development after the course (Likert scale 1-5) 170 NADIA SANSONE, DONATELLA CESARENI, ILARIA BORTOLOTTI As for possible gender differences, female students generally expressed a higher perception, but no significant differences were found in any items or scale. 2) How do students evaluate the course and its activities? Generally, students showed appreciation for the course, especially when comparing it to traditional ones. Compared to the latter, the course was recognized as exciting and engaging, capable of generating «inter- est in a pedagogical subject and a desire to go deeper into the topic» and made attractive by the idea of «working together towards a common goal», even if this was considered as requiring a greater commitment and effort «than having to just study from a book» because, on this course, «you have to attend the class and engage all through the course». y g g g In the following table (Tab. 5), we have highlighted the pros and cons the students saw in the different aspects of the course which they them- selves pointed out as most relevant. Table 5 – Pros and cons of individual aspects of the course from a student perspective. Pro Cons Group-work – a real innovation, not existing in other courses – a stimulus for individual partici- pation, as well as supporting real interdependence – issues relating to a non-homoge- neous participation by some group members Online discussions – the possibility to really get to know many points of view – engaging material and content (e.g. PROMOTING XXI CENTURY SKILLS IN HIGHER EDUCATION 171 pleasant of all my three years, because it was based on exchanging ideas, thinking all together; the opposite of the usual boring frontal lessons». Probably, the group work had this impact on the students’ perception since it worked well (as already mentioned, the designers paid great at- tention to integrating individual and collaborative agencies as stated in DP2): «For me it was reassuring that it was not a thing like, “as a group we decide what to do and then a single person does the job”, but that everyone had to and could participate». Among the activities of the course, however, some were considered more able to promote useful skills for future profession, such as the Learning Dia- ries, considered «useful also for the profession we will choose. Putting into writing and finding out what’s going on, what I feel, somehow helped me to recognize it right when it was happening and to adjust my behaviour». Weaknesses and limits were also pointed out by the students, starting from the online discussions of the first module (see Tab. 4): «At a certain point I said to my group-mates, “listen, please, could you stop doing thirty-line monologues? Could we start and have a discussion? Because in my opinion that’s what you have to do […] do you really think I’m going to go read ten posts of thirty lines that have nothing to do with each other? […]”. This complaint, however, ended with a possible solu- tion: “maybe you should present it a bit more like a debate”». In other cases, suggestions referred to more practical aspects, such as the timeframes: «In my opinion, the problem was that in the second mod- ule we discussed the added value technologies for too long (...) and then, suddenly, on the day, we were told to design a project … so […] I would redefine the timeframe, giving a whole third module to the drafting of the project […] or I would have put in a fourth module». 3. Final remarks PROMOTING XXI CENTURY SKILLS IN HIGHER EDUCATION articles and project) – the tendency of some peers to write lengthy notes on web forums, without having read others’ – lim- ited time dedicated to both the sec- ond discussion and the project Role Taking – the Synthesizer role promoting a higher focus on the discussion – the Tutor role connecting on- line and offline activities – the up-down role distribution not considering personal inclina- tions and availability – tutor’s stimuli seen as critical and therefore not implemented Evaluation and self-evaluation – ongoing evaluation seen as use- ful for self-monitoring – self-assessment considered as triggering good reflection – structure of the Learning Di- ary perceived as too binding or repetitive – also, its lack of anonymity as im- pacting authenticity Peer review – a possibility to improve, both by receiving and providing feedback – feedback given not always constructive Online environment – the chance to be connected at any time, even via smartphones – web forum not supporting proper discussions because of its structure Table 5 – Pros and cons of individual aspects of the course from a student perspective. In detail, we can see how group work was the real added value of the course, as claimed in the following excerpt: «This course was the most 3. Final remarks In this contribution we have described a university course based on the Trialogical Learning Approach and the study we performed to detect its impact on students’ appreciation of the learning activities and percep- tions relating to their skills development. Collaborative activities, continuous development of knowledge ob- jects, active use of technology, and the possibility to learn different dis- ciplines and practices, were recognized and appreciated by the students as key factors of the course, after which they perceived that they had re- inforced some skills crucial for their future career. According to the DBA method that inspired our work, and to fur- ther enhance the overall student experience, in future courses we will be paying attention to the critical areas reported by the students: partici- pation in work groups, certain tools and materials, and the timeframes. NADIA SANSONE, DONATELLA CESARENI, ILARIA BORTOLOTTI 172 Finally, we will focus on improving interdisciplinary collaboration and communication, perceived as the most underdeveloped skill. References References Binkley M., Erstad O., Herman J., Raizen S., Ripley M., Miller-Ricci M. and Rumble M. 2012, Defining twenty-first century skills. Assessment and teaching of 21st century skills, Springer, Berlin, 17-66. Brown A.L. 1992, Design experiments: Theoretical and methodological challenges in creating complex interventions in classroom settings, «The Journal of The Learning Sciences», II (2), 141-178. Cole M. 1996, Cultural psychology. A once and future discipline, Harvard University Press, Harvard. 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The concept of activity in Soviet psychology, Sharpe, Armonk. * Glenda Galeotti, PhD, Post-doc Researcher, Department of Education and Psychology (SCIFOPSI), University of Florence, Italy. Email: glenda.galeotti@unifi.it; Gilda Esposito, PhD, Post-doc Researcher in Adult Learning and Education, University of Florence, Italy. Email: gilda.esposito@unifi.it. Glenda Galeotti (University of Florence), Gilda Esposito (University of Florence)* Abstract: This paper presents a research on work-related learning through School-work Alternance in Secondary Education that involved researchers of University of Florence, ten secondary Schools, public and private entities in the Province of Arezzo and La Spezia. From the analysis of three case studies, it elic- its criteria for an educational model that integrates work-related learning with student voice perspective. Keywords: skill mismatch, learner-centred teaching, experiential learning, ca- reer guidance. 1. A challenging context: from the category of crisis to that of transition A European and Italian reasoned geography of crisis suggests that we risk answering to the many challenges originated from 2008 in- ternational crisis, and continuously changed since then, with ‘out- dated’ responses if we stick to the category of crisis, although quite developed and diversified in literature (Serres 2009; Bauman 2014). In fact, after almost ten years of profound transformation not only in the labour market and economics, but regarding overall citizens’ living styles, it might be time to shift to the transition approach (Hopkins 2012). The latter indicates that not only we are not going to leap back to the pre-2008 crisis for structural reasons, but, turned into positive terms, we face the opportunity to build something completely new, that is ecologically sound and sustainable both for human beings and the Planet Earth. Thanks to digital technology, we should work to- ward the construction of a resilient and empathic society (Rifkins 2010) where all citizens can contribute to new political and economic forms of societies that are more inclusive and fair. In such a trip from what is no longer and something which is still underway, what is the contribution that transformative education (Mezirow 1991) can offer in shaping citizens of the future, as individual and as communities, starting from the phase of their life when they prepare themselves to assume their roles in human society? 176 GLENDA GALEOTTI, GILDA ESPOSITO In this paper, we focus on how transformative education and ed- ucational models that integrate work-related learning and a student voice approach can contribute to build a diffused human and social capital that can lead transition toward new scenarios, in a framework of open networks and value chains going back and forth from the private to the public spheres of society. But where are we? How does the panorama appear to be today with such long-term vision? There are definitely plenty of clouds in the sky. Skill mismatch in national and European labour markets is one of them since it hinders young people to develop and thrive while creating stagnation in economies: it originates from insufficient correspondence between workers’ competences and the transform- ing demand of the labour market. Evidences show there is not an ac- ceptable correspondence between workers’ skills and the need of the labour market (ILO 2014). A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING The phenomenon of over-skilled is significantly increasing in some European countries (3.6% from 2002 to 2012), especially for young people and women. In Italy (ISFOL, PIAAC-OECD 2014) indicates the following data: • Under-skilled rate is 7.5%, while OECD average is 3.6% • Over-skilled rate is 12%, while the OECD average is 10% • Over-skilled rate is 12%, while the OECD average is 10% CEDEFOP (2015, 2010) records indicated that the phenomenon is still rising in Italy compared with previous surveys. In fact, hinder- ing EU and national growth, such phenomenon turned into a chal- lenge especially in Italy, where the percentage of under-qualified workers has become worryingly higher than European average. A wealth of explanations is available: workers’ poor digital and tech- nological literacy within a national framework of still fragile lifelong and work-based education and training together with new complex requirements in work organization, job design, career development that have not been met. Another compelling factor in the analysis is the impact of digital technologies on labour markets that has boosted part of the popula- tion, especially the most innovative niche, but at the same time dis- oriented the majority of workers, especially elder ones. According to the EU there may be a lack of up to 500 thousands ICT profession- als in 2020 (EU 2016). In fact, in an everyday more digital society developing a digital talent pool is crucial for the EU labour Market, both in terms of competitiveness but also for inclusion. According to the same study today, around 45% of Europeans have only basic digital skills. While all sectors of the economy are becoming digital 36% of the labour force has insufficient digital skills. Despite high unemployment rates, Europe also lacks digitally skilled persons to fill job vacancies. i j It is arguable then that EU challenge is not just to improve skill lev- els, but to match people with the right skills to the right jobs It is an European and national priority that requires policies to increase edu- cation and training responsiveness to labour market needs. Mitigating skill mismatch in an era of fast-paced digitalisation and automation re- quires a well-developed skills anticipation infrastructure in countries and an integrative approach to skills governance that should be sup- ported at the central level (CEDEFOP 2010). 1. A challenging context: from the category of crisis to that of transition European Bank indicates skill mismatch for stressed and non-stressed countries, calculated as the difference in skills between the labour force and employment. Focusing on the more recent evolution, skill mismatches remained at high levels in most euro area countries, especially in the stressed euro area countries, such as Spain, Greece, Ireland and Portugal, where increases already occurred in the first phase of the crisis. In other stressed countries– Italy, Cyprus and Slovenia – skill mismatch has recently significantly increased (ECB 2015). ( ) In such context workers are either:i • Over-skilled i.e. too qualified for available jobs. This leads to brain drain and youth migration toward more solid economics as is the case for Italy as demonstrated in the Fondazione Migrantes Re- port Italiani nel Mondo for 2016. p • Under-skilled i.e. not properly trained to insert into contempo- rary jobs, putting investors in the conditions to search for human resources abroad, often without a clear governance, or migrants and local unskilled workers compete in the same under-protected market, worsening societal disintegration. We can highlight another aspect: in a globalized rapidly changing competitive society cross-country mobility flows in the EU are still much lower than those recorded in other highly integrated econom- ic areas, notably the United States, and well below mobility within countries (EC 2015). Migrations and mobility have different social and symbolic weight in general public opinion: the first is connected to poverty and exclusion while the second to the fulfilment of life project, self-improvement and increased wealth. Nevertheless, they both represent ways to foster economies and inclusion, by matching supply and demand in an everyday more globalized labour market. A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING 177 A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING Among the best strategies that have been experimented to contain and progressively reduce such mismatch, available literature shows that strengthening relations among formal and no-formal education Agencies on one side and employers and the labour market on the other is a priority, in order to strengthen value chains from education, training and the labour market for future workers and prevent skill obsolescence or, even worse, their absence 178 GLENDA GALEOTTI, GILDA ESPOSITO from the market for those who are already employed (CEDEFOP 2015). Another field of experimentation is to overcome disciplinary borders in education and training and develop workers’ soft skills as strategic gear to enter and remain in the labour market, no matter how diverse are the features of each sector (CEDEFOP 2016). ( ) At the euro area level, skill mismatch could also be reduced by encouraging greater labour market mobility – both within national labour markets and across the euro area countries. There is a need to improve the governance of such processes that are indeed already in place and can deliver great benefits not only for autochthonous citi- zens, but also for migrants and asylum seekers, as stated in the EU Agenda for Migration (EU 2015). In Italy we have a great opportunity with the model of School Work Alternance that is contained in the Law 107/2015, known as ‘La buona scuola’. The law represents a contribution, and have great potential, to transform and improve transition paths from school to work in Italy.f y In Europe, different types of transitions from formal education to training and employment make a great difference in youth employ- ment rates as well as in gender equity (Shoon-Silbereisen 2009). In particular, the authors indicate four main approaches that match with as much welfare systems: • Continental/Central Europe with dual systems that interconnect education, vocational training and labour from early stage of life and put the labour system at the centre. • Scandinavian universalistic systems with a strong investment on inclusive education and training and open access for all, embed- ded in the idea of collective social responsiveness that should not leave anyone left behind. • Anglo-Saxon liberal regimes, that liberalize training opportuni- ties, do not protect youth from risks but bet on competitiveness of individual and market. • Lastly Mediterranean countries where there is a lack of investment in creating stronger links between vocational training, labour and innovation. A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING Vocational training is culturally seen as a second choice compared to University and there are strong unbalances of high quality courses in different territories. What is then the vision we can inspire from in facing the challeng- es? According to the New skills Agenda for Europe 2020 EU aims to: g g p • Improve the quality and relevance of skills formation • Make skills more visible and comparablef The Agenda also offers some suggestions on how to proceed that are taken into consideration in this paper, as: A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING 179 • improve skills intelligence and information for better career choices; i • making Vocational Education and Training (VET) a first choice by enhancing opportunities for VET learners to undertake a work based learning experience and promoting greater visibility of good labour market outcomes of VET; • a review of the Recommendation on Key Competences to help more people acquire the core set of skills necessary to work and live in the 21st century with a special focus on promoting entrepreneurial and innovation-oriented mind-sets and skills. • a review of the Recommendation on Key Competences to help more people acquire the core set of skills necessary to work and live in the 21st century with a special focus on promoting entrepreneurial and innovation-oriented mind-sets and skills. In our research, we put the accent on the reflection on how to go beyond not only discipline-based competence, but also on key com- petences (EU 2006) that have been partly made obsolete by social and economical change. We assume that investing in the develop- ment of soft skills as a strategic element to enter and remain in the labour market (CEDEFOP 2016). Soft skills in our model complement hard skills related to a par- ticular job or task and can be defined as a combination of transversal competences and attitudes that valuing already existing people’ capa- bilities improve their performance at work, while boosting their social and emotional intelligence (Goleman 1996). School Work Alternance should be based on a clear statement of educational goals to be attained, in terms of soft skills. Intended to be an innovative educational expe- rience that combines knowledge and know-how, School Work Alter- nance, and all stakeholders involved, should guide students in reckoning their aspirations while opening the guided and evaluated learning to the outside world. 2. Methodology When confronted with the poor attainment in school to work cooperation, seizing the opportunity of ‘La Buona Scuola’ (Italian Law 107/2015), we investigate on how to foster the development of soft skills through work-based learning, putting students at the cen- tre in their local communities. The research object is to identify cri- teria to design a pattern of work-related learning in a student voice perspective, through drawing from lesson-learned in three empiri- cal researches in Secondary Education that involved the University of Firenze and ten Secondary schools in the Province of Arezzo and La Spezia (Galeotti 2016; Esposito 2016). These were School Work Alternance projects realized through action-research and here pre- sented as three case studies. The method adopted in this qualitative research (Silverman 2002) is the case study, which is «a research strategy comprising an all-encom- passing method» (Yin 2009: 14; Yin 2011), rather than a real and proper methodology, able to deal with particular situations within their own functioning, characteristic complexity (Stake 1995). This is an analysis of phenomenon in the real context in which they occur and, as such, uses multiple sources of evidence, with an exploratory purpose and pre- liminary to future studies, also in a comparative perspective (Yin 2009). The stages of investigation developed as follows: g g p • Building the conceptual framework and identifying the variables of analysis • Building the conceptual framework and identifying the variables of analysis • Collection and analysis of documents, materials and products in the three Schools • Comparative analysis of the data collected in the three case studies. As mentioned, case studies refer to three school-work Alternance projects activated between 2015 and 2016 by schools in collaboration with the University of Florence and with a wealth of stakeholders of the territories involved. The first case study called ‘Heritage Lab’ is linked to the project Market of Cultural Heritage and the New Genera- tions, financed by the Tuscany Region and the Fondazione Cassa di Risparmio of Firenze. The aim of the project was to identify stra- tegic actions for the development of the cultural sector in terms of strengthening the skills of the various players involved, including those of young people engaged in vocational education and train- ing. A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING From the results of our research, we see at least three clear advan- tages in School Work Alternance: g • It starts to fill a gap of communication, mutual understanding and interrelation between school and the world of work. As mentioned above the Mediterranean transition model is still too characterized by a clear cut between the time to learn and the time to work that urgently needs to be overcome. • It has an inter-generational approach: in Schools the teacher are the only adults that students interact with, while in alternation they are exposed to decision makers, managers, professional, workers and a wealth of stakeholders. • It fosters local networks and alliances for change among actors who are not so used of keen to working together, especially since their organi- zational systems are so diverse. The firm work so distinctively from a secondary school: the power relations are different, are expectations in terms of commitment and results. However, after only two years of im- plementation, there are many open questions that still need a response. GLENDA GALEOTTI, GILDA ESPOSITO 180 2. Methodology Launched in 2011, the project was structured in a first round of research on the cultural sector in the Arezzo area in terms of system characteristics, service offerings, training demand (FCRF 2013) and a second phase (2014-2015) with a research-intervention with sev- eral local stakeholders (cultural sector companies, schools, museums, A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING 181 public agencies, training agencies, etc.). Research-Action on Work School Alternance was developed in the second phase of the project. p p p j Peoplefusion is a complex digital platform (<http://www.people- fusion.it>) that has been designed by the Municipality of La Spezia in collaboration with UNIFI and the firm INFOPORTO in order to facilitate the access of foreign residents to the network of social services available in the local community. Financed by Fondazione TIM in 2014, the project involved in the modality of school-work alternance around 75 young students of secondary school. Their role was not only to collect information on existing services but also to support professionals in making those information available and usable for different sectors of the population. The platform in fact proved useful not only for foreigners (it is translated into English, Spanish, Albanian, Arabic and Bangli) but also to native Italians. g ) Young Lab is part of the wider project of Observatory of Social Change realized by the Municipality of La Spezia with the Scientific support of UNIFI. Its aims are to promote a culture of data and evidence among the social workers of the Service network, through embedded learn- ing and action research. The Observatory yearly collects quantitative and qualitative data on the diverse aspects of social change elaborate them and provide decision makers with recommendations for poli- cy and practices. Young Lab was meant to be a social innovation in previous experience of Observatories: it elicits students’ perspectives and ideas on social issues, putting them in the condition of acting as ‘junior researchers’ accompanied both in training and in action by seniors. Forty-five students in fact were trained, designed and sub- mitted questionnaires, realized interviews and participated to focus groups in order to research the challenges of volunteer associations in facing new needs of the population. The Table 1 briefly summa- rizes the main features of each project. The analysis of the three case studies was based on a set of vari- ables derived from two concepts: ‘work-related learning’ and ‘student voice approach’. 2. Methodology The term ‘work-related learning’ describes a complex educational phenomenon that includes a series of educational missions, a set of ac- tivities, a collection of topics and a repertoire of teaching and learning styles (Huddleston, Stanley 2012). In literature there are also other definitions that refer to functional educational strategies for achieving career and career goals, which include times in workplaces, alternat- ing with more conventional learning instances within school insti- tutions (Coll et al. 2008); or formal education and training programs for job creation, formal and informal learning in the workplace, con- tinuing vocational training for professional development offered out- side the workplace (Dirkx 2011). While carrying a focus on work or GLENDA GALEOTTI, GILDA ESPOSITO 182 education, these definitions share a bottom line: integrating a practi- cal experience (such as work) with an educational experience (such as upper secondary education) that creates synergies and significant benefits for students and other stakeholders (Gardner, Bartkus 2014). Table 1 – The three projects of School Work Alternance, Schools, classes and students involved. [Source: Author’s own] Project Secondary schools Classes N. students involved Heritage Lab ISIS Buonarroti-Fossom- broni, Arezzo IIS “Città di Piero”, Sansepolcro IIS “Signorelli”, Cortona 3 Technical Institutes 1 Lyceum (Classic) Two class groups and two mixed groups com- ing from 4 classes 70 with a majority of female students People fusion Liceo Scientifico “Pacinotti” Istituto Tecnico Commer- ciale “Fossati-Da Passano” Istituto Tecnico “Capellini-Sauro” Istituto Professionale Al- berghiero “Casini” 1 Professional Institute 2 Technical Institutes 1 Lyceum (Scientific) They were organized in 5 mixed groups coming from 9 classes 75 with a substantial gender balance Young Lab Istituto Tecnico Commer- ciale “Fossati-Da Passano” Liceo Classico “Costa” Istituto Professionale “Einaudi-Chiodo” 1 Professional Institute 1 Technical Institute 1 Lyceum (Classic) They were organized in 2 mixed groups coming from 7 classes 45 students with a majority of female students Table 1 – The three projects of School Work Alternance, Schools, classes and students involved. [Source: Author’s own] The ‘student voice’ approach (Flechter 2017, 2014; Seal, Gibson, Haynes, Potter 2015; Grion, Manca, 2015; Grion, Dettori 2015; Cook-Sather, Grion 2013), now widely disseminated abroad but still little known in Italy, emphasizes the importance of collecting «any expression of any learner about anything related to education» (Fletch- er 2014: 2), or the perspective of students about educational activi- ties and school life in general. 45 students with a majority of female students 2. Methodology Students in fact can offer information and reflections to which teachers and others should listen to in rela- tion to their actions (Cook-Sather 2009). The recognition and legit- imacy of the contribution of the concerned stakeholders open up to the possibility of making students more involved and responsible in their school contexts while at the same time improving the quality of training (Cook-Sather 2002; Angus 2006). A literature review of ‘student voice’ perspective shows different ways to practice it, which were considered in the construction of criteria for case analysis. Spe- cifically, we identified four main areas: i yi • Learning process: when students plan educational activities and carry out student-led research, for increasing their investment, • Learning process: when students plan educational activities and carry out student-led research, for increasing their investment, A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING 183 ownership, and consequent learning (Flutter, Rudduck, 2004; Fletcher 2004; Bragg, Fielding 2003). gg g ) • School culture: involving students as partner to transform the at- titudes and systems that underlay the culture of organizations, schools and communities (Young, Sazama 2006; Rudduck 2007). gg g ) • School culture: involving students as partner to transform the at- titudes and systems that underlay the culture of organizations, schools and communities (Young, Sazama 2006; Rudduck 2007). • Diversity: engaging students to promote diversity of perspectives and the acceptance of cultural, racial, economic, and social diver- sity (Rubin, Silva 2003; Cushman 2003). y ( ) • Civic engagement: students as change agents can lead to the de- velopment of skills and abilities to be active citizenship and effec- tive members of their communities (Young, Sazama 2006; Hooks 2004; Freire 1987). As a qualitative research, data collection and analysis were carried out using the content analysis method (Semeraro 2011), applied to the empirical materials produced during the implementation of the three projects (interviews, analysis reports, self-report of the researcher, ac- tivity monitoring cards, student products, etc.), which describe prob- lematic moments and knowledge building at individual and group level (Denzin, Lincoln 2005). In order to reach a better and deeper understanding of the reality investigated (Coggi, Ricchiardi 2005), the analysis of the collected data was carried out using the interpretative approach (Trinchero 2004), which provided for the aggregation of evidence on the aspects that characterize the work-related learning, based on our reworking of Cooper, Orrell and Bowden proposal (2010). 2. Methodology The collected data was then elaborated, combining the work-related learning and stu- dent voice approaches, starting with the different declinations of the two concepts in the literature and the theoretical framework of the study to highlight the relationships between them. Subsequently, col- lected data were analysed in a comparative key to highlight common elements and differences in order to identify research criteria and de- fine an educational model that could combine work-related learning with the student voice approach in the alternance of school and work. 3. Research results Beyond the different specific objectives of the three projects (cul- tural heritage, access to services for foreigners, understanding social change and policy design) and the output of the involved teams, the didactic activities carried out within the job alternation became and indoor and/or outdoor labs, where students ‘as researchers’ were en- gaged in planning and implementing their ideas and proposals which GLENDA GALEOTTI, GILDA ESPOSITO 184 could only materialize through their full participation. The meth- odologies used in the workshops followed the logic and principles of problem-based learning (Barrows, Tamblyn 1980, Barrows, 1988; Woods, 1994) and project-based learning (Krajcik, Blumenfeld, 2006). These two approaches, in some respects superimposable, are both at- tributable to constructivism (Brandon & All, 2010; Fox, 2001) and in particular to located learning (Lave, 1988; Lave, Wenger, 1990; Brown, Collins, Duguid 1989), active knowledge building (Dewey 1959, 1973), social interactions (Vygotskij, 1980; Wertsch, 1985) and experiential learning (Kolb 1984; Kolb, Boyatzis, Mainemelis, 1999). Both are among the methods of learner-centered teaching (Weimer 2013), and move from a problem that constitutes the starting point of the learning process, which develops through the design and applica- tion of a resolution hypothesis of the same. What differentiates them is the greater or lesser attention to the design momentum, namely the search for effective and operational solutions that aim, whenever possible, to produce concrete applications or the production of prod- ucts consistent with the analysis carried out. y In the three study cases, training goals have been declined in terms of strengthening soft skills with reference to some of the ten areas of expertise deemed necessary by 2020 by the Institute for the Future of Palo Alto (2010). One aspect that has characterized all the experiences was active involvement in various forms of stakeholders in the territory, i.e. pub- lic and private actors other than University involved actively in the realization of laboratory activities. The Table 2 shows the different characteristics of the three educational proposals from their key ele- ments in a comparative way. Table 2 – Main results of comparative analysis of three case studies. [Source: author’s own] Heritage Lab People Fusion Young Lab Overall Objectives Innovate the relation- ship between higher education and labour market, with a focus on valuing local cultural heritage. Around 30 public and private service providers with hundreds of pro- fessionals involved Distretto Socio-Sanitar- io 18 of La Spezia; Vol- unteer Service Centre “Vivere Insieme” AN- TEAS; More than 50 Associations and Coop- eratives informed A 2016 profile of so- cial change that mainstreams student perspective, particular- ly in analysing the Civil Society role. 3. Research results To develop soft skills together with discipli- nary contents To capitalize students’ perspective in facili- tating foreign citizens’ access to the Local Ser- vice Network through a multi-lingual inter- active web platform. Students were asked to collect information, develop relations and create a user-friend- ly approach, valuing their experience and networks. To include youth’s per- spective and experien- tial living in the analysis of social challenges and needs within the frame- work of the Observatory of Social Change of Dis- tretto Socio-Sanitario 18 of La Spezia Table 2 – Main results of comparative analysis of three case studies. [Source: author’s own] A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING 185 A STUDENT VOICE APPROACH IN WORK-RELATED L Heritage Lab People Fusion Working/ Learning methodology project-based learn- ing and problem-based learning Involve-share common objectives among differ- ent stakeholders. – train-apply-reflect- solve problems. The strongest results developed OUTSIDE the School. – experiment meaning- ful school to work (and back) transitions Output Design of an E-com- merce website for local and traditional artisans A project on territorial marketing to improve youth tourism A project of cultural heritage valorisation to participate in Nation- al call of Ministry of Education Co-design of School Work Alternance pro- jects in cultural institu- tions and organizations A web-based platform connected to social me- dia whose contents have been collected and se- lected by students. Learning outcomes Strengthened soft skills: Social intelligence; sense-making, Novel And Adaptive Think- ing; Cognitive Load Management. Promoted the transition to Career Management Skills Strengthened soft skills and citizenship A more informed and active local community especially among youth De-construction of prejudice and preven- tion of hate speech based on ignorance Specific features In each activities phases submission of a self-as- sessment and evalua- tion questionnaire for reflecting on soft skills ‘stimulated’ during the activities Digital skills enhanced through coding Focus on ‘digital democracy’ Partners Local and regional en- trepreneurs in Cultural Heritage sector Around 30 public and private service providers with hundreds of pro- fessionals involved Heritage Lab People Fusion Young Lab Working/ Learning methodology project-based learn- ing and problem-based learning Involve-share common objectives among differ- ent stakeholders. – train-apply-reflect- solve problems. The strongest results developed OUTSIDE the School. – experiment meaning- ful school to work (and back) transitions Share objectives, value student own experi- ences, train in basic re- search methodology, ‘know where you live’, observe, collect and re- flect, cultivate informed citizenship Output Design of an E-com- merce website for local and traditional artisans A project on territorial marketing to improve youth tourism A project of cultural heritage valorisation to participate in Nation- al call of Ministry of Education Co-design of School Work Alternance pro- jects in cultural institu- tions and organizations A web-based platform connected to social me- dia whose contents have been collected and se- lected by students. A 2016 profile of so- cial change that mainstreams student perspective, particular- ly in analysing the Civil Society role. A web-based platform connected to social me- dia whose contents have been collected and se- lected by students. lls d ity uth - Strengthened soft skills, especially ‘get to know your environment’ and protagonism in promot- ing change. Understood the best use of participatory research and activation tools as world café, question- naires, interviews ed Students see themselves as ‘junior researchers’ Intergenerational edu- cation and learning Strengthened soft skills and citizenship A more informed and active local community especially among youth De-construction of prejudice and preven- tion of hate speech based on ignorance Strengthened soft skills, especially ‘get to know your environment’ and protagonism in promot- ing change. Understood the best use of participatory research and activation tools as world café, question- naires, interviews Digital skills enhanced through coding Focus on ‘digital democracy’ Students see themselves as ‘junior researchers’ Intergenerational edu- cation and learning Strengthened soft skills and citizenship A more informed and active local community especially among youth De-construction of prejudice and preven- tion of hate speech based on ignorance A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING Valuing youth perspec- tive on main contem- porary social issues, in particular active citi- zenship and solidarity and intergenerational dialogue Learning outcomes Strengthened soft skills: Social intelligence; sense-making, Novel And Adaptive Think- ing; Cognitive Load Management. Promoted the transition to Career Management Skills Strengthened soft skills and citizenship A more informed and active local community especially among youth De-construction of prejudice and preven- tion of hate speech based on ignorance Strengthened soft skills, especially ‘get to know your environment’ and protagonism in promot- ing change. Understood the best use of participatory research and activation tools as world café, question- naires, interviews Specific features In each activities phases submission of a self-as- sessment and evalua- tion questionnaire for reflecting on soft skills ‘stimulated’ during the activities Digital skills enhanced through coding Focus on ‘digital democracy’ Students see themselves as ‘junior researchers’ Intergenerational edu- cation and learning Partners Local and regional en- trepreneurs in Cultural Heritage sector Around 30 public and private service providers with hundreds of pro- fessionals involved Distretto Socio-Sanitar- io 18 of La Spezia; Vol- unteer Service Centre “Vivere Insieme” AN- TEAS M th 50 A 2016 profile of so- cial change that mainstreams student perspective, particular- ly in analysing the Civil Society role. Valuing youth perspec- tive on main contem- porary social issues, in particular active citi- zenship and solidarity and intergenerational dialogue 186 GLENDA GALEOTTI, GILDA ESPOSITO By analysing how the concepts of work-related learning and stu- dent voice have been declined in the three study cases, it is important to note that while Heritage Lab has been included in the curricular activity and has been a pre-requisite for students to enter the select- ed company, People Fusion Labs and Young Lab laboratories have developed first within the organization hosting the alternation, i.e. the Municipality of La Spezia, supported by UNIFI. Going back the work-related classification previously illustrated, Heritage Lab is a Learn for work (QCA 2003) experience that engages and inte- grates with the curriculum of studies in order to develop skills in a prospect of employability. People Fusion and Young Lab are ex- periences of Learn through work typical of the alternative training device. Considering the student voice perspective, all three cases have adopted it in the planning of educational activities and in civil engagement even if in different ways (i.e. A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING Heritage Lab has focused on the export of cultural heritage for social development – the eco- nomics of their communities, while People fusion and Young Lab deepened the aspects of social inclusion and the new social needs of communities. The Table 3 below shows the fertile convergence the two approaches. Table 3 – Connection between work-related activities carried out by the three project with student voice prospective. [Source: author’s own] Work-related Student voice Learn for work Learn through work Learn about work SV for learning. Heritage Lab People Fusion; Young Lab SV for school culture Heritage Lab SV for diversity People Fusion SV for civic engagement Heritage Lab People Fusion; Young Lab Table 3 – Connection between work-related activities carried out by the three project with student voice prospective. [Source: author’s own] If we consider the classification of the range of student-voice-ori- ented activities proposed by Toshalis and Nakkula (2012), the tree case studies are between ‘activism’ and ‘leadership’. While the ‘ac- tivism’ typology includes identifying problems, generating solutions, organizing responses, agitating and/or educating for change both in and outside of school contexts, leadership typology previews co- planning, making decisions and accepting significant responsibility for outcomes, co-guiding group processes, co-conducting activities youth are understood as leaders. Programs that prepare students to A STUDENT VOICE APPROACH IN WORK-RELATED LEARNING 187 lead tend to view youth as problem solvers, with the skills and in- sights communities require in order to move forward (Mitra 2009; Osberg, Pope, Galloway 2006). Angus L. 2006, Educational leadership and the imperative of including student voices, student interests, and students’ lives in the mainstream, «International Journal of Leadership in Education», IX (4), 369-379. Bahou L. 2011, Rethinking The Challenges and Possibilities of Student Voice and Agency, «Educate Journal», 1, 2-14. • Develop expert assessment/evaluation tools for traineeship ‘as learning experiences’ and of the soft skills acquired • Develop expert assessment/evaluation tools for traineeship ‘as learning experiences’ and of the soft skills acquired The educational model proposed integrates learner-centered teach- ing, ‘learn through work’ and ‘learn for work’ by evaluating students’ voices in negotiating curricula, in order to develop skills in a perspective of employability. Overall, the model aims at shaping new relationships between School, Enterprises and the Labor Market while integrating knowledge and disciplinary skills with soft skills. It can contribute to: • Design school-work Alternance with the direct involvement of stu- dents and the local community (Fletcher 2014; Seale et al. 2015).f • Develop training pathways that offer students the opportunity to explore employment opportunities and career development (Subra- maniam, Freudenberg 2007), within the framework of Cultural and Social Economy. • Give more visibility to the development of soft skills in curricula be- yond disciplinary skills (Freudenberg, Brimble, Cameron 2011).f y p y ( g ) • Connect between theory and practice, with positive learning effects. • Foster motivation through valuing incoming experiences, knowledge and aspirations for further learning. There is ample room for further research on potentialities and scaling up opportunities of the same model in higher Tertiary Edu- cation, especially vocational training. Some have already emerged: • Promote and disseminate a culture of mutual understanding be- tween Universities, Schools and other key stakeholders of the local community. y • Experiment entrepreneurship and innovation labs for the develop- ment of new profiles, product and services of the Transition Society. y • Experiment entrepreneurship and innovation labs for the develop- ment of new profiles, product and services of the Transition Society. Finally, for the Department of Education and Psychology, the mod- el can be exploited to train future teachers, educators, professionals, entrepreneurs and other facilitators on the methods and potentiali- ties of work-related approaches, through models genuinely based on experimentation and evidence. References Angus L. 2006, Educational leadership and the imperative of including student voices, student interests, and students’ lives in the mainstream, «International Journal of Leadership in Education», IX (4), 369-379. 4. Final remarks and lessons learnt To conclude, we would like to focus on lessons learnt and sugges- tions for implementing work-related activities with a student voice approach, even scaling up to higher education. Integrating and fos- tering synergies between formal and non-formal agencies and prac- tice results in significant advantages not only for students but also for the rest of the actors involved.i By pursuing a measurable impact of developed and diversified skills for employability, a contemporary learner-centered perspective emerges: students learn through work while teachers, trainers and ex- perts enrich content and didactic through action learning; they are all engaged to build employability paths.i g g p y y p Resuming the definition of work-related learning proposed by Huddleston and Stanley, the presented experiences can be described as the result of the integration between: • an educational mission directed at the development of professional pathways starting from the valuing of knowledge and disciplinary skills held by students and the resources of a specific territory;l • a set of activities characterized by the integration of active and reflec- tive methods directed at strengthening soft skills;i • a collection of topics that revolve around specific content but which by their nature have an interdisciplinary character and are therefore adaptable to different study paths and economic sectors; • a repertoire of learning and learning styles that develop through the connection of problem-based learning approach to project-based learning, both of which are related to learner-centered teaching methodologies. Based on the findings of case study analysis, the main criteria for implementing a work-related learning experience in a student voice perspective are: p p • Co-design paths of school work alternance in a participatory way, involving students, teachers and local stakeholders g • Stimulate collaborative research networks between social, edu- cational, economic and cultural organizations and stakeholders within the private and public sector p p • Consolidate and build students’ soft skills starting from valuing from their previous knowledge and disciplinary skills. 188 GLENDA GALEOTTI, GILDA ESPOSITO • Develop expert assessment/evaluation tools for traineeship ‘as learning experiences’ and of the soft skills acquired • Develop expert assessment/evaluation tools for traineeship ‘as learning experiences’ and of the soft skills acquired p Bahou L. 2011, Rethinking The Challenges and Possibilities of Student Voice and Agency, «Educate Journal», 1, 2-14. 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Daniela Maccario (University of Turin)** Abstract: This article presents the path and the results of research aimed at de- veloping innovative teaching approaches in the context of academic courses for in-service teachers as regards their professional training. Keywords: teacher education, professional training, didactic models. * This essay returns to the article: Maccario D. 2017, Professionalization of teachers and problematization processes, «Revista Internacional del Forlação de professores (RIPF)», II (2), 140-158. ** Daniela Maccario, PhD, Associate Professor of General Didactics, Department of Philosophy and Education Sciences Department, University of Turin, Italy. Email: daniela.maccario@unito.it. PROFESSIONALIZATION OF TEACHERS AND PROBLEMATIZATION PROCESSES* Daniela Maccario (University of Turin) Daniela Maccario, PhD, Associate Professor of General Didactics, Department of Philosophy and Education Sciences Department, University of Turin, Italy. Email: daniela.maccario@unito.it. * This essay returns to the article: Maccario D. 2017, Professionalization of teachers and problematization processes, «Revista Internacional del Forlação de professores (RIPF)», II (2), 140-158. 1. Introduction This article presents the lines and findings of a research study whose aim is the fine-tuning of an innovative, didactic training model with- in a 1st level professional Master’s degree programme in Didactics and Educational Psychology for Specific Learning Disorders introduced by the Department of Philosophy and Education Sciences at the University of Turin, in collaboration with the MIUR (Ministero dell’Università e Ricerca Scientifica – Ministry for Higher Education and Scientific Re- search) - USR (Ufficio Scolastico Regionale – The Regional Schools Of- fice) of Piedmont (A/Y 2011-12; 2012-13), addressed to teachers of any type and level of school. The author, in her capacity as director of the Master’s degree programme, assumes responsibility for the didac- tic planning of the Course, seen as a crossroads between the practical and professional knowledge areas of teachers, and the scientific and subject-specific knowledge areas offered by the formal-type teach- ing provided by the Master’s degree, a potentially strategic learning lever in professionalization management. In relation to restrictions of a general nature, University-related, and available resources) the research study lines began from the problem: how do we respond, via education and vocational training of a university nature – with specific refer- ence to traineeship activities – to the needs of the professional development of teachers in service in relation to action planning and management, contexts and didactic and educational actions such as to promote integration of all students, with specific focus on those with SLD? In this context, which areas of profes- 194 DANIELA MACCARIO sional action are most critical from the standpoint of teachers in schools and need to be monitored in the planning of university professional in-service training? PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES 195 carry out their job, professional teachers would need to be able to tap into an integrated multiplicity of references in a rational way, both of a theoretical and general nature and derived from experi- ence, to be able to contextualize, thanks to a personal effort of in- terpretation, the issues to be dealt with and the possible strategies for solutions, in a kind of dialogue with the situation which passes via the action – ‘reflection-in-action’ – and entails recognizing, re- viewing, and developing one’s own theoretical, conceptual and op- erational methods. Research contributions of an anthropological origin, based on analysing the forms of knowledge that teachers ac- tivate in doing their job, make the learning potential of theoretical and scientific knowledge areas ‘for teaching’ particularly problem- atic, since they are conditioned by the possibility of linking them to tangible, operational situations and detailed tasks (Tardif, Lessard 2004). A generic reference to experience is not being called into question but rather the setting up of training mechanisms which can enhance the job as an authentic ‘mediator’ in the building of pro- fessional knowledge. Other indications in favour of professional training, experience-based approaches, can be recognised in the theory of adult learning from a ‘transformative’ perspective, in the theoretical reasoning supporting competency-based approaches in professional training and in the research branch attributed to pro- fessional didactics of French-speaking origin. Amongst the meth- odological and operational implications of the transformative learning theory to be taken into consideration in training pro- grammes, one criterion recommended in the offering of additional content/knowledge concerns the active building of relations between new cognitive factors and previous knowledge areas, so as to pro- mote the evolution of personal, interpretative frameworks (Mezirow 2003). Elements which are also essentially aligned with implications for factors concerning curriculum planning in the development of training sequences, originate from the generation of professional training, competency-based theories. 2. Theoretical framework 2. Theoretical framework From the standpoint of primary theoretical and methodological assumptions, one of the main tasks of research in the pedagogic and didactic field pivots arounds the processing of knowledge support- ing the professionalization of teachers and educators in managing the definition of models, mechanisms, and training-based profes- sional practices in line with the complex, fixed and dynamic nature of didactic and educational actions (Van der Maren 2014; Furlong, Oancea 2008). Taking this context of a general nature to define a didactic case model structuring the traineeship path of the Master’s degree programme, exploration was undertaken of academic works that gathered elements to answer the question: given the resources and actual restrictions, in relation to the identified training objectives, what op- erational criteria need to be adopted in the development of a traineeship path directed towards supporting the professional development of teachers? Several aspects which need to be monitored came to light. These points will be briefly illustrated. Within the debate on the applicability of the con- cepts of ‘profession’ or ‘professional development’ to a teacher’s job, in the most recent academic papers, the position is commonly ad- opted attributing to teaching the nature of a professional activity, albeit with distinctive characteristics (Damiano 2004). Generally speaking, it is held that teachers may be considered professionals in that their job essentially consists of the creation of non-routine in- tellectual actions with a view to pursuing objectives in the complex situations in which they operate, with a significant degree of auton- omy and responsibility, starting with a personal and study back- ground built on multiple ‘resources’ and forms of theoretical and scientific and practical and experience-based forms of knowledge (also involving the ethical and value-related sphere) (Paquay, Altet, Charlier, Perrenoud 2006; Perrenoud 1999a). From this perspective, theoretical knowledge in the education area – ‘for teaching’ – may be resources to develop professional ‘action potential’, if acquired in a spendable form, in response to teachers’ need for professional train- ing. Reference to the ‘Reflective Practitioner’ paradigm (Schön 1983) provides the foundation for proposing a criterion of didactic transposition of ‘educating and teaching’ knowledge areas as theo- retical and conceptual frames helpful to teachers for carrying out and analysing their (own and that of others) didactic and educational practices and the assumptions underpinning them (Altet 2010). To PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES In this case, also starting by recovering experience-based learning theories, we can trace ‘spiral- shaped’ development paths, coming from lived experience (action, creation of an activity, etc.), followed by a first phase of performance- reflective practice, directed towards rebuilding events, reinterpret- ing them and transforming then via narration to render them intelligible; followed by conceptualization and modelling, via de- contextualization, which aims at identifying more general and stable strategies and models, to be reinvested at a later stage, on returning to practice in other more or less similar contexts (Le Boterf 2000). P f i l Did ti h th ti d th i f th l ti b 196 DANIELA MACCARIO sion between ‘cognitive models’ and ‘operational models’ (Pastré 2007). Professional learning would primarily consist of the acquisi- tion of pragmatic concepts or action organisers – ‘operational mod- els’, learned directly in context and referring to a class of professional situations within the ambit of a practice community. Alongside con- cepts that reference a pragmatic model, other conceptualization methods or ‘cognitive models’ come into play which concern the characteristics of the action ‘beyond’ the transformative tension felt immediately while the action is in progress. In professional learn- ing, the operational models learned in practice refer to cognitive models that may be explicit and formalized when they are based on scientific knowledge areas or, more often, are largely implicit and informal, as can occur in the case of professional activities with a high rate of complexity, such as teaching. It is important to consider the type of division which may be established in learning between cognitive and operational models. A professional learning course, to allow a certain autonomy and efficacy at an operational level, in addition to a detailed reference to specific situations, requires cog- nitive and operational models to be clearly separate and recogniz- able, while at the same time succeeding in creating a reciprocal dialogue, a dynamic that allows both to evolve and also allows prac- tice to evolve. It is desirable that cognitive models, to represent a useful basis for the building of operational models, are attributable to clearly identifiable knowledge areas validated ‘through testing in the field’, in practice and by practices, and which are offered in re- lation to problematic situations, i.e. ill-defined problems that need to be framed, defined, and handled in a partially innovative man- ner. PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES In the field of theories concerning possible didactic training models, at least in relation to the reference context (Damiano 2014), we find evidence of ‘practice didactics’, based on the alternation of direct experience – according to standard strategies, such as, for ex- ample, observation and imitation of model actions and co-operation, followed by recording them as encoding of observation and render- ing imitation explicit, complements to the execution and occasions for carrying out the actions, invaluable for promoting abstraction, i.e. for identifying the core of the didactic action in context, to ex- amine it from a decontextualized and generalizing perspective, also thanks to comparison with formal teaching knowledge areas, via forms of representation and analysis of the aspects that form them; this, as a function of a more complete conceptualization by the teacher undergoing professional, action-based training and possibly formalization in modelling terms and the appropriation/building of teaching theories that are capable of directing operational choices which are intentionally well-founded. sion between ‘cognitive models’ and ‘operational models’ (Pastré 2007). Professional learning would primarily consist of the acquisi- tion of pragmatic concepts or action organisers – ‘operational mod- els’, learned directly in context and referring to a class of professional situations within the ambit of a practice community. Alongside con- cepts that reference a pragmatic model, other conceptualization methods or ‘cognitive models’ come into play which concern the characteristics of the action ‘beyond’ the transformative tension felt immediately while the action is in progress. In professional learn- ing, the operational models learned in practice refer to cognitive models that may be explicit and formalized when they are based on scientific knowledge areas or, more often, are largely implicit and informal, as can occur in the case of professional activities with a high rate of complexity, such as teaching. It is important to consider the type of division which may be established in learning between cognitive and operational models. A professional learning course, to allow a certain autonomy and efficacy at an operational level, in addition to a detailed reference to specific situations, requires cog- nitive and operational models to be clearly separate and recogniz- able, while at the same time succeeding in creating a reciprocal dialogue, a dynamic that allows both to evolve and also allows prac- tice to evolve. 2. Educational instrument Planning restrictions provide for division of the 60 academ- ic credits (in Italian CFU – university academic credits) attrib- uted to the Master’s degree (following an overall modular system: 3 modules, each worth 20 CFUs, at ‘foundation’, ‘intermediate’ and ‘advanced’ levels), in 41 CFUs to be allocated to subject-relat- ed teaching, and in 14 CFUs (350 hours) for professional training placement. A traineeship pathway unfolds that is essentially ad- dressed towards promoting the fine-tuning/development of pro- fessional competence, understood as identification and analysis of practical and operational problems linked to integration in the class of students with SLD, with review, as a pro-active and relaunch- ing function, in relation to conceptual and theoretical frameworks offered by the subject-related knowledge areas taught during lec- tures. To promote interconnectedness with the teaching of subjects, the traineeship activities have been developed in three stages, I, II and III, respectively, according to a scheme alternating workshop traineeship at the academic location and activities in schools. As far as traineeship instructors are concerned, since we can only count on the limited involvement in mentoring tasks of staff in schools, due to organizational and management restrictions, instructors ap- pointed by the University have been given this responsibility (ac- cording to an instructor-course student ratio of 1:10), selected from amongst teachers in service, based on their educational and profes- sional curriculum in the field of teaching students with SLD. The action of the traineeship tutor is supported by a brief, initial train- ing course, ongoing co-ordination meetings, and the setting up of a structured working guide. The tutor action consists in preparing the traineeship activity in the school and in promoting integrated activation of the ‘practical’ and formal knowledge areas acquired, through analysis and discussion of the cases observed (Altet, 2000) thanks to inter-professional discussion and exchange within the small group and one-on-one tutoring actions (Wenger 1998). The subject of observation-analysis-problematization and development within the ambit of the traineeship and the didactic action of the teacher understood as «mediation action», refers to «what the teach- er does in relation to what the student does for learning cultural subjects» (Damiano 2013: 133). In the first module, work delivery includes the observation and reconstruction of a didactic action (les- son or cycle of lessons characterised by didactic and training con- sistency), according to a structured schedule. PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES 197 PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES It is desirable that cognitive models, to represent a useful basis for the building of operational models, are attributable to clearly identifiable knowledge areas validated ‘through testing in the field’, in practice and by practices, and which are offered in re- lation to problematic situations, i.e. ill-defined problems that need to be framed, defined, and handled in a partially innovative man- ner. In the field of theories concerning possible didactic training models, at least in relation to the reference context (Damiano 2014), we find evidence of ‘practice didactics’, based on the alternation of direct experience – according to standard strategies, such as, for ex- ample, observation and imitation of model actions and co-operation, followed by recording them as encoding of observation and render- ing imitation explicit, complements to the execution and occasions for carrying out the actions, invaluable for promoting abstraction, i.e. for identifying the core of the didactic action in context, to ex- amine it from a decontextualized and generalizing perspective, also thanks to comparison with formal teaching knowledge areas, via forms of representation and analysis of the aspects that form them; this, as a function of a more complete conceptualization by the teacher undergoing professional, action-based training and possibly formalization in modelling terms and the appropriation/building of teaching theories that are capable of directing operational choices h h ll ll f d d PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES 2. Educational instrument Work delivery within the third module provides – given the situa- tion-problem previously identified and also in the light of learning acquired of a conceptual and theoretical nature regarding teaching in mixed classes with students with SLD – for the definition, when- ever possible, via discussion and exchange with class teachers – of possible objectives for improvement, identifying cases for innova- tive action regarding methods to monitor impact. 2. Educational instrument Followed, also thanks to group discussion mediated by the traineeship tutor, by analysis of the actions observed, strengths and critical points, commencing 198 DANIELA MACCARIO from professional experience and theoretical elements offered in the subject-relating teaching of the Master’s programme, to be formal- ized in a project work assignment, to be developed throughout the entire traineeship study path and the subject of a final assessment at the end of the Master’s degree). In the second module, in relation to the situation observed, the course student is asked to identify sit- uations-problems of a professional nature (Pastré 2007; Perrenoud 1999b) arising from teaching in cases of students with SLD, prior to sharing the operational definition of the concept (situations pre- senting obstacles, challenges and problematic cases that raise issues – also of an ethical nature – reflection – also at a metacognitive level – in relation to the planning and management of didactic sequences, learning assessment, management of the relationship with students and relations with colleagues and families). Active involvement in class with a debriefing interview by the teacher (Vermesch, 2011) from professional experience and theoretical elements offered in the subject-relating teaching of the Master’s programme, to be formal- ized in a project work assignment, to be developed throughout the entire traineeship study path and the subject of a final assessment at the end of the Master’s degree). In the second module, in relation to the situation observed, the course student is asked to identify sit- uations-problems of a professional nature (Pastré 2007; Perrenoud 1999b) arising from teaching in cases of students with SLD, prior to sharing the operational definition of the concept (situations pre- senting obstacles, challenges and problematic cases that raise issues from professional experience and theoretical elements offered in the subject-relating teaching of the Master’s programme, to be formal- ized in a project work assignment, to be developed throughout the entire traineeship study path and the subject of a final assessment at the end of the Master’s degree). 2. Educational instrument In the second module, in relation to the situation observed, the course student is asked to identify sit- uations-problems of a professional nature (Pastré 2007; Perrenoud 1999b) arising from teaching in cases of students with SLD, prior to sharing the operational definition of the concept (situations pre- senting obstacles, challenges and problematic cases that raise issues – also of an ethical nature – reflection – also at a metacognitive level – in relation to the planning and management of didactic sequences, learning assessment, management of the relationship with students and relations with colleagues and families). Active involvement in class with a debriefing interview by the teacher (Vermesch, 2011) and subsequent description and analysis also in relation to concep- tual and theoretical elements during the Master’s degree lessons. Work delivery within the third module provides – given the situa- tion-problem previously identified and also in the light of learning acquired of a conceptual and theoretical nature regarding teaching in mixed classes with students with SLD – for the definition, when- ever possible, via discussion and exchange with class teachers – of possible objectives for improvement, identifying cases for innova- tive action regarding methods to monitor impact. – in relation to the planning and management of didactic sequences, learning assessment, management of the relationship with students and relations with colleagues and families). Active involvement in class with a debriefing interview by the teacher (Vermesch, 2011) and subsequent description and analysis also in relation to concep- tual and theoretical elements during the Master’s degree lessons. Work delivery within the third module provides – given the situa- tion-problem previously identified and also in the light of learning acquired of a conceptual and theoretical nature regarding teaching in mixed classes with students with SLD – for the definition, when- ever possible, via discussion and exchange with class teachers – of possible objectives for improvement, identifying cases for innova- tive action regarding methods to monitor impact. – in relation to the planning and management of didactic sequences, learning assessment, management of the relationship with students and relations with colleagues and families). Active involvement in class with a debriefing interview by the teacher (Vermesch, 2011) and subsequent description and analysis also in relation to concep- tual and theoretical elements during the Master’s degree lessons. 3. Methodology The research originated from the need to obtain elements pro- viding empirical confirmation of the theoretical premise, according to which a didactic professional programme based on the rationale of ‘immersion-decontextualization’ and focussing on promoting problematization processes linked to the action of teaching, based on the synergic activation of areas of knowledge of a scientific and discipline-oriented and teaching area-related nature, linked to the practice of working as a teacher, may represent a path that is po- tentially effective in the professional training of teachers in an aca- demic environment. The research also set out to gather – from an exploratory standpoint and from the perspective of teachers– items of knowledge that could prove useful for identifying particularly criti- cal areas in managing teaching practices in mixed classes. Contexts which need to be taken into consideration when planning teacher training courses and which are the focus of in-depth study as part of the research in the pedagogic and didactic field. The study re- PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES 199 fers to the system tested during the second edition of the Master’s programme in the A/Y 2012-2013 (pre-tested and fine-tuned over the first year of the course in A/Y 2011-12: sample of 71 Master’s programme students). The research sample consisted of 52 teach- ers in service in Piedmont schools (6 pre-school teachers, 32 pri- mary school teachers and 14 middle school teachers), enrolled in the professional Master’s programme. Survey of the impact of profes- sional placement was conducted by means of project work required from Master’s students. The observation context was represented by schools within the Piedmont region with agreements in place with the University of Turin, as venues for professional placement (in pre- schools, primary, middle, and high schools). To extend the experi- ence of professional knowledge areas, the Master’s students were able to choose their placement also at school levels which were not their habitual teaching level. The training instrument was thus “tested” with a random sample of graduating students, in any case, held to be significant for a first validation, as was the choice of the observa- tion environments. 3. Methodology The unit of analysis of the textual material ob- tained was represented by identifying situations/problems – SP – of a typical and recurring professional nature arising from teaching in classes with the inclusion of students with SLD, from the standpoint of Master’s students or a set of teaching actions habitually used by teachers, with the aim of creating conditions to favour learning in their students, which could be improved according to the reasoned and critically-based analysis of the Master’s course students. To ren- der the construct operational, the survey took the SPs described by course students into account in terms of ‘action flows’ observed in context and identified/explained by the actors, analysed with spe- cific reference to theoretical and conceptual elements progressively consolidated/learned during the Master’s programme. All written work produced regarding the second session of project work was subjected, by the author, to analysis of the subject matter, adopting a post-encoding process and a subsequent grouping into categories (with the support of N-Vivo 10 software), with frequency calcula- tion (Trinchero 2007; Miles, Huberman, Saldaña 2014). 3. Methodology The unit of analysis of the textual material ob- tained was represented by identifying situations/problems – SP – of a typical and recurring professional nature arising from teaching in classes with the inclusion of students with SLD, from the standpoint of Master’s students or a set of teaching actions habitually used by teachers, with the aim of creating conditions to favour learning in their students, which could be improved according to the reasoned and critically-based analysis of the Master’s course students. To ren- der the construct operational, the survey took the SPs described by course students into account in terms of ‘action flows’ observed in context and identified/explained by the actors, analysed with spe- cific reference to theoretical and conceptual elements progressively consolidated/learned during the Master’s programme. All written work produced regarding the second session of project work was subjected, by the author, to analysis of the subject matter, adopting a post-encoding process and a subsequent grouping into categories (with the support of N-Vivo 10 software), with frequency calcula- tion (Trinchero 2007; Miles, Huberman, Saldaña 2014). fers to the system tested during the second edition of the Master’s programme in the A/Y 2012-2013 (pre-tested and fine-tuned over the first year of the course in A/Y 2011-12: sample of 71 Master’s programme students). The research sample consisted of 52 teach- ers in service in Piedmont schools (6 pre-school teachers, 32 pri- mary school teachers and 14 middle school teachers), enrolled in the professional Master’s programme. Survey of the impact of profes- sional placement was conducted by means of project work required from Master’s students. The observation context was represented by schools within the Piedmont region with agreements in place with the University of Turin, as venues for professional placement (in pre- schools, primary, middle, and high schools). To extend the experi- ence of professional knowledge areas, the Master’s students were able to choose their placement also at school levels which were not their habitual teaching level. The training instrument was thus “tested” with a random sample of graduating students, in any case, held to be significant for a first validation, as was the choice of the observa- tion environments. 4. Results A first finding of the analysis concerns the nature of problematiza- tion processes regarding professional practices, to which the learning system gave rise. A positive impact was found, with a high rate of SP identification ‘in the strict sense’, attributable to precisely described professional practices (Tab. 1: 0.1). 200 DANIELA MACCARIO Table 1 0. Analysis of professional practices and problematization processes Pre- school f. Primary. school f. Middle. school f. High. school f. Total f. Total f.% 0.1 Problems focussing on profes- sional action in context (SP) 15 37 38 37 127 93 0.2 Problems focussing on the student 1 1 2 1 0.3 Problems focussing on con- text conditions 2 2 1 5 4 0.4 Problems focussing on teacher 1 1 1 3 2 Total 16 40 42 39 137 100 0. Analysis of professional practices and problematization processes Pre- school f. Primary. school f. Middle. school f. High. school f. Total f. Total f.% 0.1 Problems focussing on profes- sional action in context (SP) 15 37 38 37 127 93 0.2 Problems focussing on the student 1 1 2 1 0.3 Problems focussing on con- text conditions 2 2 1 5 4 0.4 Problems focussing on teacher 1 1 1 3 2 Total 16 40 42 39 137 100 The analysis of surveyed SPs highlighted three areas identified as critical D: defining and managing learning strategies (Tab. 1:1.1), classroom management (Tab. 1: 1.2.); problems associated with iden- tifying and taking charge of cases of children with SLD. 0. Analysis of professional practices and problematization processes The analysis of surveyed SPs highlighted three areas identified as critical D: defining and managing learning strategies (Tab. 1:1.1), classroom management (Tab. 1: 1.2.); problems associated with iden- tifying and taking charge of cases of children with SLD. The analysis of surveyed SPs highlighted three areas identified as critical D: defining and managing learning strategies (Tab. 1:1.1), classroom management (Tab. 1: 1.2.); problems associated with iden- tifying and taking charge of cases of children with SLD. 1. Problematization focussing on professional action in context (SP): environments Pre- school f. Primary. school f. Middle. school f. High. school f. Total f. 1 Compensatory tools are teaching and technological tools that replace or facilitate required performance in deficient learning ability. 4. Results Total f.% 1.1Managing didactic progression and mediation 4 15 23 23 65 51 1.2Classroom management 5 14 13 10 42 33 1.3Diagnostic practices and taking charge of students with SLD 6 8 2 4 20 16 Total 15 37 38 37 127 100 1. Problematization focussing on professional action in context (SP): environments Within the context of SPs attributable to managing didactic pro- gression (Rey 1999) and mediation, a stage indicated as highly prob- lematic is the continuation of teaching practices which are largely transmissive and abstract; a point reported concerns the limited ap- preciation of the potential of multi-modal and multi-media com- munication using teaching technologies (Calvani 2011) (Tab. 3: 1.1.1.; 1.1.2.). Of note is the finding that there is scarce contextual- ized awareness of the ways for using compensatory tools and the ex- emptive measures provided under legislation supporting learning by PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES 201 students with SLD1 (Tab. 3: 1.1.3.) and methods for managing assess- ment practices, at times scarcely perceived at a docimological level, with limited appreciation of the learning support potential and for the teaching action (Tab. 3: 1.1.4) (Maccario 2012). The subject of foreign language teaching reveals its own critical areas, (especially in high school), associated with the offering of learning activities heavily based on decoding the written language (Tab. 3: 1.1.5). At times, the source of the problems in managing teaching in class can be identified in the excessively standardized/formalized planning practices adopted by schools that are ill-adapted to the actual, con- textual conditions of classes and individual students (Tab. 3: 1.1.6). Table 3 Table 3 1.1 Management of didactic progression and mediation Pre- school f. Primary. school f. Middle. school f. High. school f. Total f. Total f.% 1.1.1 Prevalence of classroom-taught lessons (deductive-abstract evo- lution, dominance of oral medi- ation and uniformity of requests to students from the standpoint of performance). 1 6 6 9 22 34 1.1.2. Limited use of the communica- tion potential of technologies. 1 1 4 4 10 15 1.1.3. Formal/scarcely personalized use of compensatory tools and exemptive measures. 3 5 5 13 20 1.1.4 Limited explanation of assess- ment and judgement criteria; focus on results from a summa- tive standpoint. 4 5 4 13 20 1.1.5. Scarcely personalized teaching of foreign languages. 2 1 3 5 1.1.6. Formalized/lack of planning practices. 4. Results 2 1 1 4 6 Total 4 15 23 23 65 100 1.1 Management of didactic progression and mediation In classroom management, the research identified that maintaining class discipline is a problem, as are suitable conditions for involving students with scholastic vulnerabilities (Tab. 4: 1.2.1). Noteworthy, In classroom management, the research identified that maintaining class discipline is a problem, as are suitable conditions for involving students with scholastic vulnerabilities (Tab. 4: 1.2.1). Noteworthy, 202 DANIELA MACCARIO and a recurring factor, is the management of didactic communica- tion following a method which effectively selects the more prepared students, marginalizing those in difficulty (following a phenomenol- ogy already noted in the literature (Perrenoud 1997; Kahan 2010) (Tab. 4: 1.2.2) and scarce attention to building motivating relations with students (Charles 2002) and promoting in students, respect for diversity and co-operation (Tab. 4: 1.2.3). It can be seen, in several cases, that organizational conditions supporting learning are unsuit- able for encouraging the involvement of students with SLD (Tab. 4: 1.2.4) (Tomlinson 2006; Vio, Toso 2007; Cornoldi 2007). As for tak- ing charge of students with SLD, it can be seen that, at times, there is limited investment in communication/co-operation with their fami- lies (Tab. 5: 1.3.1) (Vio, Toso 2007; Epstein 2009). and a recurring factor, is the management of didactic communica- tion following a method which effectively selects the more prepared students, marginalizing those in difficulty (following a phenomenol- ogy already noted in the literature (Perrenoud 1997; Kahan 2010) (Tab. 4: 1.2.2) and scarce attention to building motivating relations with students (Charles 2002) and promoting in students, respect for diversity and co-operation (Tab. 4: 1.2.3). It can be seen, in several cases, that organizational conditions supporting learning are unsuit- able for encouraging the involvement of students with SLD (Tab. 4: 1.2.4) (Tomlinson 2006; Vio, Toso 2007; Cornoldi 2007). As for tak- ing charge of students with SLD, it can be seen that, at times, there is limited investment in communication/co-operation with their fami- lies (Tab. 5: 1.3.1) (Vio, Toso 2007; Epstein 2009). Table 4 1.2 Classroom management Pre- school f. Primary. school f. Middle. school f. High. school f. Total f. 4. Results Total f.% 1.2.1 Difficult classroom manage- ment, discipline/involvement 2 4 1 3 10 24 1.2.2 Selective dialogue and limited reciprocal communication 3 5 1 9 21 1.2.3 Limited attention to relations with and amongst students as a function of learning success 1 3 3 4 11 26 1.2.4 Organizational/management conditions and non-differen- tiated learning support actions 2 4 4 2 12 29 Total 5 14 13 10 42 100 Table 5 1.3 Diagnostic practices and taking charge of SLD students Pre- school f. Primary. school f. Middle. school f. High. school f. Total f. Total f.% 1.3.1 Limited communication strat- egies and educational alliances with families 2 3 2 2 9 45 1.3.2 Late/uncertain identification of problems at school attribut- able to SLD 4 3 7 35 Table 4 4. Final remarks The research conducted, although limited, confirms the possibility of offering university courses focused on the professional placement of teachers to enhance their professionalization, based on a dialogue between practical/experience-based areas of knowledge and an alter- nation of didactic occasions/settings, within a framework of training synergies between university and school that provide for an adequate assumption of responsibility and the training of teachers to take on mentoring functions (possibly also in context). The study also appears to indicate that the possibilities for creating inclusive and personal- ized teaching to the advantage of students with SLD – but not only limited to these – are particularly linked to a full review of teaching methods and development/fine-tuning of the competences required of teachers for ‘creating a class’ (Rey 1999) as a community for stu- dent learning. PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES 203 1.3.2. Interpretation/application dif- ficulties regarding SLD di- agnoses issued by social and healthcare services. 2 2 4 20 Total 6 8 2 4 20 100 1.3.2. Interpretation/application dif- ficulties regarding SLD di- agnoses issued by social and healthcare services. 2 2 4 20 Total 6 8 2 4 20 100 Table 4 PROFESSIONALIZATION AND PROBLEMATIZATION PROCESSES References Altet M. 2000, L’analyse de pratiques. Une démarche de formation professionnalisante?, «Recherche et formation», 35, 25-41. 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Van der Maren J.M. 2014, La recherché appliquée pour l (para)médical, travail social, De Boeck, Brussels. Vermesh P. 2011, L’entretien d’explicitation, ESF, Paris. Vio C. and Toso C. 2010, Dislessia evolutiva. Dall’identificazione del disturbo all’intervento, Carocci, Roma. Wenger E. 1998, Communities of practice: learning, meaning and identity, Cambridge University Press, New York. Nicola Andrian (University of Padua)* Abstract: The research aims to analyse and justifiably develop an innovative model for the internationalization of University social responsibility experiences through the international mobility of undergraduate students, with a combina- tion of study and internships. Keywords: university internationalization, university social responsibility (third mission), internship abroad, service learning. * Nicola Andrian, PhD Student, Department of Philosophy, Sociology, Pedagogy and Applied Psychology (FISPPA) of the University of Padua, Italy, under joint super- vision of the PhD Course in Education and Contemporaneity of the University of the State of Bahia, Brazil. Email: nicola.andrian@phd.unipd.it. , @p p 1 Degree Courses at the former Faculty of Education Sciences and at the FISPPA. INTEREURISLAND: FROM FIELD RESEARCH TO A POSSIBLE MODEL FOR INTERNATIONALIZING UNIVERSITY SOCIAL RESPONSIBILITY EXPERIENCES Nicola Andrian (University of Padua)* 2 A social and cultural exchange project promoted by the Social Promotion Association En.A.R.S. – Padua, Italia. <http://www.enars.it> (03/2018). * Nicola Andrian, PhD Student, Department of Philosophy, Sociology, Pedagogy and Applied Psychology (FISPPA) of the University of Padua, Italy, under joint super- vision of the PhD Course in Education and Contemporaneity of the University of the State of Bahia, Brazil. Email: nicola.andrian@phd.unipd.it. 1 Degree Courses at the former Faculty of Education Sciences and at the FISPPA. 2 A social and cultural exchange project promoted by the Social Promotion Association En.A.R.S. – Padua, Italia. <http://www.enars.it> (03/2018). 1. Introduction The research presented in this article originates from the desire to share a series of speculations relating to the experience of study and internship abroad of 12 Undergraduate students of the University of Padua1 (UNIPD), Italy. Between 2012 and 2015, these students par- ticipated in the BEA Project2 in the city of Petrolina, in the State of Pernambuco, Brazil. One of the peculiarities of the BEA Project is the combination of studying abroad with an internship that Italian university students do when participating in Extensão projects developed by local universi- ties and communities. The FORPROEX − Pro-Rectors National Forum of Extension in Brazilian Public Universities (2010), defines Extensão as an education- al, cultural, scientific, and political interdisciplinary process which promotes transformative interaction between universities and other sectors of society. According to Prof. Pedro Nuno Teixeira (2015), Vice Rector of Aca- demic Affairs at the University of Porto in Portugal, Extensão Universitaria is becoming increasingly important both in Latin America, where the main objective is to influence social actions and interventions promot- 206 NICOLA ANDRIAN ing citizenship and local leadership, and in developed economies where services and technology transfer receive greater attention. In Continental Europe, the most common term for Extensão is ‘Third Mission’, which is sub-divided into three areas: Further Education, Technology Transfer and Innovation, and Social Engagement. Between 2012 and 2014, in Petrolina the combination of the inter- national mobility of students for internships, their participation in social responsibility projects of local universities, and the learning through soli- darity services in the community, created a highly challenging context from diff erent standpoints. Figure 1 – The BEA Project Cycle. [http://www.enars.it>] igure 1 The BEA Project Cycle. [http://www.enars.it>] Service Learning University Social Responsibility (Third Mission) International Mobility and Intercultural Dialogue Progetto BEA (Andrian N.) International Mobility and Intercultural Dialogue University Social Responsibility (Third Mission) The positive evaluations of all the people involved in the expe- riences3 and the protagonism experienced, perceived, and reported by the university students involved prompted further analyses on the meaning and value of the BEA approach, both regarding the inno- vative educative practices, and to the University’s pathways and pro- cesses of Internationalization and Social Responsibility. Numerous studies, from the 1980s onwards, have analysed the im- pact (in terms of value, signifi cance, benefi ts, etc.) of the experienc- es of undergraduate students abroad. 3 University students, academic tutors, supervisors of the internships, and BEA Project coordinator. Data sources: Internship evaluations and Final Reports on Internship Abroad experiences. Figure 1 – The BEA Project Cycle. [http://www.enars.it>] 1. Introduction For instance, the Study Abroad Evaluation Project (SAEP), carried out in 1982 under the overall co- ordination of the European Institute of Education and Social Policy, analysed 116 diff erent study programmes abroad (Oxford Studies in Comparative Education, vol. 5, 1995, Aspects of Education and the Eu- ropean Union, David Phillips). Currently there are several results that have documented the contributions of mobility programmes on stu- dents and their educational growth. 207 INTEREURISLAND Regarding the specific BEA experience, the level of students’ re- ports on their practice and perception of their learning through the experience constantly prompted further analysis, with community referents, academics, and tutors.i It was additionally confirmed, as suggested by Milan (2007, 2009), that from the point of view of intercultural dialogue, the project ap- proach stimulated students, through the participatory-solidary travel in other cultural contexts, «a true and profitable 360-degree identity opening, that expands the mind and the existential experience, pro- moting the authentic recovery of the community dimension and the building of a world-mind, of earthly identity». «Before focusing on interculturality as a “social” – “political” – “cultural” construction, the objective of the educational perspective is the ‘intercultural per- son’, capable of dialogue and reciprocity». DECENTRALIZATION: The fact of finding myself in the situation of a foreigner, made me think a lot about the ability to get out of my world, of my way of thinking, the ability to ‘move away from me’. As a team, we worked a lot on the importance of expressing ourselves while omit- ting any judgment; we paid more attention to identifying our own in- terpretations and opinions, linked to our culture and experiences, trying to keep them separate from the descriptions of what we saw or heard4. In 2015, the development of BEA’s architecture led to a collabo- ration with the Department of Human Sciences (DCH), Campus III of the University of the State of Bahia in the nearby city of Juazeiro, Brazil. The existence of a bilateral agreement between UNIPD and UNEB and the signing of a Joint PhD Supervision agreement be- tween the Pedagogical and Educational Sciences Course, FISPPA, UNIPD, and the Education and Contemporaneity Course, UNEB, created the conditions to implement research that would be able to systematize these activities and promote further investigation. 4 Equipe BEA 2015: Final report on an internship abroad by a graduate student in Educational Sciences, FISPPA, UNIPD, Padua, Italy. 5 INTEREURISLAND: INTERsectoral, Extensão Universitária, Research, Interculture, and Service-Learning. Approaching a New Development. 6 Social and Intercultural Pedagogy Research Group-GRIPSI-FISPPA, UNIPD. 6 Social and Intercultural Pedagogy Research Group-GRIPSI-FISPPA, UNIPD 1. INTEREURISLAND5 – The Research Under the supervision of Professors Giuseppe Milan and Luca Agostinetto, FISPPA, UNIPD6 and Augusto César Leiro, PPGEduC 208 NICOLA ANDRIAN Program, UNEB, the research focused on analysing the development of an innovative model to internationalize university social responsibility experiences (Public/Social Engagement, Third Mission), through the mobility of undergraduate students, combining study and internships. The interest in summarizing the experience of the BEA Project and the possibility of replicating this experience within the Bilat- eral Agreement between the Department FISPPA, UNIPD and the DCH, UNEB, led to considering Multiple Case Studies as the most appropriate research methodology, since this was an empirical inquiry into contemporary phenomenon (i.e. a ‘case’), set within a real-world context, especially when the boundaries between phenomenon and context are not clear (Yin 2009). The research design included a Pilot Case Study and a Multiple Case Study, as follows: • Pilot Case Study: Petrolina, Brazil (March to June 2016). Case Study 1: Rovigo, Padua, Italy (October to December 2016). • Case Study 1: Rovigo, Padua, Italy (October to December 2016). • Case Study 2: Juazeiro, Brazil (March to July 2017). • Case Study 2: Juazeiro, Brazil (March to July 2017). As suggested by Yin (2005), the research involved the use of mul- tiple data sources, which made it possible to refer to a wider range of issues, and a data triangulation to develop converging lines of inqui- ry. Tools and data collection sources were the following: documents (diaries, internal and/or administrative documents, E-mail corre- spondence, etc.), interviews, questionnaires, focus groups, direct and participant observations. Due to the restrictions of the current paper, an overview of the different Case Studies is provided first. Secondly, the Process Mod- el created from the Pilot Case study data analysis is illustrated, as implemented at FISPPA, UNIPD and DCH, Campus III, UNEB and analysed through Multiple Study Cases 1 (Rovigo, Italy) and 2 (Juazeiro, Brazil). 1.1 Pilot Case Study 7 Social Educational Assistance Foundation for adolescents in conflict with the law – State of Pernambuco, Brazil. 1.1 Pilot Case Study Context: City of Petrolina, State of Pernambuco, Brazil. Period: from March 1 to June 30, 2016 Research Object: The BEA Project Hypothesis: yp • The BEA Project proposal is significant from the point of view of the University’s Internationalization and Social Responsibil- ity and from the educational point of view for the undergraduate students involved. • The BEA Project proposal is significant from the point of view of the University’s Internationalization and Social Responsibil- ity and from the educational point of view for the undergraduate students involved. 209 INTEREURISLAND • This proposal is replicable, with proper adjustments, in the con- texts of Rovigo, Italy and Juazeiro, Brazil, as poles of a bilateral agreement between the FISPPA Department, UNIPD and DCH, UNEB. Partners: l d l Partners: • Social Promotion Association En.A.R.S. Padua, Italy: BEA Project pro- moter and internship institution, through an Agreement with UNIPD; • Social Promotion Association En.A.R.S. Padua, Italy: BEA Project pro- moter and internship institution, through an Agreement with UNIPD; • UNIPD, University of Padua, Italy: promoter of international mobil- ity of undergraduate students for internships abroad; • UPE, University of Pernambuco, Campus of Petrolina, Brazil: pro- moter of the Extensão project Communication and educational relation- ships in extracurricular educational contexts; p • FUNASE7, Petrolina-PE: Progetto BEA Partner hosting degree stu- dents for internship. • UNIVASF, Federal University, Petrolina: En.A.R.S. Partner and promoter of intercultural and peer education activities with local and foreign students. g • APAE; Association dealing with people with disabilities. University students involved: 2 students from the Educational Sci- ences Course FISPPA, UNIPD in Rovigo, Italy; 45 students from the 8th grade of the Degree Course in Pedagogy, UPE, Petrolina- PE Campus; 30 students from different UNIVASF graduate courses. Analysis Sub-unit: 1) The sequence of activities envisaged by the BEA Project 2) University Social Engagement project established under the coordi- nation of the BEA Project and through the collaboration of the UPE with FUNASE CASEM Petrolina 3) Undergraduate students’ perception of their learning through the experience; 3) Undergraduate students’ perception of their learning through the experience; Analysis of the data collected from the various inquiries confirmed the initial hypothesis that the BEA proposal is significant from the point of view of University Internationalization and Social Responsi- bility and from the educational point of view for the students involved. 8 Data source: final semi – structured questionnaire to Italian undergraduate stu- dents on mobility programmes in Petrolina (March-June 2016). 1.1 Pilot Case Study I am convinced that the BEA Project proposal is extremely significant I am convinced that the BEA Project proposal is extremely significant from this point of view. I could see how important every moment I lived 7 Social Educational Assistance Foundation for adolescents in conflict with the law – State of Pernambuco, Brazil. 210 NICOLA ANDRIAN was. Daily sharing, team meetings, reflection on certain issues and on practice, the university course, the stimulus to keep me questioning. All this was essential for a continuous growth and an educational process lasting four months8. Definition of a cyclical sequence of BEA Project activities and re- flections on the possibility of replicating the experience analysed at the other poles of the agreement between FISPPA, UNIPD and DCH, UNEB, led to development of a possible process model which was implemented and analysed in Case Studies 1 and 2. 2. INTEREURISLAND – The Possible Process Model 2. INTEREURISLAND – The Possible Process Model Following analysis of the data collected during Study Cases 1 and 2 and an in-depth investigation of the studies on the University’s In- ternationalization and Social Responsibility, Figures 2, 3, and 4 sum- marize the ‘current situation’ as regards the justifi ed development of the possible model for the Internationalization of University Social Responsibility experiences, INTEREURISLAND. 1.2 Multiple Case Study Case Study 1: October to December 2016. Case Study 1: October to December 2016. Case Study 1: October to December 2016. y Context: City of Rovigo, Italy. y Context: City of Rovigo, Italy. Context: City of Rovigo, Italy. y g y Partners: DCH, Campus III, UNEB; FISPPA, UNIPD; Social Cooperatives Porto Alegre (Immigration) and Peter Pan (Children with Specific Learning Disorders), Department of Culture and Uni- versity, Rovigo. Partners: DCH, Campus III, UNEB; FISPPA, UNIPD; Social Cooperatives Porto Alegre (Immigration) and Peter Pan (Children with Specific Learning Disorders), Department of Culture and Uni- versity, Rovigo. Case Study 2: March to July 2017. Case Study 2: March to July 2017. Context: City of Juazeiro, State of Bahia, Brazil. y J Partners: FISPPA, UNIPD; DCH, Campus III, UNEB; NAENDA, FUNDAC (adolescents in conflict with the law), Pastoral da Mulher Partners: FISPPA, UNIPD; DCH, Campus III, UNEB; NAENDA, FUNDAC (adolescents in conflict with the law), Pastoral da Mulher (Women in prostitution) and Lar São Vicente de Paulo (Senior citizens). For both Cases: (Women in prostitution) and Lar São Vicente de Paulo (Senior citizens). For both Cases: Unit of Analyses: The INTEREURISLAND Process Model Theoretical postulate: Theoretical postulate: The INTEREURISLAND Process Model can be implemented in the two poles of the bilateral agreement between the FISPPA De- The INTEREURISLAND Process Model can be implemented in the two poles of the bilateral agreement between the FISPPA De- partment, UNIPD (Italy) and the DCH Department, Campus III, UNEB (Brazil) and the study of the two different cases will high- light the strengths and weaknesses of the proposal in the two poles of the bilateral agreement between the FISPPA De- partment, UNIPD (Italy) and the DCH Department, Campus III, UNEB (Brazil) and the study of the two different cases will high- light the strengths and weaknesses of the proposal. Sub - unit of analysis: g g Sub - unit of analysis: 1) The sequence of practices implemented through the INTEREURIS- LAND Process Model; q p LAND Process Model; 2) The Social Responsibility project of the University; 3) The degree students’ perception of their learning through the experience; 211 INTEREURISLAND INTEREURISLAND Process Model (Andrian N.) INTERNATIONALIZATION and INTERCULTURAL DIALOGUE Creation, Development and Institutionalization of International Partnerships: • Stipulate of Memorandum of Understanding (MoU), among the interested Universities; • Stipulate of Addendum of Memorandum (MoU), between Departments / Faculty of areas of Interest; • Stipulate of Joint PhD Agreement. International Mobility of Social Responsibility Working Groups (IN & OUT): • Constitution of the SR WG: A Professor, a researcher, two students, an administrative technician and a tutor. IN & OUT: Didactic activities, research and social responsibility (integrated projects / programs), seminars, conferences, publications etc. • Students IN: Welcome activities; orientation of study, internship activities (social/public engagement projects with the community) and research; certification/recruitment activities and credits; • Students OUT: Orientation to the proposal INTEREURISLAND - Selection and definition of the Learning Agreement (study, internship and / or research), recognition of credits (Post Mobility). • Specific training: Intercultural activities (including languages courses) and peer education; • Evaluation during and after the implementation Planning of the new INTEREURISLAND Cycle. INTERNATIONALIZATION and INTERCULTURAL DIALOGUE INTERNATIONALIZATION and INTERCULTURAL DIALOGUE Creation, Development and Institutionalization of International Partnerships: • Stipulate of Memorandum of Understanding (MoU), among the interested Universities; • Stipulate of Addendum of Memorandum (MoU), between Departments / Faculty of areas of Interest; • Stipulate of Joint PhD Agreement. International Mobility of Social Responsibility Working Groups (IN & OUT): • Constitution of the SR WG: A Professor, a researcher, two students, an administrative technician and a tutor. IN & OUT: Didactic activities, research and social responsibility (integrated projects / programs), seminars, conferences, publications etc. • Students IN: Welcome activities; orientation of study, internship activities (social/public engagement projects with the community) and research; certification/recruitment activities and credits; • Students OUT: Orientation to the proposal INTEREURISLAND - Selection and definition of the Learning Agreement (study, internship and / or research), recognition of credits (Post Mobility). • Specific training: Intercultural activities (including languages courses) and peer education; • Evaluation during and after the implementation Planning of the new INTEREURISLAND Cycle. Internationalization and Intercultural Dialogue Intersectoriality and University Social Responsibility Service Learning Intersectoriality and University Social Responsibility • Specific training: Intercultural activities (including languages courses) and peer education; p • Evaluation during and after the implementation • Evaluation during and after the implement Planning of the new INTEREURISLAND Cycle. Planning of the new INTEREURISLAND Cycle. INTEREURISLAND Process Model (Andrian N.) SERVICE LEARNING Involvement and reciprocity; Identifying academic objectives: for local and foreign students, under the supervision of the Local and Foreign Social Responsibility equipe; Identifying genuine needs / challenges of the Community: Under the supervision of the Intersectoral WG; Planning and implementation of project activities: Under the supervision of the Intersectoral WG; Systematic reflection on the experience; Assessment of the experience: during and after the implementation; Demostration of new understanding and Planning of the new INTEREURISLAND cycle. SERVICE LEARNING Involvement and reciprocity; Identifying academic objectives: for local and foreign students, under the supervision of the Local and Foreign Social Responsibility equipe; Identifying genuine needs / challenges of the Community: Under the supervision of the Intersectoral WG; Planning and implementation of project activities: Under the supervision of the Intersectoral WG; Systematic reflection on the experience; Assessment of the experience: during and after the implementation; Demostration of new understanding and Planning of the new INTEREURISLAND cycle. INTERSECTORALITY AND SOCIAL RESPONSIBILITY OF UNIVERSITY 2.1 Starting from ‘Internationalization and Intercultural Dialogue’ INTERSECTORALITY AND SOCIAL RESPONSIBILITY OF UNIVERSITY SERVICE LEARNING INTERSECTORALITY AND SOCIAL RESPONSIBILITY OF UNIVERSITY Creation, development and institutionalization of local partnerships: Stipulate of technical cooperation agreements between universities, departments / faculties and Community (public and private institutions, formal, non-formal and informal groups); Creation of the Intersectoral Working Group: Local and Foreign Social Responsibility WG with Tutors / referents of the Community; Planning and Development of Social / Public Engagement Projects: Between Departments/Faculties or Bachelor Courses and Institutions / Groups of the Communities; Development of interactive didactic workshops, within university courses of interest and among the activities carried out in the communities. Evaluation: during and after the implementation; Planning of the new INTEREURISLAND cycle. The Model presents the practices divided into three major areas that have been separated, both in the scheme presented and in the management of the proposed practices and processes. Areas which develop in close reciprocity and permeability, following a concentric sequence, each starting and developing through the other. 2.1 Starting from ‘Internationalization and Intercultural Dialogue’ 2.1 Starting from ‘Internationalization and Intercultural Dialogue’ Nowadays, universities face signifi cant challenges due to major changes in international scenarios, (Knight 2009): 212 NICOLA ANDRIAN • innovations in information and communication technologies are providing alternative and virtual ways to provide higher education; • new types of providers are emerging, such as international compa- nies, for-profit institutions, corporate universities, and IT and me- dia companies; • providers – public and private, modern and traditional – have be- gun delivering education across national borders to meet the de- mand in other countries. • alternative types of cross-border programme delivery, such as branch campuses and franchising and twinning arrangements, are being developed. In 2014, Knight remarked that ‘isolated’ student mobility is considered the first generation of Cross Border Education. The latest frontiers identify, among the key features of the third gen- eration, the Education Hub, the birth and development of Inter- national Branch Campuses and Excellence Research Centres and the development of specific mobility programmes, which are pri- marily addressed to: students, HE providers, research centres, and the knowledge industries. g Within this complex framework, the INTEREURISLAND mod- el proposes a synergy between university/community local relations and international relations between different contexts and social re- sponsibility projects/programmes. INTERSECTORALITY AND SOCIAL RESPONSIBILITY OF UNIVERSITY As indicated also by the Committee on International Relations of FORPROEX (2013), the proposal foresees the international mo- bility of a Social Responsibility Working Group (WG) composed, at the academic level, by a reference professor, a researcher (also a PhD student as joint supervision), two students, an administrative assis- tant, and a tutor. In university/community interactions, exchanges of social respon- sibility WG should be based on community approval and support for potential projects and programmes to be implemented, through the creation of an inter-sectoral team. In addition, this condition opens up the possibility for communities in different countries to develop their forms of exchange through international technical cooperation agreements and/or programmes. g p g Figure 3 illustrates the complete cycle of activities that students are required to follow in synergy with the academic Social Responsibil- ity WG and the Intersectoral WG. The plan considers the activities of the semester prior to the mobility period, carried out at the home university, the activities of the mobility semester at the host univer- sity, and those of the semester after the mobility. 213 INTEREURISLAND Figure 3 – INTEREURISLAND international student mobility process. Figure 3 – INTEREURISLAND international student mobility process. Orientation activities and specific training Intercultural activities and peer education Call for International mobility and Learning Agreement Graduation Intercultural activities and peer education Final Report presentation INTEREURISLAND International students mobility Process (Andrian N.) Social Engagement Project: Study (University) Internship (Community) Intercultural activities and peer education 1 Semester – Home University 2 Semester – Host University 3 Semester – Home University International mobility International mobility The Service Learning Cycle 2.2 Developing ‘Intersectorality and the Social Responsibility of the University’ INTEREURISLAND International students mobility Process (Andrian N.) INTEREURISLAND International students mobility Process (Andrian N.) INTEREURISLAND International students mobility Process (Andrian N.) 1 Semester – Home University International mobility Call for International mobility and Learning Agreement International mobility Intercultural activities and peer education 3 Semester – Home University Final Report presentation Graduation 2.2 Developing ‘Intersectorality and the Social Responsibility of the University’ The most signifi cant ideas contributing to the creation of a refer- ence framework for the University’s Social Responsibility projects (Fig. 4), originated from evaluation of the data collected from analy- sis subunits on social responsibility projects, developed in each of the 3 diff erent Study Cases. 9 UPE Pro-Rectorate of Extension and Culture, <http://www.upe.br/modalida- des-de-acoes> (01/2018). INTERSECTORALITY AND SOCIAL RESPONSIBILITY OF UNIVERSITY An example is provided below, based on information about the Extensão project Communication and educational relationships in extra- schooling educational contexts of the Pilot Case Study. A project to teach Interpersonal Relationships and Group Dynamics of the 8th Degree Course in Pedagogy, UPE, Campus of Petrolina, according to the following Extensão – PROEC, UPE9 actions: Knowledge Area: No. 7 – Human Sciences; Knowledge Area: No. 7 – Human Sciences; Main thematic area: No. VI – Education; Secondary subject area: No. II – Education and Citizenship; Extension Line: No. 26 – Vulnerable Social Groups. 214 NICOLA ANDRIAN For the implementation of the project, an intersectional WG was created, consisting of the General Coordinator and the socio-psy- cho-pedagogical WG of FUNASE, local students (Pedagogy Degree Course, UPE) and foreign students (Educational Sciences Degree Course, Rovigo FISPPA, UNIPD) and a deputy professor of the related issues, as researcher. A WG that planned and developed the entire intervention, which turned out to be the focus of the given case study and of the entire INTEREURISLAND proposal. y p p Academic/Educational Goals (shared and chosen by the professor and students involved: UPE and UNIPD): Acquiring/developing knowl- edge and skills regarding group dynamics, assertive communication, and educational relationships. p Community needs (expressed and shared by the FUNASE WG): creating moments of integration and developing positive relation- ships among adolescents in confl ict with the law welcomed by the institution, among teenagers and the staff of the institution itself and among teenagers and other reference people (adult) from the community. y Implemented activities: • interactive didactic laboratory on group dynamics and educational relationship, planned and managed by 45 students of the 8th Peda- gogical Period, UPE, at the UPE Headquarters and during the cur- ricular lessons with the active participation of the same students and 10 teenagers, welcomed by FUNASE. The workshop also provid- Figure 4 – INTEREURISLAND reference scheme for University Social Responsibility. Figure 4 – INTEREURISLAND reference scheme for University Social Responsibility. ACADEMIC CONTEXT Faculty, Departments or Degree Courses Practices developed by the local and foreign students involved, with the supervision of the lnternational Social Responsibility Working Group: - Study; - Interactive didactic workshops (within the courses of interest) with the participation of people from the community; - Intercultural activities and peer education COMMUNITY CONTEXT Public or Privat Institutions, Informal or non-formal groups. Practices developed by the local and foreign students involved, with the coordination of the Intersectorial Working Group: - Internship; - Interactive workshops (within internships activities) with the participation of other undergraduate students attending the course of interest - Intercultural activities and peer education INTEREURISLAND: Reference scheme for University Social Responsibility (Andrian N.) The Service Learning Cycle INTEREURISLAND: Reference scheme for University Social Responsibility (Andrian N.) COMMUNITY CONTEXT Public or Privat Institutions, Informal or non-formal groups. i pa stud The Service Learning Cycle The Service Learning Cycle Practices developed by the local and foreign students involved, with the supervision of the lnternational Social Responsibility Working Group: - Study; - Interactive workshops (within internships activities) with the participation of other undergraduate students attending the course of interest - Intercultural activities and peer education - Interactive didactic workshops (within the courses of interest) with the participation of people from the community; - Intercultural activities and peer education 215 INTEREURISLAND ed opportunities for sharing and dialogue between the students, the coordination of the department of Pedagogy and the socio-psycho- pedagogical WG of FUNASE, regarding the professional profile of the pedagogical coordinator in institutions dealing with minors in conflict with the law. Encounters on assertive communication and active listening, held weekly at the FUNASE institution and in the city park. Nine intern- ship mornings planned and conducted by local and foreign students, with the active participation of teenagers and the socio-psycho-ped- agogical WG of the Institution. Agostinetto L. 2013, Un educatore di frontiera: intuizioni, contributi e sfi de interculturali, in Cornacchia M. and Madriz E. (eds.), Educazione e 2.3 ‘Service Learning’ Service Learning (S.L.) is a pedagogical proposal that originated in the United States and has spread rapidly to different regions of the world. Many trials have been carried out in several countries over the last twenty years, accompanied by many definitions, recognized benefits, and scientific reference literature on it. ii As indicated by Maria Nieves Tapia (2006), and from a ‘Didactic Identity Card’ (Fiorin 2016), S.L. lets students (schools and/or in- stitutions of every level) be actors in the planning and implementa- tion of activities combining school learning (with precise curricular goals) and solidary service (identifying real needs together with sub- jects from the community). Service Learning seeks to engage students in activities that both com- bine community service and academic learning. Because service learning programs are typically rooted in formal courses (core academic, elective, or vocational), the service activities are usually based on particular cur- ricular concepts that are being taught (FURCO 1996). The Practical Guide, The Nature of Learning, Using Research to In- spire Practice from the OECD, Centre for Educational Research and Innovation (2010), includes S.L. among the eight different proposals for building innovative learning environments. In Italy, the Minis- try of University and Research (MIUR) has been promoting specific S.L. training for students and teachers with the Ministerial Decree No.663 of September 1, 2016, and the project ‘Introduction of meth- odologies of participation in the Italian school system’. g p p y The INTEREURISLAND model envisages that the planning and implementing of project activities follow the S.L. Cycle pro- posed by the National Youth Leadership Council (2009) as shown below (Figure 5). 216 NICOLA ANDRIAN Figure 5 – The Service Learning Cycle (National Youth Leadership Council 2009). 3. Final remarks 3. Final remarks International mobility and intercultural dialogue between players from diff erent countries, intersectorality between Universities and communities, Alternance between study, internships, and service learning proved fertile ground for exchange and enrichment, the ba- sis of the INTEREURISLAND model. The research highlighted the strengths and weaknesses of this possi- ble model that promotes the development of relationships at two levels. The research highlighted the strengths and weaknesses of this possi- ble model that promotes the development of relationships at two levels. At the international level, by creating a network of partnerships as a privileged line of exchange, mobility, intercultural dialogue, in- novative didactics, specifi c training, internships, and research. At the international level, by creating a network of partnerships as a privileged line of exchange, mobility, intercultural dialogue, in- novative didactics, specifi c training, internships, and research. At the local level, by creating intersectoral social responsibility WGs that facilitate peer-to-peer interaction between academia and their communities, building shared knowledge through innovative teaching and research activities, pro-social activities, sustainable de- velopment, and the practice of active citizenship. References References References 217 INTEREURISLAND insegnamento. La testimonianza di una realtà inscindibile Edizioni Università di Trieste, Trieste, 87-99. insegnamento. La testimonianza di una realtà inscindibile Edizioni Università di Trieste, Trieste, 87-99. 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References Fórum De Pró – Reitores De Extensão Das Universidades Públicas Brasileiras – Extensão Nas Instituições Comunitárias De Ensino 218 NICOLA ANDRIAN Superior 2013, Referenciais para a construção de uma Política Nacional de Extensão nas ICES, XX Encontro Nacional de Extensão e Ação Comunitária das Universidades e Instituições Comunitárias. Superior 2013, Referenciais para a construção de uma Política Nacional de Extensão nas ICES, XX Encontro Nacional de Extensão e Ação Comunitária das Universidades e Instituições Comunitárias Comunitária das Universidades e Instituições Comunitárias. Fórum De Pró – Reitores De Extensão Das Universidades Públicas Brasileiras 2013, Política Nacional De Extensão Universitária, Editora Universitária UFPE, Recife. 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Vigilante A. 2017, Il Service Learning: come integrare apprendimento ed impegno sociale, «Educazione Democratica, Rivista di Pedagogia politica», IV (7), January. Yin R.K. 1994, Case study research. Design and Methods, Sage, Thousand Oaks.i Yin R.K. 2005, Lo studio di caso nella ricerca scientifica, progetto e metodi, Armando Editore, Roma. * Giordana Szpunar, PhD, Assistant Professor of General Pedagogy, Department of Social and Developmental Psychology, Sapienza University of Rome, Italy. Email: gior- dana.szpunar@uniroma1.it. Wrote paragraphs 2, 4, 6; Barbara Barbieri, PhD, Assistant Professor of Psychology of Human Resources, Department of Social Sciences and Institutions, University of Cagliari, Italy. Wrote paragraphs 1, 3, 5. Giordana Szpunar (Sapienza University of Rome), Barbara Barbieri (University of Cagliari)* Giordana Szpunar (Sapienza University of Rome), Barbara Barbieri (University of Cagliari)* THE ROLE MODEL WITHIN PROFESSIONAL TRAINING. THE COMPLEXITY AND FLEXIBILITY OF EDUCATION PROFESSIONS Giordana Szpunar (Sapienza University of Rome), Barbara Barbieri (University of Cagliari)* Giordana Szpunar (Sapienza University of Rome), Barbara Barbieri (University of Cagliari)* 1. The training of professionals in the third millennium Nowadays, it is commonly agreed that the remarkable changes in social and labour organizations over the last decades have resulted in an increasing demand for professionals with high cognitive flexibility, able to constantly re-align their skills and upgrade their competences (Bauman 1999; Beck 2000a, 2000b; Forti, Varchetta 2001; Rullani, Vicari, 1999; Weick 1995). In this context, training – especially the training of professionals – has started to question its own meaning and nature, dramatically rethinking them. In fact, the training of professionals can no longer give practical competence a professional dimension, relying on sys- tematic and scientific knowledge to provide an instrumental solution to the problems: professionals must be able to reflect and act within ever-changing and unpredictable contexts, using their knowledge and skills in a flexible and effective way. lf y From a mere pedagogical and cultural point of view, it is neces- sary to analyse the most suitable training-didactic models for the de- velopment of reference prospects and schemes, as well as appropriate methods and tools for the training of ‘reflective professionals’ capable of developing the ‘artistic skill’ increasingly required to manage the ‘ever-changing’ work contexts. Despite the knowledge of the scenario where the future organiza- tional players are going to be acting, the academic world still struggles to regard the academic path as the ideal place for the accomplishment of tailor-made professional projects, thereby slowing down the entry of young graduates into employment (Zucchermaglio 2007). 224 GIORDANA SZPUNAR, BARBARA BARBIERI This scenario has led the reflection on training to believe that ap- prenticeships organized during studies might offer an opportunity to experience the future profession chosen by the students. Basically, an apprenticeship can be considered a shift from learning to doing/acting, the moment when the knowledge accumulated turns into the specific skills of that activity. However, this kind of apprenticeship is not an actual step towards employment – indeed, this is not its aim: it should instead trigger a transformational learning process where students begin to perceive themselves as future professionals, turning the knowledge gained into crucial skills for their profession. Therefore, learning still plays a key role at this stage, even though it is intended for the construc- tion of a professional role, instead of acquisition of the theories under- lying a certain profession. 2. The professional identity of educators and the importance of role models Nowadays, the stratification of the meanings underlying the con- cept of education is found in the common assumption that education has a procedural dimension covering the whole of life and all ages, including the various growth steps and difficult moments that char- acterize the construction of our personal identity. The onset of new individual needs and the resulting expansion of social policies and educational services have widened educators’ scope for action. Fur- thermore, the educational context can be regarded as a highly com- plex and dynamic system featuring a series of relational processes only partially predictable and operationalizable (Mortari 2009). Therefore, education professions, in particular, mostly act with- in unique, ever-changing and unpredictable working contexts. The large variety of professional contexts requiring the presence of educa- tors leads to an extensive branching of its operational features, which vary according to the recipients of the educational activity, their age, and specific needs, as well as to the preventive, promotional or reha- bilitation purpose of the same action. Education professions involve various skills connected to their mul- tidisciplinary profile, which help shape the operational representations and models underlying the professional activity but cannot enclose all the pedagogical knowledge required by these professions. Knowledge, the knowledge of how to do and the knowledge of how to act – intended as the box containing the necessary technical-professional tools – must complement and integrate the knowledge of how to stay within contexts and relations with a hermeneutic-transformative perspective. p p In this dimension, the Role Model acquires immense importance, since it is considered an example and a guide to give shape and meaning to the process leading to the construction of our professional identity. p g p y From a sociocultural point of view (Mclnerney, Roche, Mclner- ney, Marsh 1997; Kerka 1998), in fact, role models are applied by those professionals who – mostly unintentionally – help the student understand the many facets of their profession within specific educa- tion and work situations through observation, imitation, reflection and abstraction, thus promoting the construction of their professional identity (Cruess, Cruess, Steinert 2008). In this scenario, role-modelling acts as an important educational and training strategy, as largely underestimated as it is (Kenny, Mann, MacLeod 2003; Cruess, Cruess, Steinert 2008). The role modelling process (Fig. 1. The training of professionals in the third millennium Apprenticeship allows students to become familiar with a professional role, under the guidance of a tutor (situ- ational factor/social support), whether formal or informal (business/ in the work context/application). In fact, during this learning process, students should ideally become acquainted with the work practices of their profession under careful supervision. Thus, practitioners should benefit from specific training (Lave, Wenger 1991; Alastra, Kaneklin, Scaratti 2012) being personally involved in the activities of a practice community (Wenger 1999) and have direct experience of the constant evolution and unpredictability of the world of work within a simpli- fied and controlled professional context, through preliminary plan- ning and scrupulous supervision (Schön 1987). This would help them apply the knowledge acquired to practical professional situations with an evolving (Mezirow 1991) and reflective (Schön 1987) perspective. g ( )l ( ) p p Consequently, the achievement of professionalism is very com- plex. It requires the identification of effective teaching strategies no longer based exclusively on traditional content transmission models. As already stressed, most professional behaviours are learned through practical experience (Kenny, Mann, MacLeod 2003.) Professionals build their professional background on the job, taking inspiration from and interacting with more experienced colleagues and tackling their work experience day by day: in point of fact, successes and failures teach us ‘the way’ (Bourdieu 1980; Vino 2001). The role-modelling process begins precisely during the academic educational path as well as within didactic experiences and apprenticeships through observa- tion of the practices typical of that specific educational/work envi- ronment and the behaviour of experienced professionals. However, reflection on academic education lacks the study of role modelling in the sense of a process which dramatically influences the construction of a professional identity through observation and reflective imita- tion, while offering some interesting opportunities (i.e. apprentice- ships, and tutoring and mentoring services). THE ROLE MODEL WITHIN PROFESSIONAL TRAINING 225 THE ROLE MODEL WITHIN PROFESSIONAL TRAINING Consequently, learning through role models relies on a complex combination of conscious and unconscious actions, involving both observation and reflection. The active reflection on the process al- lows learners to turn unconscious acquisitions into conscious thoughts and transform the latter into principles and actions (Cruess, Cruess, Steinert 2008; Benbassat 2014). 2. The professional identity of educators and the importance of role models 1) shows how the establishment of professional behav- iours largely depends on the relation between a greater or lesser degree of voluntary imitation and a reflective elaboration of the behaviours observed. It is a social and critical process leading to the assessment GIORDANA SZPUNAR, BARBARA BARBIERI 226 and co-construction of content, which urges students to interpret an experience and assign a meaning to it. Any refl ection on the behav- iours observed alters (processes, creates, rejects, confi rms, questions) the meaning schemes currently used in relation to the integrated role model and, through the generalization of the conclusions, changes the meaning perspectives and the behaviours enacted (Mezirow 1991). Figure 1 – Role-modelling process. [Cruess, Cruess, Steinert 2008] Active observation of role model Making the unconscious conscious Reflection and abstraction Translating insights into principles and action Generalisation and behaviour change Unconscious incorporation of observed behaviours Active exploration of affect and values Figure 1 – Role-modelling process. [Cruess, Cruess, Steinert 2008] Figure 1 – Role-modelling process. [Cruess, Cruess, Steinert 2008] Active exploration of affect and values Generalisation and behaviour change In this learning framework, the professional reference (business or academic tutors, teachers, etc.) chosen by the students is a crucial ele- ment for at least two reasons: on the one hand, he/she represents the role model who acts as an example during the students’ approach to the professional role; on the other, he/she triggers the refl ection pro- cess and raises awareness of the experience, thus favouring ascent of the incorporation of the behaviours observed from the subconscious to the conscious level, in addition to the resulting transformation of intuitions into principles and actions. p p The ability to become aware of the behaviours observed and to re- fl ect upon them is fundamental for the effi cacy of role modelling. In fact, the refl ection in the action, on the action, and for the action (Schön 1987) allows future professionals to turn implicit contents into explicit ones (Cruess, Cruess, Steinert 2008), thus redefi ning their personal, social, and professional world (Brookfi eld 1986). Refl ection on the past experiences and behaviours observed is also important for the transformation of observations into concepts through a generaliza- tion process (Kolb 1984). THE ROLE MODEL WITHIN PROFESSIONAL TRAINING 227 3. Exploratory research 228 GIORDANA SZPUNAR, BARBARA BARBIERI More specifically, the focus groups were intended to redefine the students’ convictions about the characteristics of a good educator, as well as helping them identify any ‘reference’ people during their ed- ucational path or apprenticeship experience who acted as a model to construct their own professional identity, thus recognizing the char- acteristics necessary to become a role model. More specifically, the focus groups were intended to redefine the students’ convictions about the characteristics of a good educator, as The outcome of the focus groups was written down word for word, and examined using a conventional analysis approach.i g y pp The issues the students faced during these focuses included a defi- nition of ‘efficient’ educators as well as a description of the reference subjects met during their educational path or apprenticeship.i j g p pp p The first question was: «What comes to your mind when I say, “efficient educator?”». This question proposes to redefine the convic- tions of the Education Sciences students regarding the characteristics of an efficient educator. The second question was: «Try to recall your academic and ap- prenticeship experience. Now try to remember the people who had a major impact on your personal and professional growth path». The general stimulation and the specific stimulating questions (Who is he/ she? What are his/her personal features and professional skills? What was the most important lesson that you learned from this relationship?) aimed to analyse the kinds of professional who acted as reference models help- ing the students construct their professional identity during their ed- ucational path or apprenticeship. 3. Exploratory research The research introduced below was carried out in 2014 during the BSc and MSc courses in Educational and Training Sciences at The Sapienza University of Rome to investigate the characteristics of the role models chosen by the future educators, either in the academic context or during apprenticeships. g pp p The sample consisted of 34 students and graduates from The Sa- pienza University of Rome – 31 women and 3 men – of whom 12 were attending the 2nd and 3rd year of the three-year Degree Course in Educational and Training Sciences, while 16 were students on the Master’s Degree Course in Pedagogy and Educational and Training Sciences, who had already taken their BSc while 6 had taken both their BSc and MSc and were working in the educational field. Internship is mandatory during these courses. Consequently, all the individuals involved in the research had done one or more apprentice- ships. The most widespread sector was in services for early childhood, chosen by 12 people for their apprenticeships. The others were distrib- uted among group homes (6), universities (5), museums and educational institutions (5), residential, semi-residential or day-care facilities for peo- ple with mental or physical disabilities (4), prisons (1) or hospitals (1). p p y ( ) p ( ) p ( ) Out of the 34 subjects involved in the research, 14 were working on an occasional or ongoing basis. In particular, 5 people were ten- ured or substitute teachers at early childhood education institutes, 2 were baby-sitters, 1 was a swimming instructor, 1 an elderly caretak- er, 1 a part-time waitress, 1 a shop assistant and 1 a support teacher. Thus, 12 out of the 14 workers were operating in educational areas. Students and graduates were divided into 5 focus groups made up of 6-8 participants each. The focuses were presented by two experts and lasted about 3 hours. The focus group was used as an exploratory tool, since it helped understand the unconscious aspects of motivation and behaviours with reference to a specific issue (Krueger 1994; Morgan 1998); fur- thermore, the flexible, interactive, and dialogic nature of this tool al- lowed the individuals to express ideas and feelings which would not have emerged otherwise during an interview (Stagi 2000). 4. Representations of the professional identity of educators Analysis of the first issue discussed within the focuses shows the complexity of the professional role of socio-cultural educators. In fact, this kind of profession has ill-defined boundaries and opposes any attempt to stabilize and systematize it, more readily acting as a prototype and example of the uncertainty and unpredictability of the current employment market. p y The factors that undermine the profession of educator – thus weakening its social and legal recognition, as well as its perception of the construction of identity – are several and act at various levels. The diffusion of the educational needs of modern society, the vari- ous kinds of subjects expressing these needs, the expansion of the in- tervention scope of education professionals, their growing tasks, the quantitative and qualitative complexification of the interpersonal re- lations to be managed in the work context (organization, customer, multi-professional team, users, families), the lack of a specific and well-defined scientific background lead to the definition of an ever- THE ROLE MODEL WITHIN PROFESSIONAL TRAINING 229 changing constant professional profi le (Szpunar, Renda 2015; Tram- ma 2008; Maccario 2009). ) All these aspects have been stressed by the refl ections and ac- counts of students and graduates. The professional contexts of their work or apprenticeship experiences are very diff erent, since they range from early childhood services –the most widespread fi eld – to group homes, schools, prisons, hospitals, residential, semi-residential and day-care facilities. The subjects looked after by the professionals varied greatly in terms of age and educational needs. The students and graduates were aware of this, and they believed that educators’ skills and competences must take this complexity and unpredict- ability into account in the contexts and among the addressees of the service. The importance given to creativity, the ability to adapt, face and manage diff erent situations, to communicate and interact with vari- ous subjects, schedule and organize, plan and improvise at the same time, change action plans and successfully solve problems are a clear sign of this awareness.fi Generally speaking, the ‘effi cient’ educator outlined by the analysis of the focuses is one who can rely on a theoretical training that must remain up-to-date (the educator is experienced, informed, and conducts researches), while knowledge is not regarded as a priority. According to the students and graduates, the construction of meaning referring to education professionals (Fig. Figure 2 – Dimensions of an educator’s fi gure. 4. Representations of the professional identity of educators 2) mainly includes personal aspects and transverse skills, precisely due to the remarkable variability of contexts, subjects and assigned tasks. Figure 2 – Dimensions of an educator’s fi gure. Educator Sense of reality Organizational dimensions Events Socio-relational dimensions Procedural dimensions Proactivity Sensemaking Theoretical knowledge Emotions/ affections Values dimensions Knowledge is backed up by the knowledge of how to make and how to be. The accounts show educators able to train, take care, listen, support, accompany, help learn and understand, guide, stimulate, promote growth, pro- vide tools, lead towards a target, foster autonomy, enhance the qualities and potential of each person. This involves some personal features that turn education professions into a mission or vocation requiring a special pas- sion as well as an ability to see what others cannot notice. Figure 2 – Dimensions of an educator’s fi gure. Educator Sense of reality Organizational dimensions Events Socio-relational dimensions Procedural dimensions Proactivity Sensemaking Theoretical knowledge Emotions/ affections Values dimensions Figure 2 – Dimensions of an educator’s fi gure. Figure 2 – Dimensions of an educator’s fi gure. Values dimensions Sense of reality Emotions/ affections Theoretical knowledge Educator Events Sensemaking Procedural dimensions Proactivity Knowledge is backed up by the knowledge of how to make and how to be. The accounts show educators able to train, take care, listen, support, accompany, help learn and understand, guide, stimulate, promote growth, pro- vide tools, lead towards a target, foster autonomy, enhance the qualities and potential of each person. This involves some personal features that turn education professions into a mission or vocation requiring a special pas- sion as well as an ability to see what others cannot notice. 230 GIORDANA SZPUNAR, BARBARA BARBIERI … I intend education professions as a vocation requiring a certain attitude. Those who choose this profession must be able to help others…fii … In my opinion, efficient educators are able to combine scientific and ac- ademic knowledge with an art, the art of teaching. 4. Representations of the professional identity of educators Unquestionably, they can- not rely exclusively on formalized requirements, but must prove to be aware of their mission…fi Consistently, the skills and main characteristics of efficient educa- tors are empathy as well as the ability to understand others, actively listen to interlocutors, wait and see, reject prejudices, accept others’ ideas while also reformulating their own assumptions, be self-critical, work with others and co- operate positively, be calm, patient, serene, sympathetic, kind, and respectful with others, authoritative (and not authoritarian), possess leadership skills, strength, resolution and impartiality. In view of the above, it is easy to understand the key role played by a role model for the construction of such a complex kind of pro- fession, with special regard to the various stresses associated with it (individual, relational, professional and organizational). THE ROLE MODEL WITHIN PROFESSIONAL TRAINING 231 … He told me how to tackle some challenges… and helped me overcome my fear… of interacting with others… … He provided me with theoretical tools […] we studied many books and other things. But at the same time, at a practical level, he showed me the meaning, tasks, and missions of educators …i Therefore, Role Models give confidence and credit and strengthen respon- sibility, are able to listen, accept others, interact with others as an equal intel- lectual partner but, at the same time, can maintain a distinction between roles, are always authoritative, are humble and make room for others, are passionate and are able to communicate their passion, can mediate and negotiate, inspire confidence because they keep everything under control and are able to overcome any obstacle and solve any problem. The second dimension of Role Models as evidenced by the litera- ture (Wright 1996; Wright, Wong, Newill 1997; Wright et al. 1998; Elzubeir, Rizk 2001; Wright, Carrese, 2002; Cruess, Crues, Stein- ert, 2008; Fromme et al. 2010; Jochemsen-Van der Leeuw et al. 2013) deals with the profession-related knowledge of ‘how to be’ (value di- mension, meaning dimension, socio-relational dimension, affectiv- ity, design intentionality.) The outcome of the focuses can be summarized by the account of one of the participants, which mentions most of the features highlighted by the interviewees, in different words, but with the same content: (The RM) is authoritative, empathic, passionate. Passion is necessary… l d l l f d f l (The RM) is authoritative, empathic, passionate. Passion is necessary… Role Models act as a true example of education professional, since they help understand, stimulate, enhance our potential, explain, and guide us along our path. Therefore, as educators, they are respectful, enthusiastic, pleasant, easy, calm, straightforward, helpful, self-confident − but without being dogmatic − sensitive, human, empathic, cheerful, strong, resolute, charismatic, steadfast, able to find positive characteristics, let go, and motivate. … (RM) has dramatically changed my life […]. I was guided by her ex- ample and was encouraged by the strength that she gave me. During the ap- prenticeship she came to the facility only once, but she was always there for me. I could see her everywhere, with her attitude and ability to involve me in the activities. […] Her behaviour, teachings, competence and expectations and the texts that she suggested were always mirrored in my experience […]. 5. The choice of role models All the participants identified at least one reference figure met dur- ing their academic studies (professor) or apprenticeship (business tu- tor.) Basically, their accounts confirmed the literature dealing with students’ assessment of the characteristics observed in positive mod- els (Wright 1996; Cruess, Crues, Steinert 2008; Fromme et al. 2010; Jochemsen-vander Leeuw et al. 2013). Moreover, the focus analysis showed that the meaning dimensions assigned by the future educa- tors correspond to those attributed to recognizing the prototype ed- ucator (Fig. 2).i The first dimension, concerns technical knowledge associated with the education professions (theoretical knowledge, value dimension, meaning dimen- sion, socio-relational dimension). The result of this analysis is a skilful professional endowed with knowledge, great problem-solving and decision-making skills, and the ability to adapt his/her competence to the various contexts (RM can manage time and different situations, solve problems, keep everything un- der control and overcome any obstacles and is open-minded). However, spe- cial emphasis was given to the ability to guide and orient: basically, positive role models can orient, support, and promote critical reflec- tion on practical actions. They are a reference.i p y f … The RM is a guide who identifies your talent and potential… … directs your choices on the most appropriate path then allows you to gain experience… THE ROLE MODEL WITHIN PROFESSIONAL TRAINING THE ROLE MODEL WITHIN PROFESSIONAL TRAINING She could find a potential in me of which I was not aware… … (RM) guided me by showing me how he was able to construct his pro- fessionalism and career. Somehow, this cooperation enhanced my creativity and my ability to develop an idea. … I spent a lot of time with this person, who helped me make my own decisions. He did not supplant me, but taught me how to manage my choices for my career and future. 232 GIORDANA SZPUNAR, BARBARA BARBIERI The third dimension of role modelling includes the physical and symbolic places of the educational process (organizational or proce- dural dimension, events, reality elements and value dimension.) The context and the place are crucial aspects for the focus par- ticipants. It is possible to assume that the role model would act in a different way in another context. The place is described as a home, a familiar location (both well-known and cosy), quiet, tidy, and protected. Thus, the place is the privileged context where the role model urges to reflect in the action, on the action, and for the action, thus allow- ing the future professionals to reinterpret their experience, promoting the transformation of experience into professional skills. The places are the framework within which the organization, process manage- ment, teamwork, complexity, and unpredictability of this profession are experienced. 6. Final remarks In line with the literature on role modelling, the focus analysis stresses that positive role models and the places intended for learn- ing are key throughout the whole educational process, thus influ- encing the personal and professional growth of future professionals. Students, in fact, select and choose the characteristics of their refer- ence models, by internalizing a mix of values, behaviours, attitudes and competences consistent with the ideal image of the profession. Sometimes the model chosen does not carry out the specific profes- sional role assigned to the future educators. Teachers are often re- garded as reference models thanks to their personal and professional qualities, which are identified, internalized, and then projected by students onto their future profession. p The accounts of the participants show the important support func- tion played by role models for the students at various levels. First, the role model stimulates the ability to interact with action- related knowledge, thus promoting the learning processes associat- ed with space and the willingness to reflect on concrete professional practices. Then, the role model helps the students recognize and reinterpret the unconscious processes underlying the construction of their own professional identity, manifesting motivations, skills, implicit apti- tudes and fostering orientation and professional choices. In order to promote and support positive modelling processes, the institution must be characterised by an organizational culture oriented towards the development of reflective practices, the defini- tion of spaces and times intended for the re-elaboration of the stu- THE ROLE MODEL WITHIN PROFESSIONAL TRAINING 233 dents’ educational and didactic experiences, intended as a relevant moment for the construction of their professional and individual career (Zucchermaglio 2007; Salerni, Sposetti, Szpunar 2013) and the ‘growth’ of interpersonal relations (Cruess, Cruess, Steinert 2008). 6. Final remarks In other words − being a multidimensional instrument ap- plied to the place where theory and practice meet − the educational and training potential of role modelling could be promoted, devel- oped, and enriched by reflective dimensions and personal elabora- tions, as well as by the activation of some pedagogical models and patterns sharing some significant aspects with it, such as cognitive apprenticeship, situated learning, and reflective practice (Kenny, Mann, MacLeod 2003).f Eventually, for role modelling to be used as an effective teach- ing strategy and to reduce the impact of negative models, it would be necessary to identify and, if necessary, to train professions featur- ing those characteristics that students tend to seek in their reference models. This would promote an informed support to the modelling process, thus enhancing the positive results (Wright 1996; Jochem- sen-Van der Leeuw et al. 2013). 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Kolb D.A. 1984, Experiential learning: experience as the source of learning and development, Prentice Hall, Englewood-Cliffs. p gf Krueger R.A. 1994, Focus groups: a practical guide for applied research, Sage Publications, Thousand Oaks. Lave J. and Wenger, E. 1991, Situated learning. Legitimate peripheral participation, Cambridge University Press, Cambridge.fi Maccario D. (ed.) 2009, L’educazione difficile. La didattica nei contesti socioculturali e trasversali, Carocci Faber, Roma. Mclnerney D.M., Roche, L., Mclnerney V. and Marsh H.W. 1997, Cultural perspectives on school motivation. The relevance and application of goal theory, «American Educational Research Journal», 34, 207-236. Mezirow J. 1991, Transformative dimensions of adult learning, Jossey-Bass, San Francisco. Morgan D.L. and Krueger, R.A. 1998, Planning focus groups (Vol. References 2), Sage, Thousand Oaks.l Mortari L. 2009, Ricercare e riflettere. La formazione del docente professionista, Carocci, Roma. 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Senso e complessità del lavoro educativo, Carocci Faber, Roma. Vino A. 2001, Sapere Pratico, Guerini e Associati, Milano. Weick K. 1995, Sensemaking in organizations, Sage Publications, Inc, Thousand Oaks. Wenger E. 1999, Communities of practise: learning, meaning, and identity, Cambridge University Press, Cambridge. Wright S., Wong A. and Newell C. 1997, The impact of role models on medical students, «Journal of General Internal Medicine», 12, 53-56. THE ROLE MODEL WITHIN PROFESSIONAL TRAINING * Cristina Palmieri, PhD, Researcher in General and Social Pedagogy, Department of Human Sciences “Riccardo Massa”, University of Milano-Bicocca, Italy. Email: cris- tina.palmieri@unimib.it; Marina Barioglio, PhD, Post-doc Researcher in Education, Department of Human Sciences “Riccardo Massa”, University of Milano-Bicocca, Italy. Email: marina.barioglio@unimib.it; Andrea Galimberti, PhD, Post-doc Researcher in Education, Department of Human Sciences “Riccardo Massa”, University of Milano- Bicocca, Italy. Email: andrea.galimberti1@unimib.it; Maria Benedetta Gambacorti- Passerini, PhD, Post-doc Researcher in Education, Department of Human Sciences “Riccardo Massa”, University of Milano-Bicocca, Italy. Email: maria.gambacorti@ unimib.it; Tania Morgigno, PhD, Master Degree of Pedagogical Sciences, Traineeship Tutor, Department of Human Sciences “Riccardo Massa”, University of Milano- Bicocca, Italy. Email: tania.morgnigno@unimib.it. THE ROLE MODEL WITHIN PROFESSIONAL TRAINING 235 Wright S. 1996, Examining what residents look for in their role models, «Academic Medicine», LXXI (3), 290-292. ( ) Wright S. and Carrese J.A. 2002, Excellence in role modeling: insight and perspectives from the pros, «CMAJ», CLXVII (6), 638-643. Wright S., Kern D.E., Kolodner K., Howard D.M. and Brancati F.L. 1998, Attributes of excellent attending-physician role models, «The New England Journal of Medicine», 339, 1986-1993. J Zucchermaglio C. 2007, Il tirocinio in psicologia: la costruzione di interazioni ai confini, «Psicologia sociale», II (3), 441-444. THE ‘SECOND LEVEL’ EDUCATION PROFESSIONAL: A TRAINEESHIP PROGRAMME FOSTERING A PROACTIVE APPROACH TO EMPLOYABILITY Cristina Palmieri (University of Milan Bicocca), Marina Barioglio (University of Milan Bicocca), Andrea Galimberti (University of Milan Bicocca), Maria Benedetta Gambacorti-Passerini (University of Milan Bicocca), Tania Morgigno (University of Milan Bicocca)* Abstract: In this chapter, we outline the process that led to the development of the current traineeship programme − Tirocinio Formativo e di Orientamento (TFO) related to the Master’s Degree Course in Education at the University of Milan- Bicocca. We focus particularly on the interconnection between developing pro- fessional competences and addressing the issue of employability. Keywords: ‘second level’ education professionals, traineeship, employability, professional skills. 1. Introduction On the introduction of the Master’s Degree Course in Education at the University of Milan-Bicocca in the 2008-2009 academic year, its regulations already included a practical training component. This initial provision was based on feedback received from the students of the former Master’s Degree Course in Educational Counseling and Research, who had forcefully emphasized the importance of includ- ing a component which up to that point had not been part of the ac- ademic programme, and whose function was to give students insight into the role and functions of the ‘second-level’ education professional in real-life educational settings and allow them to compare their ac- ademic learning with the educational practices encountered in spe- cific workplaces. By ‘second-level’ education professional we mean 238 C. PALMIERI, M. BARIOGLIO, A. GALIMBERTI, M.B. GAMBACORTI PASSERINI, T. MORGIGNO a professional who is trained to act as a supervisor, trainer, evalua- tor, coordinator, or counsellor in the field of education, and who has therefore acquired a ‘second-level’ perspective on educational work. q p p A time of 150 hours was allotted for practical training activities during the Degree Course, and the academic staff with responsibil- ity for this component immediately began to reflect on how to struc- ture and present the training placement, initially opting for a focus on educational research. The theoretical and epistemological view underlying this initial strategy was that research is an instrument that fosters a heuristic, criti- cal, and transformative approach to educational work and the way it is thought about, organized, conducted and evaluated in education services (Ground 1992; Schön 1993; Mortari 2007; Fook, Gardner 2007). The first edition of the traineeship, labelled “research-training activity”, was initially the responsibility of individual lecturers teach- ing on the Degree Course and was tied in with the undergraduate thesis that the students were required to produce under the regula- tions of the Degree Course. g After some years, the Degree Programme Noard and the Review Board concluded that this model was no longer satisfactory, among other reasons because the students were finding it difficult to com- plete their research activities and theses on schedule. Thus, in the 2013-2014 academic year, a pilot tutoring service was introduced. 1. Introduction The students who opted to avail themselves of this service were as- signed to a training group, with a tutor whose role was to support them and monitor their progress through the research-placement programme (via face-to-face meetings on campus and on a virtual platform, following a blended-learning approach). The experimen- tal service was constructed from a set of assumptions regarding the students’ characteristics. The students were expected to be able to identify their research questions and strategies and to draw on their academic knowledge to inform their decisions and analyse the data collected. Students were also assumed to be: • accessible: present on campus, or available virtually;fi • accessible: present on campus, or available virtually;fi • appropriately active: self-sufficient; not merely passively following instructions and absorbing contents; • responsible for their own course of studies: able to make their own decisions; • professionally oriented: aware of their professional and personal aspirations, and interested in the world of educational work. Subsequent monitoring of the pilot service suggested that this rep- resentation was over-idealistic and continually challenged by the stu- dents’ actual characteristics. THE ‘SECOND LEVEL’ EDUCATION PROFESSIONAL 239 From April 2014 to July 2015, the pilot service was used by 85 students, who were divided into six groups and supervised by three tutors. One of the tutors coordinated the entire service and had responsibility for setting up and managing its online component. Continuous monitoring of the pilot service by the group of tu- tors and the representative of the Degree Programme Board with responsibility for the traineeship component, showed that it had had a disorienting and “displacing” effect on both the students and the tutors. It was deemed necessary to conduct a critical review of the de- scription of the student that had informed the design of the ex- perimental service: in the context of research carried out by two students for their theses, data was gathered on the thoughts and reflections of both students and tutors, and accordingly the train- eeship programme was once more revisited and redesigned. In the 2015-2016 academic year, a new programme was launched, labelled Tirocinio Formativo e di Orientamento (TFO) or Traineeship and Guid- ance Programme which was no longer optional but compulsory for all students, and was informed by the experience and observations of the preceding years. 2. Traineeship programme and employability In the learning society, it is becoming increasingly vital to preside over the transition from academic training to the learning that takes place in work settings (Stokes 2015). The relationship between uni- versity training and learning on the job poses a key challenge for all higher education and implies that there is a constant and continuous relationship between knowledge and knowledge in action (Cambi 2004), such that any structured experience may become a source of learning (Mortari 2003).l g ( ) The market seems to require flexibility and entrepreneurship (OECD 2015) for everyone taking their first steps outside the univer- sity towards professional contexts. Individuals are increasingly con- sidered responsible for their professional projects and are requested to imagine, project, and realize their career autonomously. Universities are requested by policy makers to take a stance in relation to these issues by implementing educational processes with clear pedagogical theoretical frameworks and coherent actions. This means not only focusing on the relation between competences and knowledge (Dozza 2012) but also developing a general approach, critical and productive at the same time, in relation to issues such as employability, entrepreneurship, flexibility: namely all those compe- tences considered key factors for future (OECD 2015). Flexibility, for example, can be considered on the one hand as a fundamental disposition to acquire but, on the other, as a concept that is questionable. Various critical voices have highlighted the risk of a passive adaptation to the market’s rules and requests (Sennett 1999). Flexibility is often connected to employability, a concept that rep- resents one of the principal areas in which the difficulty of academic institutions and professional sectors in engaging in constructive dia- logue is most visible. The underlying cause of this is their contrast- ing views of what it means to combine academic knowledge, critical thinking, and professional action (Stokes 2015). The current mainstream interpretation of the concept of em- ployability emphasizes individual responsibility for constructing the ‘right’ competences to enjoy a satisfactory professional career (Field 2006). This is what we might term a merely ‘adaptive’ interpretation of employability, according to which, once an appropriate profile has been developed, the careers market will automatically respond posi- tively. This social representation is still very widespread and should be borne in mind since it frequently implicitly informs the attitudes and expectations of university students. 240 C. PALMIERI, M. BARIOGLIO, A. GALIMBERTI, M.B. GAMBACORTI PASSERINI, T. MORGIGNO 240 C. PALMIERI, M. BARIOGLIO, A. GALIMBERTI, M.B. GAMBACORTI PASSERINI, T. MORGIGNO 1. Introduction Under this new programme, students were to be divided into work groups, each with a dedicated support tutor, and invited to orient themselves in the world of professional educational work by first studying the figure of the second-level education professional, before then going on to conduct research and documentation ac- tivities in an educational service setting with a view to exploring the professional education roles being implemented there. Subse- quent participation in seminars delivered by experienced education professionals would allow them to further explore key aspects of educational work. Finally, in the light of these experiences, the tu- tors would guide the students to reflect on their own professional identity with a view to producing a self-assessment of the skills ac- quired to date, areas for improvement, and potential future actions, on the basis of which to define their personal professional develop- ment plans for entering the workplace. The path, implemented for the first time in the 2015-2016 aca- demic year, has been further refined in the current academic year, including focus group discussions conducted with the students at the end of the full year of operating the TFO. Given this background, we now focus on the interconnection be- tween identifying and developing professional competences on the one hand and dealing with employability issues on the other, since these factors come into play in the TFO Traineeship and Guidance Programme. 2. Traineeship programme and employability Alongside or overlapping with this adaptive perspective, we increasingly find a representation of employability as more proactive. This more recent interpretation is THE ‘SECOND LEVEL’ EDUCATION PROFESSIONAL 241 being promoted by leading international organizations (OECD 2015) that dictate the education agenda and policies of their member coun- tries; it calls for an active approach to employability and self-placement, so that not only is it necessary to adapt but also to construct suitable spaces for the exercising of one’s profession. While on the one hand this way of looking at employability helps to overcome the illusion of an academic curriculum that can provide automatic access to the job market, on the other it shares one of the greatest risks of the lifelong learning paradigm, that of an increasing trend towards individualism (Dozza 2012; Biesta 2006). But what do we mean when we refer to new professional possibilities? Does pro- activity still risk being interpreted merely as an updated adaptation to market needs? Different researches (Yorke 2006; Boffo et al. 2017) show the possibility of interpreting the notion of employability not in an instrumental and reductionist way but enhancing its learning potential. If we do not consider employability only as an individual ability to be promoted among students it could, in fact, become a category able to activate reflection on the educational setting acti- vated (e.g. classroom didactics, fieldwork, etc.). In this sense, it could also represent an interesting lens through which to activate reflec- tions about how universities create relationships and dialogues with external stakeholders (for example, the organizations that host stu- dents during traineeship programmes). The notion of employability can be described, within this frame- work, as a meta-competence that emerges from a specific context in relation to the ability of the academic actors (professors, traineeship tutors, students) to contribute, on the basis of their own role and their own characteristics, in enhancing reflexivity about the profes- sional world. At the same time, the notion of entrepreneurship can be inter- preted through the representation of an ‘entrepreneur-student’ − as suggested by a much-criticized utilitarian strand (Fairclough 1993) − but also with the meaning of critical thinker, able to analyse the professional scenario and take a stance. This entails avoiding some illusions that the word ‘entrepreneurship’ fosters (e.g. 2. Traineeship programme and employability the focus only on individual abilities to meet market needs) in order to structure aptitudes and practices that can deal with what really takes place in professional contexts, and particularly in educational ones. p p y In designing the TFO, therefore, we strove to achieve a middle ground by encouraging a constructive and creative approach that seeks to transform the notion of flexibility (a term currently grossly abused in the work context), redefining it as moving from merely adapting to the requirements of the context to cultivating a receptive and attentive outlook that is also critical-reflective and proactive. This has meant re- 242 C. PALMIERI, M. BARIOGLIO, A. GALIMBERTI, M.B. GAMBACORTI PASSERINI, T. MORGIGNO jecting a pure ‘apprenticeship’ model and no longer offering training placements that almost exclusively involve shadowing qualified educa- tion professionals, as has typically been the case on undergraduate in- ternship schemes in the past. Students tend to remain firmly anchored to the earlier model. The new model is underpinned by the conviction that the sensitivity and skills required by an educator to be construc- tively employed in an educational service, particularly, in a ‘second- level’ capacity, go beyond the transverse skills usually labelled as ‘soft skills’, such as group work or communication skills (Alberici 1999). g p The tutors’ various interventions and their positioning with re- spect to the learning path of the students were thus designed with the aim of drawing out the link between the undergraduates’ future employability and their ability to identify, using the knowledge they have acquired, their own specific field of action, clearly define it, and communicate it to other professionals. In redesigning the train- ing placement, we have also sought to bring to light the cooperative dimension of this programme: it is a truly collective enterprise that involves not only the students but also the tutors and the coordinator of the service in managing the relationship with the outside organiza- tions that receive the students on placement. Implementing the new edition of the traineeship programme has often meant increasing the level of dialogue between the university and the host institutions and revisiting the partners’ mutual expectations. Indeed, the implicit ex- pectation of the partner institutions is usually that they are taking on an ‘apprentice’ to be initiated in working in the field. 2. Traineeship programme and employability Hence, they are generally taken aback by the proposed new format: a relatively small proportion of hours in the field (at least 55), a research perspective that questions functions rather than specific situations, an in-depth exploration of educational issues that in some cases have never been identified or addressed by those running the education service. THE ‘SECOND LEVEL’ EDUCATION PROFESSIONAL Prior to being sent on placement, each group of students, under the guidance of their tutor, is invited to reconstruct the figure of the second-level education professional (functions, role, knowledge base, training etc.). They do this by sharing their own previous professional experience (where present), reworking the knowledge acquired dur- ing their studies and engaging in exploratory, narrative, imaginative, and analogical activities designed to bring to light the many facets of the figure of the educator, as it has been formed in individual and collective imaginaries. A blended learning approach is implemented, in that the work ini- tiated in face-to-face sessions with the tutor is continued on the vir- tual platform Moodle, facilitating continuous interaction among the participants in the work groups and the collection of a large volume of documentary material that is then reworked and interpreted from an educational perspective. The examination of the material produced, reflecting on it, and seeking to identify its explicit and implicit edu- cational meanings has proved to be a key learning moment during the TFO programme. p g This phase of the programme has helped students stand back from the urgency of being in the thick of educational action, encourag- ing them to shift their perspective onto a more critical and thought- ful plane. This in turn allows them to call into question the image of the educator, deconstructing stereotypes, and ideas that are naive or over-idealized or merely consist of a list of duties. In many groups, an interesting outcome was the students’ new- found awareness of a significant difference between the figure of the educator portrayed in academic discourses, and echoed in the new draft education bill, or that emerges from one’s personal imagery, and the concrete figure encountered in real life. i g Becoming aware of this gap, and the group discussions that it elic- ited, impacted the students’ perceptions of employability, encouraging an attitude of readiness to familiarize themselves with the educational services they will work with in the future and to offer an active con- tribution in terms of conducting enquiries and facilitating improve- ments, rather than expecting to consolidate the skills demanded by the different services by gaining experience in the field. 3. In the field: from disorientation to learning Our experience with various groups of students over the past num- ber of years suggest that very few students are capable of fully and consciously bringing to bear a truly ‘second-level’ educational per- spective, or of seeing themselves as the active protagonists of their own training placement (Palmieri et al. 2015; Galimberti et al. 2016). g p To address this issue, it is therefore desirable that the tutoring func- tion comprises ad hoc individual and group training activities, deliv- ered both face-to-face and via distance modes.i The decision to precede field work with a preparatory phase has proved to be particularly effective in this regard. 243 THE ‘SECOND LEVEL’ EDUCATION PROFESSIONAL THE ‘SECOND LEVEL’ EDUCATION PROFESSIONAL In other words, a process of displacement was underway, whereby they had begun to abandon a concept of employability as passive adaptation to one par- ticular work setting, to employability as a fertile and proactive pres- ence in a given context. Furthermore, choosing to give the students a mandate to conduct research, and not only to shadow the on-the-job activities of a pro- fessional educator bore the specific educational purpose of reinforc- ing this new stance; a courageous decision, not without its dangers 244 C. PALMIERI, M. BARIOGLIO, A. GALIMBERTI, M.B. GAMBACORTI PASSERINI, T. MORGIGNO and rich with the potential for learning on the part of the students firstly, but also for the partner institutions and the tutor. The delicate phase of beginning the placement proper, although carefully planned, and carefully negotiated with the host services and supported by the tutors, has often required the students to deal with complex situations that are sometimes difficult, but potentially transformational. Emblematic, for example, is the initial encounter between the student and their contact in the host organization, since this is when the trainee has the opportunity or obligation to reiterate the need for the presence of a second-level function in an educational service. This passage often bears the characteristics of an initiatory experi- ence, in which trainees find themselves in a disorienting situation and must go through a series of tests: from the need to justify their mission within the host organization, by negotiating their research aims, to avoiding collusion with frequent requests on the part of the host organizations to carry out first-level placement activities. This is a process under construction, currently offering students interesting ‘incidents’ that can however, if well thematized, offer valuable op- portunities for learning. The students, who expect to be ‘accepted’ by the placement organization often find themselves having to cope with scepticism about the possible value of their training assignment: «We have no second-level professionals here», «We have no educators here, what are you coming to observe?»; «We don’t do educational work here, just entertainment». THE ‘SECOND LEVEL’ EDUCATION PROFESSIONAL This is a moment that can be daunt- ing, however, the cooperative bases of the traineeship module are designed to transform it − with the help of the tutor and the group −into a moment of reflection that allows the student to see beyond − into a moment of reflection that allows the student to see beyond the immediate difficulty, and to lay the foundations for a meaning- ful experience within this particular service and for this particular service. The presence of a tutor who can receive and contain the stu- dents’ discouragement, allowing them, nonetheless, to fully live out this experience of disorientation, and to draw on their own resources is especially important. It is hoped that one of the main outcomes for the students will be precisely that of acquiring the ability to identify, articulate and defend their knowledge, even in settings that do not immediately appear ‘suited’ to their professional profile. THE ‘SECOND LEVEL’ EDUCATION PROFESSIONAL This is a moment that can be daunt- ing, however, the cooperative bases of the traineeship module are designed to transform it − with the help of the tutor and the group − into a moment of reflection that allows the student to see beyond the immediate difficulty, and to lay the foundations for a meaning- ful experience within this particular service and for this particular service. The presence of a tutor who can receive and contain the stu- dents’ discouragement, allowing them, nonetheless, to fully live out this experience of disorientation, and to draw on their own resources is especially important. It is hoped that one of the main outcomes for the students will be precisely that of acquiring the ability to identify, articulate and defend their knowledge, even in settings that do not immediately appear ‘suited’ to their professional profile. Emblematic, for example, is the initial encounter between the student and their contact in the host organization, since this is when the trainee has the opportunity or obligation to reiterate the need for the presence of a second-level function in an educational service. This passage often bears the characteristics of an initiatory experi- ence, in which trainees find themselves in a disorienting situation and must go through a series of tests: from the need to justify their mission within the host organization, by negotiating their research aims, to avoiding collusion with frequent requests on the part of the host organizations to carry out first-level placement activities. This is a process under construction, currently offering students interesting ‘incidents’ that can however, if well thematized, offer valuable op- portunities for learning. The students, who expect to be ‘accepted’ by the placement organization often find themselves having to cope with scepticism about the possible value of their training assignment: «We have no second-level professionals here», «We have no educators here, what are you coming to observe?»; «We don’t do educational work here, just entertainment». 3. Final remarks 3. Final remarks In our experience, the TFO traineeship programme is increasingly proving to be a key space in which students can identify and experi- ment with applying the skills of ‘second level’ education professionals. 245 THE ‘SECOND LEVEL’ EDUCATION PROFESSIONAL As we move forward with the programme, we plan to continue ex- ploring the interrelationships between guidance, skills development, and employability. To this end, we are introducing a self-reflexive approach into the training placement experience on an increasingly systematic basis, and are also designing a cooperative enquiry project, with the participation of the students, to enrich our theorizing with the perspectives of those who experience first-hand the constraints and the opportunities inherent in the programme. pp p g We interpret the construct of employability as a meta-competence to be developed, a framework within which to locate different pos- sible kinds of professional activity. For trainee educators, seeking out the peculiarly educational dimension of the professional settings ana- lysed on the placement programme means, at a different level, actively seeking to identify and legitimate a professional identity of their own. The challenge has been to encourage proactive approaches, given that trainee educators’ future employability will depend on their capac- ity to identify, using the knowledge they have acquired, their own specific field of action, clearly define it, and communicate it to other professionals. At the same time, we aim to create a context that can enrich all the various stakeholders involved in the programme. In fact, while on the one hand, we aim to help students in approaching a fu- ture professional identity, on the other, and at the same time, we be- lieve in the potential to enrich also the professional contexts that offer their contribution in this effort. This stance is based on the idea that promoting employability, entrepreneurship, and flexibility in students’ curricula is not an effort related only to university: it is a process that can be activated by all the actors (students, university professors, train- eeship tutors, educational services) involved in the programme. If stu- dents have the opportunity to explore their competences in relation to professional contexts, observing existing practices and processes, on the contrary, hosting organizations have the possibility to receive student researches: an analysis of part of their educational functions. 3. Final remarks We believe that this encounter can be generative: students do not only ‘receive’ training but play an active role, representing a fundamental opportunity to observe the existing practices and processes from an- other point of view, less experienced but, at the same time, potentially able to generate reflections. Professional contexts, in fact, are always at risk of fixing their practices in procedures and reducing the education complexity in linear organizational thinking: for them, the possibil- ity to open up and listen to different point of views is fundamental. We interpret the construct of employability as a meta-competence to be developed, a framework within which to locate different pos- sible kinds of professional activity. For trainee educators, seeking out the peculiarly educational dimension of the professional settings ana- lysed on the placement programme means, at a different level, actively seeking to identify and legitimate a professional identity of their own. Th h ll h b i h i h All this learning potential is not assumed, sometimes it represents an ideal, and needs to be continuously monitored and interrogated through the feedback that emerges during the programme’s various stages. This is not a painless process to trigger: a real interest from 246 C. PALMIERI, M. BARIOGLIO, A. GALIMBERTI, M.B. GAMBACORTI PASSERINI, T. MORGIGNO professional contexts, as well students’ ability to realize interesting and effective research cannot be taken for granted. Traineeship tutors have a key role in creating ‘good-enough’ conditions that can foster a constructive dialogue, attempting to deal with the feeling of not be- ing recognized as interesting and valuable interlocutors that has oc- curred to both students and hosting educational services. Adopting the stance described in this chapter has allowed us to construct and manage a traineeship that offers the opportunity to experience a wide reflection about what adult educators are, their functions, their employability in real situations, inviting students to adopt a critical and proactive stance, sharing their ideas with the larger systems (university, professional contexts) they will have to connect with in order to start or develop their career. * Alessandra Romano, PhD Researcher of General Didactics and Special Education, Department of Scienze della formazione, scienze umane e della comunicazione inter- culturale, University of Siena, Italy. Email: alessandra.romano2@unisi.it. PROFESSIONAL DEVELOPMENT TUTORING: COMPARING ITALIAN AND INTERNATIONAL MODELS Alessandra Romano (University of Siena)* abstract: The paper presents the first outcomes of a comparative research of the incoming, on-going and outgoing practices of tutorship. The purposeful sample of universities extracted consisted of 18 Italian universities and 18 US universities. A tutorship concept in line with the transversal cross-curricular skills required for undergraduate and graduate students (Green Paper 2016, Dublin descriptors 2004) exceeds the vision of assistential tutorship and student tutoring practices, exercised by teachers and/or offered by services devoted to different types of in- tervention. The tutorship can be conceived as systemic and organizational action coherent in all phases with professionalising approach, starting from the earliest initiatives between school and university classrooms. Keywords: tutorship, soft-skills, work-oriented approach, comparative study, best practices. References Alberici A. 1999, Imparare sempre nella società conoscitiva: dall’educazione degli adulti all’apprendimento durante il corso della vita, Paravia, Torino.l Allen J. and Van der Velden R. 2011, The flexible professional in the knowledge society: new challenges for HE, Springer, The Netherlands. Biesta G. 2006, Beyond learning: democratic education for a human future, Routledge, London.f Boffo V., Fedeli M., Lo Presti F., Melacarne C. and Vianello M. (eds.) (2017), learning for employability: new strategies in higher education, Pearson, Milano-Torino. Cambi F. 2004, Saperi e competenze, Laterza, Roma-Bari. Dozza L. 2012, Apprendimento permanente una promessa di futuro, in Id. (ed.), Vivere e crescere nella comunicazione. Educazione permanente nei differenti contesti ed età della vita, FrancoAngeli, Milano, 15-30. Field J. 2006, Lifelong learning and the new educational order, Trentham Books, Stoke on Trent. Palmieri C., Gambacorti Passerini M.B., Galimberti A., Zannini L. and Riva M.G. 2015, Un’esperienza di tirocinio per professionisti educativi di secondo livello: da un approccio centrato sulla ricerca allo sviluppo di un modello tutoriale di blended-learning, «CQIA RIVISTA», XV (1), 113-127. g Fairclough N. 1993, Critical discourse analysis and the marketization of public discourse: The universities, «Discourse and Society», IV (2), 133-168.l Fook J. and Gardner F. 2007, Practising Critical Reflection. A Resource Handbook, MacGraw Hill, Maidenhead. Galimberti A., Gambacorti-Passerini M.B. and Palmieri C. 2016, Formare il professionista di secondo livello. Quali sfide per l’università?, «Scuola Democratica», 3, 667-686. Massa R. (ed.) 1992, La clinica della formazione. Un’esperienza di ricerca, FrancoAngeli, Milano. THE ‘SECOND LEVEL’ EDUCATION PROFESSIONAL 247 Mortari L. 2003, Apprendere dall’esperienza. Il pensare riflessivo nella formazione, Carocci, Roma. Mortari L. 2007, Cultura della ricerca e pedagogia. Prospettive epistemologiche, Carocci, Roma.f Mottana P. 1998, Formazione e affetti, Armando, Bologna. OECD 2015, Skills outlook 2015. Youth Skills and Employability, OECD Publishing, Paris. Olesen H.S. 2013, Beyond the current political economy of competence development, «European Journal for Research on the Education and Learning of Adults», IV (2), 153-170. Sennet R. 1998, The corrosion of character, the personal consequences of work in the new capitalism, Norton, New York-London. Stokes P.J. 2015, Higher education and employability. New models for integrating study and work, Harvard Education Press, Cambridge.l Schön D. 1993, Il professionista riflessivo. Per una nuova epistemologia della pratica professionale, Dedalo, Bari. Yorke M. 2006, Employability in higher education: what it is – what it is not, The Higher Education Academy, York. 2. The context of the research 2. The context of the research 2. The context of the research In this institutional framework, the Department of Education, Humanities and Intercultural Communication needs to face a chal- lenging problem: the data on the latest cohorts of students enrolled in the three-year Bachelor degree programs in Education and Train- ing Sciences (cohorts 2013-14, 2014-2015 and 2015-2016) showed a percentage of around 20% of formal study abandonment and drop- out, a percentage of 48% of regularly graduated students, about 10% of students being suspended for non-paying university fees, and a percentage of around 20% of students registered but that didn’t pass all the exams. 1. Introduction Universities are making efforts to build and strengthen close ties with businesses, and country’s productive sectors, to catch up with emerging and challenging learning needs with respect to current job scenarios and dialogue with stakeholders (Fabbri, Melacarne 2016: 320). The Department of Education, Humanities and Intercultural Com- munication at the University of Siena promoted profound transfor- mations in the last decade (Fabbri, Melacarne 2016). The diffused instances of innovation in university teaching testify the crisis of a teaching model lesson-centered. The training of professionals who have skills not only tied to knowing, but also to knowing how to do and how to stay in contexts, are recognized as paradigms that are now located in the university mission. The ongoing changes privi- leged multidisciplinary approaches to research and teaching, paying close attention to relationships with multi-stakeholder belonging to the enterprises and business labor market by involving them from the beginning in the redesign of more and more professional gradu- ate courses. 250 ALESSANDRA ROMANO PROFESSIONAL DEVELOPMENT TUTORING An organization that learns creates proactively, acquires and transfers knowledge and changes its behavior on the basis of new knowledge and new suggestions. Learning organizations actively try to infuse new ideas and information. To do this, they look closely at the outdoor environ- ments, take on the new talents and experts they need and invest impor- tant resources in the training and development of their collaborators. Subsequently, the new knowledge created must be transmitted to the whole organization. Learning organizations are committed to reducing structural, procedural and interpersonal factors that hinder the sharing of information, ideas and knowledge among members of the organi- zation. Behavior must also change as a result of a new way of learning (Paparella 2009: 176). The first mandate of the research resulted in the activation of an exploratory-euristic route designed to accompany expert systems of teachers-tutors, carriers of implicit and explicit knowledge, in a pro- cess of co-production and sharing of knowledge. The research group accepts the task to search and to collect ex- amples and extracts of tutoring practices (a) to create a literature of examples that could also encode mainstream trends, (b) to accompany a professional community towards the construction and sharing of a common repertoire of good tutorship practices. The need to bring new elements to the scripts shared between tu- tors and students in the community of the department, the need to improve the tutoring services offered, and to unpack tutorship prac- tices in more differentiated segments and specific, has pushed the commission in the field of new investigative trails. How to deal with good practice in national and international universities? How to deal with diverging, challenging, non-familiar models to open to the is- sue of students’ drop-out? How to develop systems action to face stu- dents’ careers abandonment? The comparative research on the tutorship practices draws out from these research questions. 2.1 The actions of the Tutorship Committee An elective group of teachers meets forming the Department’s Tu- torship Commission. The framework of community of practice build- ing (Wenger, McDermott, Snyder 2007) helps to conceptualize and offers interpretative lens to read the processes of the aggregation and management of the Commission. The members of the community of the Department Commission share patterns, protocols, languages and follow a common purpose. The common interest is to address the problem of the abandonments and of the so-called ‘suspensions’ of university careers to counteract the delays in the university suc- cess and the phenomena of drop-out. The numbers provided repre- sented a disorienting dilemma (Mezirow 2003) for the department, so the commission has been questioning how to make the Degree in Education Sciences more attractive and more distinguished from the viewpoint of the profession practices and actions, in order to support the academic careers and perfomances of students. In addition to this, there is a question of methodological order: how to study actions in transformative practice systems and trajectories that involve and en- gage key stakeholders (students, faculty, departmental governance)? g g y y p g Faced with these problems, the community of teachers, as research- ers interested in the educational issues, chooses to start an inquiry process aimed at making decisions, solving problems and supporting transformative trajectories. The recurrence of the phenomenon in the three cohorts examined elicited some reflections: it was not possible to consider the problem as an individual question, but it was understandable under the socially distributed, collective and organizational dimensions of the depart- ment as educational organization. How to themathize the issue of students’ drop-out in terms of organizational learning? Paparella at this regard (2009) writes: 251 PROFESSIONAL DEVELOPMENT TUTORING 1 About the geopolitic division, the reader may see the ISTAT protocols: <http:// www3.istat.it/strumenti/definizioni/comuni/ripartizioni_regioni_province.csv> (01/2018). 2 the division of the universities according to their greatness was developed on the basis of the statistic data released by MIUR, about the students registrations with the update of the last academic year (2014/2015). The data are available at <http://statistica. miur.it/scripts/IU/vIU0_bis.asp> (03/2018). Based on the available data, a categori- zation has been performed on the basis of the absolute number of students enrolled, dividing the sample: the first third has been highlighted as BIG (B), the second third has been highlighted as Medium size (M), the last Third has been highlighted as Small (S). 3. Comparing good practices and non-family models In the first step of the comparative study, in-coming, on-going and outgoing tutorship practices are investigated through documental analysis using online information and documentation sources. The first bottom-up categorization of the tutorship practices distinguishes six different branchs: 1. tutoring between peers, organizational actors and stakeholders/ mentors; 1. tutoring between peers, organizational actors and stakeholders/ mentors; 2. peer-tutoring practices; 252 ALESSANDRA ROMANO 3. didactic tutorship;i 3. didactic tutorship;i 4. tutorship for specific services (disability, information); 5. professional/vocational tutoring; p 6. information tutoring. 6. information tutoring. 18 Italian universities and 18 European and US universities were selected. The selection criteria for universities were: a. geopolitics (North, Center, South Italy)1 g p ( y) b. greatness (small, medium and large universities)2 g ( c. state, public and private c. state, public and private d. traditional and telematic (online and blended tutoring systems) d. traditional and telematic (online and blended tutoring systems) d. traditional and telematic (online and blended tutoring systems) The focus was on tutorship practices which the universities have put in place the policies for promoting the employability and acqui- sition of soft-skills (Boffo 2017). The data analysis allows to craete an ad hoc modeling on the Rahin model to explore customization and personalization of diversified tutoring practices according to the categorizations indicated. g The goal was not to develop a theory of tutorship systems, the ‘winning formula’ for the most attractive university, but to describe tutorship practices in academic communities. The choice of research- ers was to contribute to highlighting some aspects of ‘family resem- blance’ (Fabbri 2007) between various tutorship activity systems. The purpose was to define a formal modeling that enables to capture the processuality and the potential impact of the tutorship in the aca- demic training path, especially in preventing drop-out phenomena. 3.1 Comparative analysis of the existing: the normative references 3.1 Comparative analysis of the existing: the normative references The tutorship system is part of the Student Support Services at the University (Law No. 19, 1990, No. 341, Article 13). The Tutor is an effec- tive means to support university teaching as can act as (a) mediator be- tween students and the university demanding tasks, (b) supervisor and (c) facilitator in communication and learning (Article 13 of Law 341/1990). 253 PROFESSIONAL DEVELOPMENT TUTORING There are different ratings for tutorship practices. Lázaro (1997) formalizes four types of university tutoring (bureaucratic functional, academic, personal consultant and didactic). Zabalza (2003) speaks of three different levels of tutorship in the light of the objectives pursued: 1. the level of teacher function; 2. the level where tutorship provides advice on study skills or work systems; 2. the level where tutorship provides advice on study skills or work systems; y 3. the level where distance learning is provided, with job monitoring and job experience, etc. Álvarez and González (2008) proposed three major models of university tutoring (academic tutoring, didactic tu- toring and service tutoring). According to this latter classification, academic tutoring is done in the classroom or in groups for each of the subjects taught by the faculty. It takes place at the classroom level and aims to improve students learning. The didactic tutoring is aimed at students from the enrollement to the end of the course. The Services tutorship is generally carried out by university service technicians who provide technical advice to students on issues relat- ed to their training (scholarships, mobility, transportation, housing, legal advice, etc.). The tutorship model follows the prospect of holistic and continuous training, as proposed by the European Convergence Model (Álvarez, González 2008; Coriat, Sanz 2005). The tutorship course consists of transversal activity, it is not tied to a single subject. It is programmed and longitudinal, throughout all the course of the study, and aims to provide students with support for decision making and organizing their own “Training and professional project”. 4. ‘The rector who greets families’: comparing models and practices of tutoring in comparison 4. ‘The rector who greets families’: comparing models and practices of tutoring in comparison The comparative analysis of tutorship practices requires first of all a work of disambiguation: often similar tutorship practices have ma- ny contact points but different denominations. f Sometimes the same label is used in different contexts to describe different services. Orientation services aimed at attracting new stu- dents at their university, showed the educational and organizational structures of the university, underlining why potential clients-stu- dents had to choose that university. y The in-coming tutorship is commonly performed in those ways (present in 32/36 universities): • Open Day: In these ‘open doors’ days, the university shows its own structures and training plans of all the degrees programs. The tutors • Open Day: In these ‘open doors’ days, the university shows its own structures and training plans of all the degrees programs. The tutors 254 ALESSANDRA ROMANO often are required to work in front office and accompanying poten- tial students through guided tours. Most universities repeat the pres- ence of teachers at the presentation of the courses and oft he degree programs. p g • Test Lesson: Potential students are involved in lesson for showing the most concrete aspects of university education through open lessons and laboratory sessions. p g • Test Lesson: Potential students are involved in lesson for showing the most concrete aspects of university education through open lessons and laboratory sessions. y • Connections with high schools: universities hold contact with local schools in two ways, (a) proposing open workshops where university activities are presented and (b) hosting high school students in con- ferences and activities ad hoc. The most emerging promising practices are: • Online platforms and self-evaluating questionnaires: With these tools, the potential students are able to navigate and learn about university activities and make a self-assessment of their attitudes by addressing university choice.f y • Operative consultancy: special tutors (often tutor and guidance staff personnel) develop an orientation profile for the future students in the degree course most adhering to their knowledge and skills. • Presentation of current students: this activity, which is frequent in the case of private universities, involves tutor students considered experts who have their own university experience. y p • Skills balance and students competences portfolio. In private universities, the families of the matriculates take part into the open days and the inauguration day of the academic year. 4. ‘The rector who greets families’: comparing models and practices of tutoring in comparison The example of Ivy League American universities is paradigmatic: rectors meet matriculates’ parents personally, welcoming them as participating members of the academic community. The rite is re- peated at the graduation ceremony when the rector hosts the families of graduates and thank them and congratulate them on the achieve- ment. These are marketing strategies that promote the development of sense of participation in students and families. In the contexts of American campuses, where students space out of the home, parental guidance becomes a supportive practice in the experience of transi- tion that involves the entire family system. 5. First emerging results 5. First emerging results The activities of the various levels of tutorship (incoming, on-go- ing and out-going) are managed autonomously and decentralized by departments in universities sized as large and medium (Fig. 1). 255 PROFESSIONAL DEVELOPMENT TUTORING Figure 1 – Diagram of tutoring path from School to Job. [Source: author’s own] In the case of the ongoing tutorship, the major sections are three: 1. Didactic tutoring (support, individual, group), 2. Disability Services, 3 Counseling and Psychological Assistance Figure 1 – Diagram of tutoring path from School to Job. [Source: author’s own] Figure 1 – Diagram of tutoring path from School to Job. [Source: author’s own In the case of the ongoing tutorship, the major sections are three: In the case of the ongoing tutorship, the major sections are three: Didactic tutoring (support, individual, group), In the case of the ongoing tutorship, the major sections are three: In the case of the ongoing tutorship, the major sections are three: 1. Didactic tutoring (support, individual, group), 2. Disability Services, g ( 2. Disability Services, y 3. Counseling and Psychological Assistance. The fi rst activity is generally assigned to the support tutor, which is a continuation of the incoming tutoring (orientation) and helps stu- dents to orient themselves in the organizational didactic plans and in the creation of individual study plans. The support tutor helps also students in the logistics organization and in the retrieval of informa- tion and materials necessary for access to courses. y Didactic tutoring services used to manage students’ requests indi- vidually, through appointments (often organized online) or in groups. y g pp ( g ) g p In all universities examined (36/36), the student tutoring is al- ways attributed to the availability of teaching support services for students with disabilities and/or specifi c learning disabilities. In this case, the activities are mostly related to the logistic issues and are fi - nalized to make the campus and its spaces ‘accessible’. Through this service, the conditions of equal opportunities needed to enhance the autonomy of students with disabilities and to ensure their active par- ticipation in university life are promoted. Psychological counseling coincides with teaching tutoring and is designed to ensure students’ well-being and prevent drop-out by providing tangible support. Psy- chological Counseling services have a higher percentage presence in public universities, less in private ones. 5. First emerging results In telematic universities, counseling meetings, of individual nature, have the task of intercept- ing those troublesome moments during university careers to prevent students’ drop out. Career counseling takes place in the last period of academic ca- reer, creating a link between student and job market. The people who manage these tasks are in most cases centers for job placement. 256 ALESSANDRA ROMANO The services are: • Career Day: Days in which companies are presented to graduates and at the same time they can interact with stakeholders and business actors by proposing themselves as candidates and understanding the profiles required by companies. In these events, the participation of former students is marginal while the administrative offices manage the organization.ff g • Job Guidance Services: this service is offered in different branchs: CV and resume writing assistance, teaching strategies for interview management, specific consultations to determine the winning profile for hiring. In highly professional services, personalized consulting is proposed and human resource stakeholders are involved. The aim is to match the job offer with the job requests. fi • Placement Office and Platforms for Job Vacancies and CVs building: These online services have virtual desks in which both those who are looking for new graduates and those who have completed a degree and aspire to a job can publish. The Career Orientation Trails are divided into two moments: • a group work in which the participants collaborate for decision making • an individual interview with the facilitators to explore some aspects related to attitudes in order to trigger more informed professional choices. Lastly, business presentations are organized to have the specific time to meet the actors of the industry companies, such as Investment Bank Weekends and Recruiting Dates. The tutoring is stimulated through the participation of peer tutors (such as brilliant alumni and neo-graduates), as in the American model. We can itemize: techni- cal-administrative services in specific offices, services involving ex- students, and counseling coaching. In particular, there are tutorings that work both in groups and through interviews with the tutors, experiential methods are adopted in presence and in virtual environ- ments by working on real case studies or simulation architectures. Informative tutoring is a type of cross-service across the all the fields, as it is provided at all levels by the offices of the various uni- versities. 5. First emerging results Obviously it has a more pronounced feature in the case of the orientation where the request for information orients the inclu- sion of students in the university. y In the following picture they were represented by the diagrams of Eulero-Venn’s own semantics, the mingling of the tutoring prac- tices in the three bands: 1) orientation and in-coming tutorship, 2) on-going tutoring, 3) outgoing tutoring (career counseling) (Fig. 2). PROFESSIONAL DEVELOPMENT TUTORING 257 Figure 2 – Eulero-Venn Diagram for a mapping of Tutoring practices. [Source: author’s own] Figure 2 – Eulero-Venn Diagram for a mapping of Tutoring practices. [Source: author’s own] Regarding digitization of student and didactic services, tutoring with E-Learning platforms is widespread in both public universities and private universities. The massive use of MOOC and MOODLE platforms facilitates familiarization of students with technological de- vices through access to the information via video meeting or skype. This kind of service is typical of universities with great dimension or otherwise with a telematics nature. In telematic universities, the tutor, often called E-Tutor, has the task of supporting the student, fl uidifying the learning process and assisting students. Less used is the Summer Week, imported by the American mod- el, which represents events in which future students can immerse themselves in culture and university mechanisms. Compared to the geographic location, the Northern Universities seem to have well structured tutoring systems based on peer tutoring, with extensive student attendance, while Southern Universities centralize this task on Orientation and Tutor Offi ces. Regarding to internationalization and welcoming to foreign students, the analysed universities have services to facilitate the in- clusion of international students. In most cases these services are provided by specifi c university offi ces, through technical-admin- istrative staff . 6. Modelling the tutoring practices Based on this comparative study I propose a conceptual model for describing tutoring practices. The conceptual model is borrowed from Rahim’s model for negotiation and leadership profi les. 258 ALESSANDRA ROMANO The two parameters identifi ed for the categorization of tutoring practices are: p 1. Customization, which expresses the property of being designed ac- cording to the characteristics of the single user; 1. Customization, which expresses the property of being designed ac- cording to the characteristics of the single user; 2. Individualization, which expresses the property of being designed to be available to all applicants. 2. Individualization, which expresses the property of being designed to be available to all applicants. The two dimensions are related to the characterization of tutor- ing practices. If a service is both personalized, and then perfectly tailored to meet the needs of the individual student and at the same time is highly individualized, that is, it is provided to all students as individuals, we talk about effi cient service. fi In the case of a little personalized and little individualized service we call it as a generalized service. If the service or the practice is highly personalized, but weakly indi- vidualized is defi ned as customized service. If it is in the opposite quarter, it has a high degree of individualization and is therefore spread (Fig. 3). Figure 3 – Model of tutoring practices. [Source: author’s own] Figure 3 – Model of tutoring practices. [Source: author’s own] Figure 3 – Model of tutoring practices. [Source: author’s own] The proposed model allows to identify the most eff ective prob- lem-based tutoring practices. For the orientation services: the School-University Joint services and the active connections with the parents are highly customized and individualized practices, while activities such as Summer Week and door-to-door services even with the use of unregistered les- sons are not customized and then spreaded. Orientation as a coun- seling service is classifi ed as Customized because it is particularly personalized (Fig. 4). The proposed model allows to identify the most eff ective prob- lem-based tutoring practices. The proposed model allows to identify the most eff ective prob- lem-based tutoring practices. For the orientation services: the School-University Joint services and the active connections with the parents are highly customized and individualized practices, while activities such as Summer Week and door-to-door services even with the use of unregistered les- sons are not customized and then spreaded. Figure 3 – Model of tutoring practices. [Source: author’s own] 6. Modelling the tutoring practices Orientation as a coun- seling service is classifi ed as Customized because it is particularly personalized (Fig. 4). For the orientation services: the School-University Joint services and the active connections with the parents are highly customized and individualized practices, while activities such as Summer Week and door-to-door services even with the use of unregistered les- sons are not customized and then spreaded. Orientation as a coun- seling service is classifi ed as Customized because it is particularly personalized (Fig. 4). PROFESSIONAL DEVELOPMENT TUTORING 259 Figure 4 – Orientation tutoring practices in the Individualization/Personalization Model. [Source: author’s own] Regarding the tutoring services Group lessons are services that we could rank as Generalized, which are neither personalized nor individualized. On the opposite side, we have peer-tutoring practic- es and dedicated tutoring. Practices such as tutoring with e-learning and international student services are individualized for the refer- ence category but do not have a particularly relevant personalization nature. Personalization is decisive in services such as psychological counseling and university-specifi c projects for students with special needs (disabled students, DSA, student workers) (Fig. 5). Regarding the tutoring services Group lessons are services that we could rank as Generalized, which are neither personalized nor individualized. On the opposite side, we have peer-tutoring practic- es and dedicated tutoring. Practices such as tutoring with e-learning and international student services are individualized for the refer- ence category but do not have a particularly relevant personalization nature. Personalization is decisive in services such as psychological counseling and university-specifi c projects for students with special needs (disabled students, DSA, student workers) (Fig. 5). Regarding the tutoring services Group lessons are services that we could rank as Generalized, which are neither personalized nor individualized. On the opposite side, we have peer-tutoring practic- es and dedicated tutoring. Practices such as tutoring with e-learning and international student services are individualized for the refer- ence category but do not have a particularly relevant personalization nature. Personalization is decisive in services such as psychological counseling and university-specifi c projects for students with special needs (disabled students, DSA, student workers) (Fig. 5). Figure 5 – Tutoring practices in the Individualization/Personalization Model. [Source: author’s own] Figure 5 – Tutoring practices in the Individualization/Personalization Model. 6. Modelling the tutoring practices [Source: author’s own] 260 ALESSANDRA ROMANO Regarding the outgoing tutoring (career counseling), services such as Job Placement are categorized as Effi cient, while Career Day services and Online Platforms for CVs collection having a low relation with single and / or newly graduated students are classifi ed as widespread. Finally, the counseling service aimed at specifi c professional profi les is a very personalized service, which is therefore defi ned as Customized (Fig. 6). Figure 6 – Job Orientation tutoring practices in the Individualization/Personalization Model. [Source: author’s own] Figure 6 – Job Orientation tutoring practices in the Individualization/Personalization Model. [Source: author’s own] 6.1 Diff erential peer-tutoring 6.1 Diff erential peer-tutoring Starting from the American university model, a formal distinction is being made between the personal tutor and the professional tutor who accompanies the internship of the students and advises them on choices regarding their professional future and the most eff ective in- sertion into workplaces. ii p Beside the institutional fi gure of the lecturer there are three fi g- ures related to the teaching-learning activity: the ‘resident tutor’, the ‘senior tutor’ and the ‘junior tutor’. The Resident tutor is a research specialist or PhD directly involved in teaching and coordinating the course activities, especially support- ing the doctoral and master dissertations. The senior tutor, graduate and expert in the subject, has the role of managing the integrative teaching and co-ordination of the junior tutor’s study assistance services. y The junior tutor was a degree student able to assist younger students in their learning path during study sessions, exercise activities and labs. 261 PROFESSIONAL DEVELOPMENT TUTORING The interesting news is that the training course is articulated from the entrance of the student enrolled as a succession of tutorial relation- ships (teacher-tutor, resident tutor, senior tutor and junior tutor) in which each intermediate fi gure is simultaneously the object and sub- ject of the tutorial relationship in a training responsibility that comes to involve the students themselves. The students actively co-operate in the learning of the younger students, in the extent and within the limits of their specifi c skills. The peer-tutoring practices are present in 7 of 18 universities. 6. Modelling the tutoring practices In this fi eld, some of the services of excellence are evident: among them the presence of tutor students (with annual contracts), the ‘Pact with the Student’, systematic practices of peer-tutoring both in presence and online, the group-tutor in peer tutoring practices; the excellence- oriented tutor and the formalization of the presence of the Junior Tu- tor, both a research fellow or a PhD student. Following the American university analysed-for-the-research model, the tutoring center for excellence is a tutoring course aimed at the development of cross-disciplinary and transversal skills. Its ac- tivity resides in particular in: • linguistic tutoring • coupled linguistic tutoring • informatic tutoring Below is the timeline of the services off ered to students since their entrance in the university (Fig. 7). Figure 7 – Timeline of Tutoring practices from school to job. [Source: author’s own] Figure 7 – Timeline of Tutoring practices from school to job. [Source: author’s own] Figure 7 – Timeline of Tutoring practices from school to job. [Source: author’s own] 7. Final remarks Starting from a real problem, perceived as an organizational prob- lem, the comparative analysis of tutoring practices described aimed at diff erentiating practices depending on the training moment in which 7. Final remarks 7. Final remarks Starting from a real problem, perceived as an organizational prob- lem, the comparative analysis of tutoring practices described aimed at diff erentiating practices depending on the training moment in which 262 ALESSANDRA ROMANO they are located. The analysis allowed to distinguish between sup- portive and empowering approach. Based on the practices of excel- lence identified in the analysis of national and international models, proposals for professional tutoring practices are advanced for both tools and procedures, such as tutoring practices through badge and e-portfolio, longitudinal tutoring practices, coupled tutoring and work-oriented tutoring practices. g p Practice-based practices are mostly focused on individual student’s needs, are individual-based practices aimed at filling in formative and informational gaps, reducing student learning load, or facilitating ac- cess to university bureaucratic maze. What is and what does a support- ive tutor is still overly a heteronomous concept, while the empowering tutoring is aimed at creating learning platforms for soft-skills and for the professional skills that are useful and attractive in the labor market. The need to tie more and more what is being studied with what is going to make in the work market, presents the need to bring stu- dents into the employability criteria that are validated in the profes- sional worlds. For this reason, comparison with the models of didactic innovation and the practices of foreing universities allows the open- ness to forms of tutoring of excellence where the interest is in sup- porting the learner in the process of acquiring entrepreneurial and managerial connotations. Tandem tutoring also allows conceptualizing students participa- tion in university life and their progression of academic careers as collaborative relationships that characterize potential development areas and collaborative research. The modeling proposal did not drive to the transformation-imple- mentation of new teaching practices, but was more connected with the purpose of understanding tutorship impact on the negotiation of change processes. g p Tutoring practices become objects that are being constructed, ne- gotiated and shared, which can not be defined from beginning be- cause their plausibility and applicability criteria are right in the ability to be able to converse with situations and to be situated in local or- ganizational contexts. The tutoring becomes one of the ‘didactic management’ activi- ties (Paparella 2009: 23), towards students, particularly in the fol- lowing areas: g a. Promotion and Incoming Information […] b. Ongoing consulting for the university careers […] c. PROFESSIONAL DEVELOPMENT TUTORING 263 Instead of attempting to implement action models extracted from other realities and imposed, a process of negotiation between two ob- jects – the tutoring practices situated in place and the tutoring prac- tices of other universities – has been opened up. This process could result in a transformative path whose goal is to define shared reposi- tories to assume them as common commitments by a part of the de- partment community. 7. Final remarks Integrative business management […] d. Detection of levels of satisfaction […] e. Cultivating employability and outgoing job orientation […] g p y y g g j f. Managing special educational needs […] (Paparella 2009: 23-24). PROFESSIONAL DEVELOPMENT TUTORING ** Anna Salerni, Associate Professor, Sapienza University of Rome, Italy. Email: anna.salerni@uniroma.it; Silvia Zanazzi, Fellow Researcher, Sapienza University of Rome, Email: silvia.zanazzi@uniroma.it. * The present article is the result of joint work and reflection. However, Anna Salerni is specifically responsible for Paragraphs 1, 3 and Silvia Zanazzi for Paragraphs 2, 4, 5. References Álvarez P., González M. 2008, Los planes de tutoría en la Universidad: una guía para su implantación, Servicio de Publicaciones de La Universidad de la Laguna, San Cristóbal de la Laguna.f Boffo V., Fedeli M., Lo Presti F., Melacarne C. and Vianello M. 2017, Teaching and learning for employability. New strategies in higher education, Pearson, Milano.f Boffo V., Federighi, P. and Torlone F. (eds.) 2015, Educational Jobs: Youth and Employability in the Social Economy, Firenze University Press, Firenze.́ Coriat M. and Sanz R. (ed.) 2005, Orientación y Tutoría en la Universidad de Granada. Granada, Editorial Universidad de Granada, Granada. Á Da Re L., Álvarez P.R. and Clerici R. 2016, Le attività e gli strumenti del programma di Tutorato Formativo per i nuovi iscritti all’università: una guida operativa, Rapporto Tecnico. l p pp Fabbri L. 2007, Comunità di pratiche e apprendimento riflessivo, Carocci, Roma. , p pp fl , , Fabbri L. and Melacarne C. 2016, Didattica dell’innovazione e innovazione didattica. L’apprendimento come condizione per il cambiamento, in Fedeli M., Grion, V. and Frison D. (eds.) Coinvolgere per apprendere. Metodi e tecniche partecipative per la formazione, Pensa MultiMedia, Lecce, 319-339. Giuliani A., Moretti G. and Morini A. 2016, Didactic tutoring services and obligations for additional learning,, an empirical exploratory research: the case of the Department of Educational Science at Roma Tre University, «Italian Journal of Educational Research», 15, 63-78.f Mezirow J. 2003, Apprendimento e trasformazione, Raffaello Cortina, Milano. J , pp f ,f , Paparella N. (ed.) 2009, Il progetto educativo, vol. III, Armando Editore, Roma. Topping K., Duran D. and Van Keer H. 2015, Using peer tutoring to improve reading skills: a practical guide for teachers, Routledge, London. Paparella N. (ed.) 2009, Il progetto educativo, vol. III, Armando Editore, Roma. Paparella N. (ed.) 2009, Il progetto educativo, vol. III, Armando Editore, Roma. Topping K., Duran D. and Van Keer H. 2015, Using peer tutoring to improve di kill ti l id f t h R l d L d Topping K., Duran D. and Van Keer H. 2015, Using peer tutoring to improve reading skills: a practical guide for teachers, Routledge, London. Wenger E., McDermott R. and Snyder W.M. 2007, Coltivare comunità di pratiche. Prospettive ed esperienze di gestione della conoscenza, Guerini e Associati, Milano. Zabalza M. 2003, Competencias docentes del profesorado universitario: calidad y desarrollo profesional, Narcea, Milano. INSIGHTS FROM THE FIELDS: THE ROLE OF REFLECTION IN ‘LEARNING HOW TO LEARN’* Anna Salerni (Sapienza University of Rome), Silvia Zanazzi (Sapienza University of Rome)** abstract: In experiential learning, on-field experience needs to be processed consciously in order for learning to take place. Reflection plays a crucial role by providing a bridge between practical experience and conceptualization. Despite being a protected environment, university traineeship is a form of experiential learning that offers students a chance to learn from the fields and reflect on a possible future profession. In this paper we present and discuss a research project whose goal is the development of a methodology to educate trainees’ reflective thinking and writing. Keywords: university traineeship, experiential learning, reflective thinking, re- flective writing, reflective practitioner. 1. Introduction No one is born fully-formed: it is through self-experience in the world that we become what we are. (Paulo Freire) In experiential learning, personal experience is the focal point for learning, bringing «life, texture, and subjective personal meaning to abstract concepts» and providing «a concrete, publicly shared reference point for testing the implications and validity of ideas created during the learning process» (Kolb 1984: 21). Experience alone, however, is not sufficient for learning. For learning to take place, any in-the-field experience needs to be processed consciously. l Reflection plays a crucial role by providing a bridge between prac- tical experience and theoretical conceptualization (Schön 1983). It helps to activate a circular process between thought and action, es- sential to avoid acting in a mechanical way based on habit or merely applying theories and procedures (Dewey 1933). In the Education Science courses at the Sapienza University of Rome, traineeship is considered an essential formative step for stu- 266 ANNA SALERNI, SILVIA ZANAZZI dents’ personal and professional growth, as well as a valuable chance to integrate knowledge acquired in formal contexts at a theoretical level with skills developed in professional settings (Salerni, Sposetti, Szpunar 2014). Despite being a somehow ‘protected’ environment, university traineeship is a form of experiential learning that offers students a chance to learn from the fields and think about a poten- tial future profession. Trainees participate in concrete projects and learn, along with the help of tutors, to reflect critically, developing the necessary competence to enter the world of work and professionals. l y p p We all acknowledge the importance of reflection. However, we should not assume that individuals are naturally born with the abil- ity to reflect. 1 Logbooks need to be kept daily. The trainees should briefly note what happens and what catches their attention every day, so as to have material for reflection later on. Therefore, logbooks are different from learning journals, which are usually a weekly requirement. INSIGHTS FROM THE FIELDS: THE ROLE OF REFLECTION IN ‘LEARNING HOW TO LEARN’* More realistically, reflection requires adequate knowl- edge and skills, as well as practice and exercise (Harris et al. 2010) to analyse facts in depth and adopt new perspectives. As Mortari (2003) emphasizes, we really learn only when we can attribute meaning to our experience.i A final traineeship report is required in our Education Science courses to obtain academic credit. Beyond the formal requirement, we ask students to produce a report because writing about experiences is recognized in the literature as a useful tool to enhance reflective prac- tice. In fact, writing the report obliges trainees to re-elaborate their experience, hopefully activating a process of «reflection on action, from a distance» (Schön 1983) that fosters a growth of understand- ing and knowledge (Salerni, Sposetti, Szpunar 2014). Along the way, academic tutors recommend that trainees regularly keep logbooks1 during their field experience, since these increase learners’ awareness and provide material upon which to reflect afterwards (Gibbs 2013).l As we said, writing can be a way to fuel reflective practice by re- processing and interpreting experience. According to Moon (2006), there are three types of writing with respect to the level of reflec- tion displayed. Based on her classification and on other contributions (Kember at al. 2000; Mezirow 1991), we developed our own defini- tion to use for the analysis of final reports: yi p • Descriptive writing is merely centred on ‘what happened’ and con- tains few reflective elements. There is little attempt to focus on some selected issues, giving similar ‘weight’ to all topics. Gener- ally, stories are told from only one point of view. Ideas are linked by a sequence of facts rather than by meaning; there might be ref- erences to emotional reactions, but they are not explored in depth. yi p • Descriptive writing is merely centred on ‘what happened’ and con- tains few reflective elements. There is little attempt to focus on some selected issues, giving similar ‘weight’ to all topics. Gener- ally, stories are told from only one point of view. Ideas are linked by a sequence of facts rather than by meaning; there might be ref- erences to emotional reactions, but they are not explored in depth. 267 INSIGHTS FROM THE FIELDS The students do not seem to have any doubts or critical incidents that they want to bring up for discussion, nor do they seem to question their behaviour and actions in any way. INSIGHTS FROM THE FIELDS: THE ROLE OF REFLECTION IN ‘LEARNING HOW TO LEARN’* l • Descriptive writings with some reflections tend to be more focused and signal points for further reflection: beyond an account of ‘what happened’, there are doubts, questions and issues to be discussed. However, this type of writing still shows little analysis of the events: the reflection is not deep enough to let learning happen. There are statements showing awareness about learning, but they are very generic and often sound rhetorical. The students do not show sufficient ability to ‘distance’ themselves from the concrete situations and look at them critically.l However, this type of writing still shows little analysis of the events: the reflection is not deep enough to let learning happen. l p g g pp There are statements showing awareness about learning, but they are very generic and often sound rhetorical. The students do not show sufficient ability to ‘distance’ themselves from the concrete situations and look at them critically.l Reflective writing contains some descriptions, but it focuses on some relevant aspects to be analysed in depth. In this type of writing, one might be able to appreciate the existence of several alterna- tive points of view on the same facts. There is a certain ‘distance’ from the events, a willingness to be critical and to question one’s own actions and those of others. Emotions are recognized, and so is their significance and impact on behaviour. The students reflect on their learning referring to theoretical knowledge (to know), practical knowledge (to know how) and behaviour (how to be). Connections between formal and informal learning are established and explained. There are comments and reflections on how field experience might impact future career choices and why. ll Reflective writing contains some descriptions, but it focuses on some relevant aspects to be analysed in depth. In this type of writing, one might be able to appreciate the existence of several alterna- tive points of view on the same facts. There is a certain ‘distance’ from the events, a willingness to be critical and to question one’s own actions and those of others. Emotions are recognized, and so is their significance and impact on behaviour. The students reflect on their learning referring to theoretical knowledge (to know), practical knowledge (to know how) and behaviour (how to be). Connections between formal and informal learning are established and explained. INSIGHTS FROM THE FIELDS: THE ROLE OF REFLECTION IN ‘LEARNING HOW TO LEARN’* There are comments and reflections on how field experience might impact future career choices and why. ll p g p y Critical reflection. This type of writing contains reflections that might lead to a change in one’s basic assumptions and ‘frames of reference’. Mezirow (1991) defined this as «premise reflection». This is a very deep and ‘transformative’ re-elaboration work that implies a revision of premises deriving from previous learning and its ‘consequences’, both in terms of meanings and behaviour. This type of reflection might also be related to the concept of «deutero- learning» (Bateson 1972), which challenges one’s existing learning framework as well as one’s mental assumptions. This is a superior level of learning, a meta-cognition process that concerns ‘learning to learn’ rather than simply learning an object or a specific skill. We are fully aware that critical reflection might be very rare in students’ reports, since we deal with very young people (around 20 years old) who are in many cases experiencing the work envi- ronment for the first time. Despite this, we know from our class discussions that some students have the ability to reflect critically on the purpose and meaning of education in society, raising philo- sophical issues that might be ‘challenged’ and investigated through experience. Therefore, we consider that there is the possibility of finding pieces of critical reflection in their reports. 268 ANNA SALERNI, SILVIA ZANAZZI We strongly believe that a traineeship model based on reflection contributes to preparing reflective practitioners, able to engage in a process of continuous learning. Since reflective practice can be a very effective source of personal and professional development (Schön 1983, 1987), and given that writing on experience fosters reflection on it, we think it is important to invest time and energy in improving the final traineeship reports in terms of ‘reflective content’. In fact, in this paper, we present and discuss a research project carried out dur- ing the academic year of 2015-2016 in a research lab on the theme of traineeship. In our Education Science courses at Sapienza, in fact, first-year students are required to take an Educational Research Lab class, while traineeship involvement begins in the second year. The goal of our work was, initially, to have an assessment of the trainees’ capability to evaluate and report their traineeship experience in writ- ten form, going beyond a mere description of ‘what happened’. INSIGHTS FROM THE FIELDS: THE ROLE OF REFLECTION IN ‘LEARNING HOW TO LEARN’* Then, when we realized that reflective writing was so lacking in most of the reports, we started discussing a strategy to improve them. We began a journey whose destination is the development of a ‘methodology’ to educate reflective thinking and train reflective writing. Like any research process, this is a cycle where provisional results generate fur- ther questions, becoming new material for subsequent steps. 2. Theory 2. Theory In traditional ‘teacher-directed’ approaches based on lessons, learn- ing takes place mainly at an intellectual level. The students are passive recipients of information, often unaware of their own emotional re- sponses to the objects of learning. According to more recent theories of learning, this ‘sterile’ approach can lead to an inadequate application of knowledge in authentic real-life occasions and work contexts. A very clear example is provided by Hobbs (1992) in the nursing sphere: If student nurses or doctors are taught about how to encounter dying patients or their relatives and the information is imparted to them in a ‘teacher-directed’ mode, they do not have the necessary opportunity to reflect on their own thoughts and possible fears of death and examine such deep feelings together with their peers in the first place. In such cases they do not learn how their own fears of death might affect the quality of their work with such people (Hobbs 1992: XIV). In fact, experts in learning have argued that theoretical concepts will become part of an individual’s knowledge only after he/she has experienced them meaningfully at an emotional level. 269 INSIGHTS FROM THE FIELDS The theoretical framework of our project is the vast corpus of works on experiential learning, from which emerges a new perspec- tive on work-based learning and the creation of knowledge in gen- eral. While more traditional approaches give primary emphasis to the acquisition of knowledge, brick after brick as if a wall were being built, the experiential learning viewpoint sees knowledge as a flex- ible network of ideas and feelings (Kolb 1984). Based on this assump- tion, learning is not the mere accumulation of knowledge, but entails changing assumptions and conceptions, transforming oneself in the process (Mezirow 1991). A fundamental proposition in the experien- tial learning theory is that learning is a holistic process which fully in- volves human beings: thinking, feeling, perceiving, and behaving are all integrated functions (Kolb 1984). In this view, learning becomes the major process of human adaptation to the world, occurring in all settings, from school to the workplace, as well as in personal rela- tionships and everyday activities: it is a continuous, lifelong process that involves transactions between the person and the environment. 2 Lewin’s model of Action Research and Laboratory Training; Dewey’s model of Developmental Learning; Piaget’s model of Learning and Cognitive Development (Kolb 1984). 2. Theory As Dewey (1938) wrote, experience influences the formation of atti- tudes of desire and purpose, since every experience changes to some extent the objective conditions under which subsequent experiences takes place. In this respect, he made a distinction between educative and miseducative experiences, the latter having «the effect of arrest- ing or distorting the growth of further experience» (Dewey 1938: 25). f ll l h h fl l h g g g p ( y ) If we critically analyse the three most influential theories on expe- riential learning (Dewey 1938; Lewin 1946; Piaget 1971) we can eas- ily find similarities between them. The three models2 taken together form a unique perspective on learning (Kolb 1984), conceived as a process rather than in terms of outcome: «ideas are not fixed and im- mutable elements of thought but are formed and re-formed through experience» (Kolb 1984: 26). Consequently, knowledge continuously emerges from experience, implying that «all learning is relearning» (Kolb 1984: 26), since everyone faces new experiences drawing on ideas and knowledge stemming from previous ones. According to Kolb, to be effective, learners need four kinds of abilities: concrete experience («involving themselves fully, openly, and without bias in new experi- ences»), reflective observation («reflecting on and observing their expe- riences from many perspectives»), abstract conceptualization («creating concepts that integrate their observations into logically sound theories») and active experimentation («using these theories to make decisions 270 ANNA SALERNI, SILVIA ZANAZZI and solve problems») (Kolb 1984: 30). Thus, the learner can alternate between being an actor or an observer, going from active involve- ment to analytical detachment: he/she needs to act and reflect, and to be concrete and theoretical at the same time. This model shows very clearly the relationship between experiential learning and reflection, a pillar concept in the literature that frames our work. Reflection plays a key role in the experiential learning cycle by providing a ‘bridge’ between experience and theoretical conceptualization. p p Learning, especially in work contexts, requires an introduction to theory, together with the opportunity to apply theory to real situations in the field. Professional situations are often complex and multifaceted, meaning that «they possess multiple solutions and contain uncertain- ty» (Harris et al. 2010: 4). Reflection, then, represents a fundamental process for the practitioner who must draw on theoretical knowledge, previous experience, and knowledge of the current situation to deter- mine the most appropriate solution. 2. Theory In fact, John Dewey defined re- flection as a process aimed to «transform a situation in which there is experienced obscurity, doubt, conflict, disturbance of some sort, into a situation that is clear, coherent, settled, harmonious» (Dewey 1933: 101-102). He saw reflection as a process of inquiry that leads to a more thoughtful consideration of facts. Many years later, Schön recognized reflection as an essential method of acquiring professional knowledge. He introduced the concepts of reflection-in-action, spontaneous and im- mediate, and reflection-on-action, consisting in an analysis of the circum- stances of the event, from a distance, and planning of future actions, based on careful consideration of all information (Schön 1983). Later on, other scholars added the proactive aspect of reflection-for-action (Kil- lion, Todnem 1991; van Manen 1991, 2008), that implies planning fu- ture actions based on one’s experience and previous learning. p p g In sum, the experiential learning theory is an educational per- spective that aims at integrating theoretical and practical elements of learning using reflection as a ‘bridge’, adopting a holistic approach and emphasizing the meaning attributed to experience. This theory of learning is well known in various informal settings, such as curricular traineeships in service organizations. Experiential learning techniques include a rich variety of interactive practices whereby the participants have opportunities to contribute actively to their own learning, rather than being passive recipients in a hetero-directed process. 3. Methodology In our work we have adopted a phenomenological approach to qualitative research. Phenomenology is a philosophical orientation 271 INSIGHTS FROM THE FIELDS that emphasises people’s subjective experiences and interpretations of the world. It is based on a strong belief in the «importance of sub- jective consciousness» and on «an understanding of consciousness as meaning-bestowing» (Cohen, Manion, Morrison 2011). From a meth- odological viewpoint, in the project presented in this paper, we have used the grounded theory, developed by Glaser and Strauss (1967) to build theory, «an abstract analytical schema of a phenomenon» (Cre- swell 1998) from data «systematically gathered and analysed» (Strauss, Corbin 1994: 273). A grounded theory is a theory that emerges from data rather than being predefined and tested: theory generation origi- nates from systematic data collection and analysis; patterns and theories are implicit in data, waiting to be discovered (Cohen, Manion, Mor- rison 2011). A grounded study begins with the raising of generative questions, which help to guide the research but are not intended to be either static or curbing. As the researcher begins to gather data, theo- retical categories are identified, and, through open coding and con- stant comparison, tentative connections are developed between the categories and the data. As stated by its founders, grounded theory is a complex iterative process where data collection and analysis go hand in hand, continually informing one another (Glaser, Strauss 1967). The researcher can use several different methods to collect data, both quantitative and qualitative: from in-depth interviews, to participant observation, analysis of documents, collection of artefacts and texts, and questionnaires. A very important aspect of the grounded theo- ry approach, as of most qualitative research, is the researcher’s direct involvement in the social environment being studied, which often requires extensive fieldwork. qi Our project began when we acknowledged a problematic situa- tion: while reading students’ traineeship reports, we wondered why the writings were so limited in terms of reflection despite the variety and ‘richness’ of the field experiences. At first, we thought of analys- ing a number of final reports, to verify whether our impression cor- responded to reality. While we gathered and read more than 30 final reports written by trainees in many different fields, we built, step by step, and using an iterative process, our framework for content anal- ysis, identifying core concepts and, consequently, substantive codes to summarize empirical evidence. ANNA SALERNI, SILVIA ZANAZZI pation is concerned, while it was not possible to observe traineeships directly in the organizations where they took place, our involvement as researchers came from long-standing experience as academic tu- tors, a deep knowledge of the programme’s educational goals and the practice of supervising students’ experiences closely and thoroughly. Interviews and informal meetings with students were a way for us re- searchers to further ‘immerse’ ourselves in the reality of traineeships. 3. Methodology The second phase of the projects, partially overlapping with the first, consisted of the administration of semi-structured interviews to students in the final phase of their traineeship, as a strategy to fos- ter reflective thinking. In line with the grounded theory approach, which requires a context to be studied from different perspectives and using different methods, these interviews can also be seen as a tool to collect data on reflective processes. As far as our own partici- 272 ANNA SALERNI, SILVIA ZANAZZI 4. Research project To begin, we delved into the analysis of 32 final traineeship re- ports. Since the goal of our traineeship programme was to enhance students’ learning through field experience, we wanted trainees to re- flect mainly on their learning outcomes. Final reports should, in our opinion, contain reflections about learning at various levels. Which is why we used the following 5 criteria during the analysis process: • Knowing: content referring to theoretical knowledge and ‘theoreti- cal know-how’ learned simply by observing others. For example, learning about methods to teach children foreign languages builds theoretical knowledge; observing how English language teach- ers apply one specific method during daily activities in a nursery school leads to developing ‘theoretical know-how’; • Knowing how to do: content referring to growth in practical abili- ties developed through active participation. An example of this category is learning how to approach a student with Down syn- drome simply by doing it; • Knowing how to be: content referring to growth in terms of behav- iour and attitudes relating to a professional role. Learning how to be gentle and authoritative at the same time with children is an example of this category; p g y • Theory and practice: content referring to the student’s ability to cre- ate linkages between formal and informal education, between ac- ademic and work-based learning. An example could be observing the Montessori method applied to children and being able to dis- cuss a child’s daily activities of referring to an authoritative theo- retical framework; • Vocational guidance: content referring to the educational value of the traineeship experience, in terms of understanding of personal aptitudes and aspirations and their concrete applicability in a work field or specific profession. As an example, a student wrote that the traineeship in a nursery school taught her how infant education was not her primary goal for a professional future and explained why, based on her field experience. In addition to this, she imagined a 273 INSIGHTS FROM THE FIELDS different placement for her future and motivated her choice based on expected work characteristics that were ‘in contrast’ with her experience at the nursery school. 4. Research project Going back to our objective assessing the level of reflective writ- ing in the students’ final reports, an analysis of the first 32 pieces con- firmed that they were basically lacking in terms of ‘reflection on action’: most of them could have been defined, using Moon’s categorization, as merely descriptive, while a minority of them could have been named descriptive writings with some reflections (Moon 2006). Therefore, we de- cided to test a strategy to help students re-elaborate their experience. In the second phase of our project, we created a protocol for semi- structured interviews and subsequently administered 13 interviews to students who had just finished their traineeship and were about to write their final report. The interviews were designed to ensure that the trainees could take a critical look at themselves and their actions and explain the results of this reflection. The interview protocol was based mainly on the four-stage model of experiential learning, where the four stages are: concrete experience, reflective observation, ab- stract conceptualization, active experimentation (Kolb 1984). Some questions were inspired by the Behavioural Event Interview (BEI) model (McClelland 1973). This technique, which originates from the critical-incident method (Flanagan 1954) enriched with the Thematic Apperception Test (TAT) of aspects concerning the person, his/her motivation, learning style, and ways of thinking (Murray 1943), is used mainly in human resource management for the selection of per- sonnel, because it helps in evaluating behaviour activated by critical situations, discovering what people do concretely and how they face work-related challenges. Although we are not working in human re- sources, we believe that starting from behavioural events can be useful to help students reflect on concrete experience and on the learning de- rived from it. In the end, these were the inputs used for the interview: • Can you briefly describe your traineeship experience? p • Can you briefly describe your traineeship experience p • Can you briefly describe your traineeship experience? • Do you remember a situation during your traineeship when you encountered a problem? Can you describe it and explain what you did and how you felt, what others did, and how in the end the problem was approached and eventually solved? • Can you recall a moment during your traineeship experience when you felt particularly useful? Can you describe the circumstances, what you did, how you felt and the reasons why you felt that way? 4. Research project y y y y y • Thinking about three ‘areas of competence’, that is, ‘knowing’, ‘knowing how to do’ and ‘knowing how to be’, can you bring ex- amples from your field experience that show your personal growth and skill development? 274 ANNA SALERNI, SILVIA ZANAZZI • At the end of your traineeship experience, what do you think about your professional future after university? Did the traineeship help you reflect on this and if so, how? • At the end of your traineeship experience, what do you think about your professional future after university? Did the traineeship help you reflect on this and if so, how? Trainees responded positively to the interviews, sharing a great deal about their experiences and, in general, demonstrating that they possessed some, albeit limited, reflective abilities. Afterwards, they were invited to write their final report within 15 days, or, for those who had already written it, to review it, so as to include the reflec- tions that emerged during the interview. g g The third and last phase of our work was a comparison between the reports produced without any ‘support strategy’ and those produced after an interview, to assess whether there had been an improvement in terms of the ability to think reflectively about experience. In some cases (3 out of 13), we realized that the traineeship reports written before the interview had not been modified in any way: an arbitrary choice of the students, going against our suggestion to review them in light of the reflections that emerged during the interviews. In the majority of the cases (8 out of 13), the report had been written after the interview. However, contrary to expectations, we had to recog- nize that descriptive accounts (Moon 2006) had remained essentially the same: these reports focused mainly on a description of the host or- ganization and the activities performed, without much reflection on outcomes in terms of learning. We frequently found extremely ge- neric statements about personal growth and skills development, with- out explanations of how learning had been achieved and what specific knowledge or skills had been developed: This traineeship experience helped me grow both professionally and as a person. During my traineeship I found many connections between the theories learned in class and the development of the child observed during eve- ryday life at the kindergarten. 4. Research project Only in one case did we note an improvement from descriptive writ- ing to descriptive writing with some reflections, where the student, whose traineeship took place in a foster home for disadvantaged minors, went beyond merely reporting facts and included some personal evalua- tions of her learning achievements: Every time the staff intervened, they would explain to me why they had acted in a certain way and remind me that every ward has his/her own past and personality that need to be respected. Therefore, it is necessary to understand when it’s the best time to intervene to tackle a problem. 275 INSIGHTS FROM THE FIELDS It’s been a positive experience from all viewpoints, for the close relation- ships that I’ve developed, and also for the knowledge, both technical and personal, that I’ve acquired […]. What I learned from university exams was extremely useful, but not enough. You need to have experience, a lot of experience, to really understand how everything works in these environments, how to face difficult moments and how to intervene and support the wards in a constructive way. The relationship with children and adolescents made me understand that we should never take anything for granted. In every attitude, every word, every action there is a world to discover. We need to understand that world in order to make our contribution which, though small, is nonetheless very important. I didn’t expect to feel so comfortable in the foster home. Before starting my traineeship, I thought it would be much ‘heavier’ from an emotional viewpoint, but in the end, for me, it wasn’t. Obviously, there were diffi- culties, but, as in any other experience, it is important to know that you can make mistakes. From mistakes we learn, we learn that we should never give up, we should always be honest with ourselves and under- stand if what we’re doing makes us happy and proud. Finally, only one report could really be described as reflective writing and, to some extent, also as critical reflection. The student did her traineeship in a nursery and maternal school and in her report reflected a great deal on the connections established between theoretical learning and work-based learning. 4. Research project In addition, she elaborated on the ‘conflict’ between commercial and educational goals and about her own efforts to find a personal balance, accepting reality and, at the same time, refusing to compromise: Before my traineeship, I was advised to read Secrets of the Baby Whisperer by Tracy Hoggs. This book made me immerse myself in the complex- ity and beauty of care-giving, providing concrete instruments for daily practice with the infants. There’s a passage in Hogg’s book that captured my attention, where, in my opinion, the author makes the reader reflect about a pillar of education: that the ‘newly arrived’ should be considered as people, in their wholeness and particularity. […] Based on this scien- tifically valid assumption, I prepared myself for a traineeship in a school for 0-6-year-old children. The organization of spaces and materials in the nursery school where I worked was based mainly on commercial criteria, even if some more sophisticated games were available and captured my attention. […] My experience was a reality check. The institutions that operate in the field of infant education are many and diverse. They adopt educational mod- els that are not always based on pedagogical studies. Seeing all this with my own eyes conflicted, on the one hand, with the theoretical knowl- 276 ANNA SALERNI, SILVIA ZANAZZI edge developed during my university studies; on the other, it made me struggle, and forced me to find adequate solutions to live and act in that specific organizational context. My background in philosophy and education contributed to shaping my profile as a ‘reflection-prone’ individual. It contributed to my inclination towards individual and group reflection, to my attention in choosing acts and words dedicated to others, based on the situation and the context. […] I put all of myself into being there in the most serene and relational way possible, overcoming initial difficulties, willing to get the best out of a place where they offered entertainment rather than genuine educational activities. 5. Final remarks There can be no knowledge without emotion. We may be aware of a truth, yet until we have felt its force, it is not rs. To the cognition of the brain must be added the experience of the soul. (Arnold Bennett) An informed use of knowledge is possible only when we put in- to play personal interests, emotions, and affection. The experiential learning theory emphasizes non-cognitive aspects of learning. There can be no knowledge without emotion. We may be aware of a truth, yet until we have felt its force, it is not rs. To the cognition of the brain must be added the experience of the soul. (Arnold Bennett) 4. Research project Ac- cording to Tomassini (2007), in the social sciences, many feel a need to fill the void created by the predominance of cognitivism in learn- ing theories. Many are starting to oppose the idea that reflection is a cognitive activity and that the couple cognition-rationality should be considered ‘superior’ to emotions. From this viewpoint, a sort of ‘emotionalization’ of reflection is the necessary premise of every strategy aimed to strengthen reflection itself. Writing, if we wish it to become a reflective exercise, must focus on real experiences, ex- perienced with emotion, sometimes even with ‘suffering’. The sto- ryteller must look at his/her experience as something from which it is possible to learn, something that has a moral. He/she must be able to reflect on ‘what happens’ outside of him/herself (reflection) as well as on his/her own actions, as if he/she were looking in a mirror (re- flexivity) (Tomassini 2007). Those who tell of their experience must be able to seek and find a meaning within it. A continuous search for meaning is the root of reflection (Mortari 2003). gl We tried to stimulate the search for meaning through the inter- views. At first, the results of our research work were ‘disappointing’: we expected to receive final reports much improved and richer in reflections. However, if we refer to the experiential learning theory, 277 INSIGHTS FROM THE FIELDS these results become clearer and can be viewed as a starting point for future action. As already discussed, experiential learning must be seen as a continuous process, rather than as an outcome. Refl ective think- ing is a ‘fuel’ for experiential learning, and if we do not provide it constantly, the learning process is going to be interrupted. We knew it was unlikely that students would learn how to ‘refl ect on action’ with only one, albeit in-depth, interview (Schön 1983). The inter- views were undoubtedly very useful for them to look at their expe- rience from a distance, but they cannot be the only support off ered. At the end of this project, what we do know is that, despite the scar- city of resources, we must do our best to strengthen and enrich the support given to trainees, especially in terms of refl ective practice. 3 The concept of ‘community of practice’ was fi rst elaborated at the beginning of the ’90s in the realm of studies on apprenticeship conducted by Etienne Wenger and Jean Lave (Lave, Wenger 2006). These studies overturned the common belief that apprenticeship is based on a relationship between master and novice, affi rming instead that learning a skill is based on a social process of participation in a practice. Such practice entails a system of relationships between the novice and other members of the group, between the novice and the practice itself, and between the novice and the culture of the group. 4. Research project We also know that we should encourage students to see the trainee- ship report not merely as a formal requirement for credit, but also as an occasion to re-elaborate their experience. We are currently con- sidering setting up student-managed refl ection groups, where older students help younger peers to activate a refl ection process on their fi eld experience. Ultimately, learning is never a merely individual phenomenon concerning only one person, but involves a community of practice (Lave, Wenger 1991)3 (Fig. 1). Figure 1 – Experiential learning cycle: goals, instruments, refl ective practice, and refl exivity. Figure 1 – Experiential learning cycle: goals, instruments, refl ective practice, and refl exivity. 3 The concept of ‘community of practice’ was fi rst elaborated at the beginning of the ’90s in the realm of studies on apprenticeship conducted by Etienne Wenger and Jean Lave (Lave, Wenger 2006). These studies overturned the common belief that apprenticeship is based on a relationship between master and novice, affi rming instead that learning a skill is based on a social process of participation in a practice. Such practice entails a system of relationships between the novice and other members of the group, between the novice and the practice itself, and between the novice and the culture of the group. 278 ANNA SALERNI, SILVIA ZANAZZI The group work could benefit from using our interview questions, based on Kolb’s experiential learning cycle, as prompts for sharing ex- periences and discussion. In fact, the experiential learning cycle (Kolb 1984; Gibbs 2013) begins with concrete experience (question: «Can you briefly describe your traineeship experience?»), continues with reflec- tive observation (questions: «Do you remember a situation during your traineeship when you encountered a problem? […]; Can you recall a moment during your traineeship experience when you felt partic- ularly useful?») and subsequently abstract conceptualization (questions: «Thinking about three “areas of competence”, that is, “knowing”, “knowing how to do” and “knowing how to be”, can you bring ex- amples from your field experience showing your personal growth and skill development?; At the end of your traineeship experience, what do you think about your professional future after university? […]»). The fourth stage of Kolb’s cycle, active experimentation, happens when the trainee applies what he/she has learnt − and reflected on − to his/ her future actions. 4. Research project This is precisely what Killion and Todnem (1991) called reflection-for-action, the desired outcome of the first two types of reflection. We propose, therefore, to add a question for the trainees: «How can I improve next time? What can I do differently?». In this way, the experiential learning cycle never ends: the last stage flows into the first one, translating into a practice that is more reflective, more aware, and more competent. p Among the materials we provide on the Moodle page are guide- lines on how to write logbooks. In talking to students, we realized that logbooks are considered useless and a ‘waste of time’, and are rarely used. Instead, daily or weekly logbooks could become the main source of reflective learning for trainees. Noticing facts, beliefs, emo- tions, and critical incidents when they happen is fundamental to re- membering and, later on, using as material for reflective thinking. Logbooks could also offer input to be discussed during classes, trying to establish connections between theory and field experience. Re- flection, Dewey wrote, is a process of inquiry (Dewey 1933), and in- quiry means asking questions. It is very important to elicit students’ questions in all phases of their academic and work-based experience: not only questions on what we tell them in class, or on the reading materials for our courses, but also on what they see and experience at their placements. Logbooks could also be the place in which to gather all their questions, waiting for the occasion to share and search for answers. In sum, keeping logbooks regularly pushes the learner to alternate between being an actor or an observer, to go from active involvement to analytical detachment, exercising critical thinking by entering a cyclical experiential learning process. Moreover, while writing is widely recognized as a tool to enhance reflective practice 279 INSIGHTS FROM THE FIELDS by re-processing and interpreting experience, we should not make the mistake of taking writing abilities for granted. In some of our interviews, we noticed reflective thinking skills that unexpectedly ‘disappeared’ in the written reports. Reflective writing should also be taught and practised. One way to do this might be to show students the difference between descriptive accounts and reflective writings, or perhaps to share some of the best student work from previous years. 4. Research project h h fi d For the moment, we can say that our first intervention initiated a process of simultaneous training and inquiry. We plan to continue our laboratory for reflective practice, providing students more and diversified occasions to exercise reflection on action. Every strategy, to become ef- fective and real, needs to deal with a scarcity of resources, but as Dewey would say, «a genuine purpose always starts with an impulse» (1938). References Bateson G. 1972, Steps to an Ecology of Mind, University of Chicago Press, Chicago. g Cohen L., Manion L. and Morrison, K. 2011, Research methods in education, Routledge, London. Creswell J.W. 1998, Qualitative Inquiry and Research Design. Choosing among five traditions, Sage, London. l Dewey J. 1933, How we think: A restatement of the relation of reflective thinking to the educative process, Houghton Mifflin Company, Boston. Dewey J. 1938, Experience and Education, Collier, New York. Flanagan J. 1954, The critical incident technique, «Psychological Bulletin», 51, 327-358. Gibbs G. 2013, Learning by doing. A Guide to Teaching and Learning Methods, Oxford Brookes University, Oxford. Glaser B. and Strauss A. 1967, The discovery of Grounded Theory: strategies for qualitative research, Aldine de Gruiter, Hawthorne-New York. Harris A.S., Bruster B., Peterson B. and Shutt T. 2010, Examining and Facilitating Reflection to Improve Professional Practice, Rowman and Littlefield Publishers Inc., Lanham. Hobbs T. (ed.) 1992, Experiential training: practical guidelines, Tavistock- Routledge, London. Killion J.P. and Todnem G.R. 1991, A process for personal theory building, «Educational Leadership», XLVIII (6), 14-16. Kolb D.A. 1984, Experiential Learning: Experience as the Source of Learning and Development, Prentice Hall, Englewood Cliffs. p gf Lave J. and Wenger E. 1991, Legitimate peripheral participation, Cambridge University Press, Cambridge. y g Lewin K. 1946, Action research and minority problems, «Journal of Social Issues», 2, 34-36. 280 ANNA SALERNI, SILVIA ZANAZZI McClelland D.C. 1973, Testing for competence rather than intelligence, «American Psychologist», 28, 1-14.l y g Moon J. 2004, A Handbook of Reflective and Experiential Learning, Routledge, London.l Moon J. 2006, Learning Journals. A handbook for reflective practice and professional development – 2nd edition, Routledge, London. Mortari L. 2003, Apprendere dall’esperienza, Carocci, Roma. Murray H.A. 1943, Thematic apperception test, Harvard University Press, Cambridge. g Piaget J. 1971, Biology and Knowledge, Edinburgh University Press, Edinburgh. Salerni A., Sposetti P. and Szpunar G. 2014, Narrative writing and University Internship Program, «Procedia – Social and Behavioral Sciences», 140, 133-137. Salerni A., Sposetti P. and Szpunar G. 2013, La narrazione scritta come elemento di valutazione del tirocinio universitario. Narrative writing as an evaluation methodology of University Internship Program, «RPD, Ricerche di Pedagogia e Didattica – Journal of Theories and Research in Education», VIII (2), 9-26. Schön D.A. 1983, The Reflective Practitioner: how professionals think in action, Basic Books, New York. Salerni A., Sposetti P. and Szpunar G. References 2013, La narrazione scritta come elemento di valutazione del tirocinio universitario. Narrative writing as an evaluation methodology of University Internship Program, «RPD, Ricerche di Pedagogia e Didattica – Journal of Theories and Research in Education», VIII (2), 9-26.l Schön D.A. 1983, The Reflective Practitioner: how professionals think in action, Basic Books, New York.l Schön D.A. 1987, Educating the Reflective Practitioner: Toward a New Design for Teaching and Learning in the Professions, Jossey-Bass, San Francisco. Strauss A. and Corbin J. 1994, Grounded theory methodology: an overview, in Denzin N.K. and Lincoln Y. (eds.), Handbook of Qualitative Research. Sage, Thousand Oaks, 273-285.l Tomassini M. 2007, La riflessività dei professionisti della formazione: verso lo sviluppo di pratiche riflessive in contesti di formazione professionale, in ISFOL, La riflessività nella formazione: modelli e metodi, ISFOL, Roma. Patrizia Sposetti (Sapienza University of Rome)* Patrizia Sposetti (Sapienza University of Rome)* Abstract: This paper focuses on the relationships between academic, profes- sional and personal writing among Italian university students. The specific focus is on educators trained as part of the degree course in Education and Training Science. A solid, evident link exists between educational professions and writ- ing practices, as shown by the over two decades of research on this topic: writing facilitates the objectivization needed for sharing and, at the same time, makes it possible to distance oneself from the effort involved in the teaching professions. Keywords: academic didactics, educational writing, professional writing, edu- cational professions, active learning. * Patrizia Sposetti, Fellow Researcher, Sapienza University of Rome, Italy. Email: patrizia.sposetti@uniroma.it. 1. Writing and education professions In our knowledge-oriented society, writing is a key element, since it helps the creation of the various kinds of knowledge (Starke-Mey- erring, Paré, Artemeva, Horne, Yousoubova 2011) and the perfor- mance of human activities. The intrinsic meaning of writing lies in its contribution to human relationships and to the enactment of tex- tual actions. As stressed by Bazerman and Russel (2003): Writing is alive when it is being written, read, remembered, contemplat- ed, followed--when it is part of human activity. Otherwise it is dead on the page, devoid of meaning, devoid of influence, worthless. The signs on the page serve to mediate between people, activate their thoughts, direct their attention, coordinate their actions, provide the means of re- lationship. It is in the context of their activities that people consider texts and give meaning to texts. And it is in the organization of activities that people find the needs, stances, interactions, tasks that orient their atten- tion toward texts they write and read. So, to study text production, text reception, text meaning, text value apart from their animating activities is to miss the core of a text’s being (p. 1). Within a professional context, these aspects gain an indisput- able importance. Starting from the assumption that professional writing may take different forms (Tab. 1) − within the polarization between functionality to the performance of an activity, and reflection 282 PATRIZIA SPOSETTI on the activity carried out, with these two targets likely to overlap − we can extend this multi-dimensionality also to so-called «educatio- nal writing», a series of writings all «connected to the performance of one’s didactic, administrative, organizational and institutional task» (Biffi 2014: 35). Table 1 – A model for professional writing. [Source: reviewed by Sposetti 2011] Macro-target Subject Target Recipient Text type Communica- tion of contents to improve the practical per- formance of the professional activity Reports of specific activities Improving internal communication Internal Presentation. Persuasion. Description. (Technical re- ports, minutes, school register, in- dications etc.). Improving external communication External I m p r ov i n g t h e practical organiza- tion of the activity (simplification of practices and pro- cedures, streamline of times, etc.). Internal/ External Achieving recogni- tions for the activity carried out. 1. Writing and education professions Internal/ External Improvement of the professional activity through reflections on the experience Professional experience Divulgating the ex- perience (Cognitive function.) Internal/ External Narration Argumentation (Logbook, indi- vidual diary, life stories, reports, surveys, notes, etc.) Reflecting on the experience (Infer- ential function.) Internal/ The subject Sharing the expe- rience External Showing and con- trolling feelings and emotions (Restraining emo- tionality.) The subject Table 1 – A model for professional writing. [Source: reviewed by Sposetti 2011] Since the early 20th century, the depth and evidence of the re- lationship between writing and professional activity has led to in- teresting reflections resulting from assorted studies and researches on the field. The general subject is the use of the writing process regarded as a professionalizing tool in different sectors and areas requiring the supply of services to other individuals: caretaking, EDUCATORS IN TRAINING AND WRITING 283 teaching, training, etc., i.e. all the contexts falling within educa- tion and training sciences.i The dimensions of professional writings in the educational fields can easily be investigated by highlighting the ability of written com- munication to provide useful help within professional educational contexts. In the introduction to a recent study on reflective writing, Cros (2014) offered an overview of the studies in this field starting from an engagement for training through the analysis of profes- sional practices, which was developed in the Nineties. This context also fostered an interest in writing intended not only as a subject of communication but also as a training tool for educators. Logbooks, school works, narrations, reports, portfolios and any other written production act as general research tools and elements. Simultane- ously, the concept of training changes as it is considered not only a mechanical learning of operational procedures necessary for the per- formance of a specific professional activity, but also a «development of an adaptive, creative, and critical intelligence, a distancing and an analysis, a development taking place on the same level of practice» (page IX, translated from the French by the author.) As a result of the onset of new needs and training requirements, writing seemed to be the most suitable tool to provide an answer, giving subjects the opportunity to confront both themselves and the external ever- changing reality. 1 In the attempt to assess this assumption, various researches have been conducted on the writing-based training instruments in various professional contexts (training of trainers and educators, teaching, training of social workers, etc.) with the purpose of understanding whether writing can actually generate professional competences, wheth- er a specific kind of writing can lead to a professionalisation process and, if so, what are the peculiar characteristics of these writing forms. The main international and inter- cultural results were collected in two works carried out by Cros together with Louise Morisse and Martine Lafortune, published in 2009 and 2011. 1. Writing and education professions The characteristics of writing promote analysis, imply the possibility to take a certain distance and turn experience into words by reducing the implicit, unstable, and feeble element typical of oral transmission and allowing for a ‘deferred’ communi- cation: thus, writing becomes a tool of knowledge and profession- alization. Moreover, writing is also an economic instrument, since it is easily manageable and immediately available to the individuals being trained: everyone can write and conceivably they have already tackled professional writing. l p g Over the last decade, reflection on the features of professional writing in the educational context has been extended to the pos- sible specificity of this professional means1. This extensive and transverse research activity has emphasised two typical elements 284 PATRIZIA SPOSETTI of writing regarded as a professionalizing tool: a) writing belongs to a well-defined training context in terms of deadlines, support and clear identification of the targets; B) writing implies a reflec- tion and an intensification of professional activity, in the form of ‘reflective writing’. 2. The functions of educational writing Writing has several functions; for the purposes of this paper, it is worth mentioning three of them in particular: communicative, epis- temic, and heuristic function (Cros 2011). The communicative function is immediately clear. The purpose of communication is to share something with the others such as in- formation, experiences, and moods, which thus become known to others. Moreover, writing also has an epistemic function, i.e. the pro- duction of thoughts and knowledge: Through writing, experience – turned into the subject of reflection − can interact more easily with individuals’ knowledge and competencies and be integrated by them. Thus, a form of epistemic writing can be re- covered, namely a writing leading to a change in and growth of knowl- edge (Bereiter 1980), ‘Where composition helps the writer acquire a higher comprehension’ (Bereiter, Scardamalia 1987, 1995: 86) (Salerni, Sposetti, Szpunar 2013: 12). As stated by Cros (2011) We may say that we write to know our thought […] We are our first read- ers and the words written on the paper ask this reader (ourselves) some questions and suggest interpretations other than the original intentions towards the others. Through the reading of our own writing, we reveal ourselves to ourselves. The sense of a text is never ultimately reached; therefore, we must carry out an exegetic activity on our own texts. This activity can be scary and may result in split thoughts ... Actually, it is wrong to say that writing consists of two different steps: first the pro- cessing of thoughts and then the correct transcription in writing of the thoughts (p. 2, translated by the author). The heuristic function of writing is connected to the epistemic one, since it helps identify innovative ideas and the links between facts and thoughts, culminating with important discoveries. After all, heuristics is one of the seven functions of language as defined by Halliday (1975), through which men perform a linguistic explora- tion of the surrounding world based on questions such as «Why…?» EDUCATORS IN TRAINING AND WRITING 285 or «How…?»2 This function is further enhanced by the written ex- pression, especially among adults, with the purpose of recalling, tell- ing, and sharing experiences. Furthermore, within the learning of writing, Halliday (1993) refers to a specific linguistic shift from the general to the abstract, which helps transform experience into sys- tematic knowledge. Writing − learned as a secondary symbolic sys- tem − offers a synoptic insight into reality. With reference to our study of professional writing3, this aspect is connected to the specific ability to shape the world around us by putting down in writing the flow of events through their selection, combination, and a creation of new possible meanings. Therefore Writing proves to be […] a tool suitable – probably the most suitable – for a reflective elaboration and sharing of experiences, since writing helps distance oneself from the past, just as it enables a symbolic representation of the experience through thought, with reference to the emotional and cognitive sphere (Mortari 2003). Therefore, writing fosters the objecti- fication process underlying sharing and, at the same time, helps distance oneself from the adversities of the education professions. 2 The other six functions are: instrumental (meeting needs: «I want»); regulatory (controlling others’ behaviour: «Do this»); interactional (interacting with the persons around us and creating a dialogue: «hello»); personal (introducing oneself and one’s tastes: «This is me»; «I like»); imaginative (being creative and building worlds: «let’s pretend that»); informative (sharing information: «Today is sunny»). The scholar has identified these functions based on an investigation of the development of language among children. As stated by Cros (2011) As evidenced by Fiamberti (2006: 3), ‘The purpose of a worker’s writing is to rely on a mirror (reflective need) as well as on a bank (containment need) to face the complex situations and emotional involvement in everyday life’ (Sposetti 2011: 267). g 3 Halliday (1993) reflects on the writing learning process among children. His thought focuses on the relationship between learning and language and, in particular, on evidence suggesting the interpretation of the former (learning) based on the latter (language). 2 The other six functions are: instrumental (meeting needs: «I want»); regulatory (controlling others’ behaviour: «Do this»); interactional (interacting with the persons around us and creating a dialogue: «hello»); personal (introducing oneself and one’s tastes: «This is me»; «I like»); imaginative (being creative and building worlds: «let’s pretend that»); informative (sharing information: «Today is sunny»). The scholar has identified these functions based on an investigation of the development of language among children. 3 Halliday (1993) reflects on the writing learning process among children. His thought focuses on the relationship between learning and language and, in particular, on evidence suggesting the interpretation of the former (learning) based on the latter (language). 5 The interviews were conducted with students who attended a workshop within the PSEF Degree Course, coordinated by the author of this paper. The research meth- od consisted of training the interviewers through theoretical references and simula- tions. Then interviews were conducted in pairs with a single speaker, so that notes on the interview could be written down without disturbing or interrupting the ad- vancement. In the 2013-14 academic year, the interviews were conducted by: Chiara Cacciotti, Marta Cecalupo, Elena Cefaloni, Piera Del Prete, Federica Flammini, Ilaria Frabetti, Valentina Maddion, Elisa Toni, Simona Trombetta, Roberta Magoni, Federica Pezone, Martina Pasquali, Lidia Tavani; in the following academic year more in-depth interviews were carried out by: Alessia Ballato, Tiziana Bonanni, Giulia Caccia, Marco Cadavero, Arianna Chiaravalle, Erica Cozzolino, Martina Ferretti, Alessia Giacomini, Maria Cristina Grosso, Roberta Guidano, Claudia Iacovacci, Laura, Masala, Giulia Rocchi, Sara Sannella, Martina Squadrilli, Annamaria Strabioli and Elena Trevisan. 4 The survey was conducted using mixed methods, by using specifically designed grids, T-Lab 9 software for text analysis, and an examination of the contents of the in- terviews, which required an initial brainstorming with the students culminating in the 2015-2016 academic year with a more detailed interview draft to help the interviewees give their opinion on their relationship with writing. 3. Research on educators being trained and writing In line with the issues concerning writing within the educa- tion professions, this section presents the data collected during the 2015-2016 year during an interview with some students enrolled in the Master’s Degree in Pedagogy and Educational Sciences at The Sapienza University. The interviews fall within a broader investi- 286 PATRIZIA SPOSETTI gation of written productions and of the attitude towards writing among the students attending the first year of the Pedagogy and Educational Sciences (PSEF) course, started in the 2011-2012 aca- demic year. These students had taken a three-year degree in Peda- gogy and Education Techniques, thanks to which they were able to work as educators with children aged 0-3 years at group homes, post-school, and social institutes as well as with people of various ages and unique needs (the disabled, the elderly, foreigners, immi- grants, etc.) g The investigation relied on the assumption that these students were fully aware of their relationship with scientific and professional writ- ings, including any evolution, changes, and difficulties. In fact, they had followed a growth path during their academic studies together with a considerable experience in scientific writing during the draft- ing of their final dissertation and apprenticeship reports.i gi pp p p Based on the outcome of the first years of the research, in the 2013-14 and 2014-15 academic years, the author of this paper con- ducted analysis on the entrance exams that the PSEF students had to take. This analysis was backed up by short interviews on the academic writing4. In 2015-2016, detailed interviews were con- ducted to better understand the meaning of the writing process for the individuals and have an insight into this phenomenon based on the participants’ perspectives5. The students interviewed account- ed for almost two-thirds of the people enrolled in the first year of the Master’s Degree Course (42 students), equal to 25 students plus 4 students enrolled in the second year. They were all professional educators since they had taken a three-year Degree in Education; EDUCATORS IN TRAINING AND WRITING 287 27 had attended a Degree Course in Educational and Training Sci- ences at the Sapienza University and 2 a Degree Course in Train- ing Sciences at the Roma Tre University; over two-thirds of these professional educators being trained already had work experiences. This was a sub-group made up of 21 students, 8 of whom regularly work as educators. 6 For a description of the apprenticeship in this course refer to the contribution provided by Anna Salerni and Silvia Zanazzi to this paper. 6 For a description of the apprenticeship in this course refer to the contribution provided by Anna Salerni and Silvia Zanazzi to this paper. 7 This essay could not dwell on the series of data collected. For an insight into this issue, see Brusco, Lucisano, Salerni e Sposetti (2014) and Sposetti (2017b, forthcoming) also for an explanation of the tools used. 7 This essay could not dwell on the series of data collected. For an insight into this issue, see Brusco, Lucisano, Salerni e Sposetti (2014) and Sposetti (2017b, forthcoming) also for an explanation of the tools used. 3. Research on educators being trained and writing Thirteen participants had professional writing experiences at work, 6 interviewees had gained professional writ- ing experience for the drafting of the apprenticeship report required by the three-year Degree Course in Educational and Training Sci- ences6, while 10 students declared that they had no writing experi- ence, neither for work nor for apprenticeship purposes. l pp p p p The interviewees were asked to reflect on three fundamental is- sues: their relationship with a) writing in general, b) academic writ- ing, c) writing specifically for educational activities7.i As for the last aspect, they had to define professional writing by mentioning the differences with respect to so-called ‘leisure writing’ so as to gather information on the perception of specificity and the recognition of a specific text form. The participants largely agreed on the peculiarity of leisure writing, since it is characterised by emo- tionality, confidential contents, personal expressions, spontaneity, low formality and attention to correctness, familiarity with the in- terlocutor, who is treated as a friend. Only one interviewee found a slight difference between these two forms of writing, while anoth- er assumed that it might be equally challenging; finally, one student provided no answer to this question. p q According to the participants, the intimacy of leisure writing contrasts with professional writing, which is speculatively intend- ed as more accurate in terms of compliance with the rules and text form, with some differences being observed between those who had writing experiences within their educational work or apprenticeship and those who had none (Tab. 2). Educators subjected to training with writing experiences mainly believed that professional writing is characterised by compliance with language rules (9 students, on- ly 1 of whom had no writing experience), the use of a specific ter- minology (4), the role of the addressee, the work context, and the subject in question. Those who had no writing experience tended to provide generic answers: professional writing is more immediate (4), more accurate and requires a formal register (4). 288 PATRIZIA SPOSETTI Table 2 – Characteristics of professional writing (28 participants). 3. Research on educators being trained and writing Professional writing Experience of professional writing At work For the uni- versity apprentice-ship No experi-ence Total strictly complies with spelling and syntactic rules and well-struc- tured form 6 2 1 9 deals with different topics, has a more spe- cif ic context and a well-defined addressee 5 2 - 7 is more accurate and “reflective” [sic] - - 4 4 must refer to a precise field and feature a for- mal register - - 4 4 has a more complex lan- guage and terminology 2 2 4 Total 13 6 9 28 e 2 – Characteristics of professional writing (28 participants). In most cases, the educators being trained who were interviewed for the purposes of this research believed that writing is an important work tool; as for usefulness, the answer provided by those who had writing experiences was different from those who had no experience at all (Tab. 3) 5 participants said that writing is almost or totally useless, while 4 people had never written texts for professional purposes. The latter tended to give quick and assertive answers like «It’s not impor- tant» (int. nos. 1 and 28) or «It’s unnecessary» (int. 29); those who had already had writing experience believed that educators should be able to face specific kinds of writing (such as a logbook), whose drafting does not require exceedingly difficult skills. The other reflections recognized the importance of writing for the acquisition of important instrumental skills (9 interviewees, 1 of whom had never had writing experience), relational competences (8, 2 of whom without any writing experience) or professional growth (7, 3 of whom with no writing experience.) p g ( g p ) Finally, educators were asked to interpret their relationship with professional writing in relation to the three-year Degree Course, in- cluding the fulfilment of sufficient professional writing criteria by the graduates in the educational sector (Tab. 4). The answers mostly indicated the need to acquire further competences, both general and specific. This was the opinion of 17 interviewees, 14 of whom with EDUCATORS IN TRAINING AND WRITING 289 writing experiences at work or during their apprenticeship; 6 of these stressed that the burden of the acquisition of these skills should not be on universities. Only 4 participants − equally distributed among the two categories − believed that the new educators did not have the writing skills necessary to meet professional obligations. 4. Final remarks The data relating to the interviewees’ opinion on writing for ed- ucation professions should be seen in the context of the relationship with writing per se, and with writing as part of a training path. The use of writing was regarded as positive by most of them with refer- ence to a generically expressive or functional (writing for study pur- poses) and more complex dimension, when it came to coping with the different writing opportunities included in the academic path, together with the drafting of the final dissertation. g gi These students saw writing as a sort of double-headed herm, re- sulting from the formal vs. informal polarization, where formal writ- ing is associated with compliance with spelling, grammatical and syntactic rules as well as proper terminology. They regarded formal writing − whether intended for university or a work context − as ‘accurate writing’, a kind that complies with morphosyntactic and lexical rules. Yet the forms taken by writing within the education professions can fall within divergent genres, ranging from scientific writing to a more confidential writing of a diary; sometimes multiple forms coexist in the same text. Within educational writing contexts, it is easier to find texts where narration, description and argumenta- tion tend to overlap. Professional writing often shows features asso- ciated with spontaneity and poor planning which the interviewees attributed exclusively to personal writing, defining a dichotomy be- tween the latter and professional writing which emphasise produc- tion in one case and planning in the other. In this regard, the educators being trained involved in the survey seemed unable to perceive the complexity and versatility of this tool or the continuum between formality and informality, especially con- sidering the marginality of stylistic aspects in the professional educa- tional writing: what really counted was not the aesthetic properties of the writing, but its ability to report experiences in a detailed man- ner. To achieve these goals, educators must undertake a complex path which starts from their experience and culminates with its verbaliza- tion, thus leading to the production of texts that must be readable by external readers and rely on narration and description, on one hand, and argumentation and reflection, on the other (Cros 2014). These writing forms are crucial for educators’ work and their ac- quisition requires training and support, as stressed also by the inter- viewees. 3. Research on educators being trained and writing Table 3 – Interpretation of the importance of professional writing for the educators (29 interviewees). Is professional writing important for educators? Experience of professional writing At work For the university apprentice-ship No experi-ence Total Scarcely or no impor- tant at all for work purposes 1 – 4 5 Very or sufficiently important since educa- tors must have writing skills suitable for their reports and projects 6 2 1 9 Very important, since it improves compe- tence and fosters hu- man interactions 4 2 2 8 Important also per professional and per- sonal growth 2 2 3 7 Total 13 6 10 29 Table 4 – The competences of the graduates (29 participants). Are the professional writ- ing skills of the new ed- ucators suitable for their activity? Experience of professional writing At work For university apprenticeship No experi-ence Total Absolutely 2 – 2 4 Yes, in general 1 2 5 8 These skills are gained over time and do not depend on academic studies 2 4 6 Some specific skills are necessary for profes- sional activities 8 – 3 11 Total 13 6 10 29 Table 3 – Interpretation of the importance of professional writing for the educators (29 interviewees). Table 4 – The competences of the graduates (29 participants). 290 PATRIZIA SPOSETTI 4. Final remarks 4. Final remarks The professional writing skills described above do not and cannot result exclusively from a curricular academic course: similar- ly, they neither manifest spontaneously during professional activities nor do they arise from the routine drafting of documentation. Writ- ing intended as a professional growth and development tool includes 291 EDUCATORS IN TRAINING AND WRITING some rules, and its learning is based on a path consisting of deadlines, assessments, discussions, and support from experts or the group itself. pp p g p This means that professional writing involves both the institu- tion requiring the production of a certain kind of writing and the path for the acquisition of writing skills. Professional writing cannot be decontextualized or abstract. For educators, writing should be a means and not an end in itself, since it should act as a professional and practical support to concrete situations. The relation between uni- versity and work (Pollet 2001, 2004; Reuter 2004; Chartrand 2006; Ganobscik-Williams 2006; Lillis 2006; Hyland 2007; Blaser 2008; Lillis, Scott 2007; Lea, Jones 2011; Baudet, Rey 2012) as well as be- tween academic education and leisure and work contexts (Ivanič, Edwards, Barton, Martin-Jones, Fowler, Hughes, Mannion, Miller, Satchwell, Smith 2009) is key, and requires reflection on the necessity to create specific and well-structured learning paths. Within educa- tional Degree Courses, apprenticeship is regarded as a special occa- sion also for training aiming at the production of ‘professionalizing’ writings closely connected to the work, and acting as a connection and shift from training to work. The reflections of the participants moved precisely in this direction, recognizing the importance of the apprenticeship report for the orientation of professional writings. References Baudet C. and Rey V. 2012, De l’écrit universitaire à l’écrit professionnel: comment favoriser le passage de l’écriture heuristique et scientifique à l’écriture professionnelle?, «Scripta», Belo Horizonte, XVI (30), 169-193. Bazerman C. and Russel D.R. (eds.) 2003, Writing Selves/ Writing Societies. Research from Activity Perspectives, The WAC Clearinghouse Colorado State University, Fort Collins. y Bereiter C. 1980, Development in writing, in Gregg L.W. and Steinberg E.R. (eds.), Cognitive Processes in Writing, Erlbaum, Hillsdale, 73-93. Bereiter C. and Scardamalia M. 1987, The Psychology of Written Composition, Lawrence Erlbaum Associates, Mahwah, New York. Biffi E. 2014, Le scritture professionali del lavoro educativo, FrancoAngeli, Milano. 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Reuter Y. 2004, Analyser les problèmes de l’écriture de recherche en formation, «Pratiques», 121-122, Les écrits universitaires, June 2004, Metz, CRESEF, 9-27. Starke-Meyerring D., Paré A., Artemeva N., Horne M. and Yousoubova L. 2011, Writing in Knowledge Societies, Parlor Press, Anderson. * Pierluigi Malavasi, PhD, Full Professor of General and Social Pedagogy and Director of the School of Advanced Studies in Environmental Studies (ASA) - Catholic University of the Sacred Heart, Italy. Email: pierluigi.malavasi@unicatt.it. CHAPTER III GUIDANCE AND CALLING CHAPTER III GUIDANCE AND CALLING INTERPRETATIONS. MARKET, WORK, TRAINING Pierluigi Malavasi (Catholic University of the Sacred Heart)* Pierluigi Malavasi (Catholic University of the Sacred Heart)* Pierluigi Malavasi (Catholic University of the Sacred Heart)* Abstract: What of the job market in the era of Brexit and in the interna- tional arena of the Trump presidency? Naturally, there are various perspec- tives, but forms of emotive or propagandist rhetoric threaten to hold sway here. Above all, Guidance Towards the Job market means recognizing the com- plexities of geopolitical change within our information-sharing and collab- orative networks, as well as the various and contrasting interpretations that arise. The current decline of the European Union − of which the UK pub- lic referendum result is symptomatic − and the ‘America First’ slogan, now a distinguishing feature of the current US government, are only two of the multiple forces interacting and defining pervasive transformations in eco- nomic, labour, and educational policies. Keywords: guidance, market, work, training. What of the job market in the era of Brexit and in the interna- tional arena of the Trump presidency? Naturally, there are various perspectives, but forms of emotive or propagandist rhetoric threaten to hold sway here. Above all, Guidance Towards the Job market means recognizing the complexities of geopolitical change within our in- formation-sharing and collaborative networks, as well as the various and contrasting interpretations that arise. g p The current decline of the European Union − of which the UK public referendum result is symptomatic − and the ‘America First’ slogan, now a distinguishing feature of the current US government, are only two of the multiple forces interacting and defining perva- sive transformations in economic, labour, and educational policies.l Conflicting views abound. Take, for example, the challenge of agreeing on multilateral interventions to address the issue of immi- gration. Or the relationships between market, work, and training con- sidering Europe’s infringement procedures for excessive debt, which affected Greece so drastically, and the lack of any formal rebuke by EU authorities regarding Germany’s excessive trade surplus. Globally, professions and employment dynamics ‘are coming to terms’ with interpretations that the official statistics reports summa- rize with expressions such as «the slowing of the world economy», «increasing inequality and the acceleration of change», «quality of 296 PIERLUIGI MALAVASI human resources and attraction of investment» and «oversupply and low-cost labour». human resources and attraction of investment» and «oversupply and low-cost labour». 1 CENSIS, Rapporto sulla situazione sociale del Paese 2016, FrancoAngeli, Milan 2016, p. 159: “Lo slowdown nell’economia mondiale. World growth was 3.1% in 2015 and 2016, and will not exceed 3.4% in 2017. World trade grew on average by 6% between 1990 and 2011, and by 2.7% annually between 2012 and 2014. Global unemployment is growing. In 2015 global unemployment reached 197.1 million, which is around 30 million more than the pre-crisis figure. Between 2016 and 2017, the figure is expected to rise by 3.4 million. The unemployment rate is stated to be around 5.8%. In 2017, the number of unemployed is expected to exceed the 200 million mark. Poor and vulnerable worker numbers are increasing. Precarious employment, with limited access to protection, af- fects 1.5 million workers, around 46% of the total employed. The “rest” in the informal economy, forced labour and child labour Informal employment in non-agricultural sectors has reached 50% globally. 21 million people are in forced labour, whilst child labour is estimated to account for around 167 million people.” INTERPRETATIONS. MARKET, WORK, TRAINING Today, Pedagogy contributes iuxta propria principia to develop cul- tural sensitivity and lead the political consensus towards a concept of change governance. Such a perspective focuses on the relationship between education and work (Federighi 2010), and between the in- dividual and the sense of community. This is a regulatory objective still to be reached and never fully achieved globally or substantially on the ground; including the concept of stakeholder in this scenario personalizes the responsibility of companies, public entities, and edu- cational institutions towards society, identifying the specific groups or individuals to be taken into consideration in the actions associated with transforming the labour and education ‘markets’ (Vischi 2011). Pierluigi Malavasi (Catholic University of the Sacred Heart)* When the CENSIS Foundation processed the latest data from the International Labour Organization, the International Monetary Fund, and the Bank of Italy, it observed a global increase in unemployment and in the number of poor and vulnerable workers, confirming the con- tinued use of forced and child labour1. In many respects, the data on Employability and Competencies was unsettling. p y y p g In light of these trends, it is logical to refer to a document that had a global resonance fifty years ago. In his Encyclical Letter Populorum Progressio, Pope Paul VI noted: «An ever-more effective world soli- darity should allow all peoples to become the artisans of their des- tiny», «every human life is called to some task» (Pope Paul VI 1967: nos. 65 and 15). This cautionary message from 1967 was echoed in the words of Pope Francis, both in his United Nations Address and in the Apostolic Exhortation Evangelii gaudium: «The misuse and destruc- tion of the environment are also accompanied by a relentless process of exclusion» (Pope Francis 2015). And «we are not simply talking about ensuring nourishment or a dignified sustenance for all people, but also their general temporal welfare and prosperity. This means education, access to healthcare, and above all employment, for it is through free, creative, participatory, and mutually supportive labour that human beings express and enhance the dignity of their lives. A just wage enables them to have adequate access to all the other goods which are destined for our common use» (Pope Francis 2013). Since the 1950s, we have seen the acceleration of technological progress, an increase in gross domestic product, and economic-finan- cial interdependence on a global scale. These have become part of a concept of development preoccupied with production, oriented to- wards increasing profit, and not to the general improvement of living conditions, nor the creation of a supportive society. INTERPRETATIONS. MARKET, WORK, TRAINING 297 1. Market, stakeholders, educational planning Examining organizational structures and procedural regulations to achieve better results in terms of service efficiency, and/or product quality becomes a point of reference for a system of reasoning based on the interdependency between striving for competitiveness and ethical tension: the civilian community as a whole is the stakeholder when it comes to finding the best possible allocation of resources and redistributing the benefits. In cases of inefficient and wasteful services, or expen- sive, sub-standard products, then expenditure must be reviewed, and, at the same time, the investment must be evaluated from an ethical perspective. The effectiveness of the objectives pursued and efficien- cy in human work and in the use of material resources are cardinal elements of an approach to educational project design that is atten- tive to the ethical and economic interconnections of organizational action and the governance of its development.i g p No choices are merely technical or justifiable through recommen- dations or guidelines laid down by specialists in organization. It is in- appropriate and misleading to uncritically accept arguments deduced from economic and financial publications and studies on manage- ment, to make decisions on spending cuts and cost saving, prioritiz- ing certain administrative or corporate departments over others. Good deliberation involves well-founded, practical wisdom and takes on a political, ethical, and educational character that no corporate management theory should underestimate. Much as it is welcome, the work of specialist commissions and technical support must be scrutinized by the stake- holders to be adopted appropriately. Educational planning for organizational change takes place within a context rooted in history, which can be visualized as a group of interacting stakeholders, with the names and faces of members of society. The challenge is to build an authentic dialogue, without forgetting to decide which stake- 298 PIERLUIGI MALAVASI holders to engage and how they can best be involved in the delibera- tion process. In all cases, there are multiple groups of interested parties (shareholders, communities, consumers, suppliers, social activist groups etc.), who require the management’s attention. Educational planning, in pursuit of the common good, must be tailored to consider the multiplic- ity of the rights represented by the principle stakeholders identified and to manage mediation in all its complexity, to encourage the achievement of coherent objectives and the balanced development of the community. j p y Human capital formation is consciously and intentionally an ongoing project. 1. Market, stakeholders, educational planning From a systemic perspective, educational planning requires the responsible engagement of the various stakeholders in developing the cur- ricula and the strategies of the training plan. Its success or failure not only has consequences on the ultimate shareholding stakeholder − society as a whole − but on all stakeholders, corporations, institutions, etc. l At this dynamic historical juncture, it is essential to reflect on the challenges of matching competitiveness with social cohesion, technological progress and economic development in order to explore new routes for ed- ucational planning. In order to plan meaningful interventions to man- age change, we must find an appropriate new way to define development, a prospect that requires integrity and the skill of political representation, deals with growth and equality in economic reforms, and concerns the qual- ity and productivity of investments in teaching and human capital formation. y p y f g p f The banking crisis, which has caused such alarm since late 2007, reveals the danger posed by the distorted dynamics of the markets and credit management systems, by inadequate regulation on new high- leverage ‘derivative’ products, and by change governance models that have related poorly to ethical and moral values and have been ethi- cally inadequate in a number of ways, and therefore not very trans- parent in terms of the close link between the management’s performance and the disingenuous sale of risks to the investor. The crisis, the con- sequences of which can still be sensed in the social fabric of many countries, must inspire constant vigilance with regard to the need to introduce the question of the moral, political and economic responsibilities of proprietors and senior management in the principal areas of multidisciplinary investigation and public debate. The emphasis placed on the crisis, which has had serious and pro- longed effects, may also stimulate suitable decisions regarding a dif- ferent way of structuring knowledge (Morin 1999). Today, a thorough reinterpretation of the culture of work and management training is strategically important to guide change and promote innovation. Many success stories demonstrate that it is not by luck that a product or process triggers growth, but is rather by creating a culture based on creativity and the willingness to invest in the fusion of human capital, participative leadership and financial resources. INTERPRETATIONS. 1. Market, stakeholders, educational planning MARKET, WORK, TRAINING 299 An authentic business culture generates wealth if it is part of a supportive citizenship structure and pursues education on legality as one of the key values in planning and governance. One theory on human development as freedom (Sen 1999) binds together the market, the stakeholder, and educational planning. f The pedagogical debate in the last decade in Italy offers various contributions to support the hypothesis that investment in educational planning can bring about an increase in value and beneficial participa- tion on the part of the stakeholders. The diversity of cultural orienta- tion and the juxtaposition of methods in relation to the planning of education and training in the knowledge-based society, with refer- ence to theories of organization, research into communities of prac- tice, social responsibility, and the relevance of social pedagogy, are all essential heuristic elements, which generate reflexivity and stra- tegic development (Alessandrini 2007; Fabbri 2008; Malavasi 2007). Is it a realistic ambition for educational planning to develop a struc- tured understanding of governance and guidance towards the job market in line with the perspective of the International Conference Employabil- ity and Competencies. Innovative Curricula for New Professions?fi An affirmative response is closely linked to the relevance assigned to the culture of education as a vehicle for human development. The texture of solid inter-institutional models, and planning that respects the various partners’ prerogatives and competences, are cooperative approaches that generate value i.e. human dignity and economic profit, in a long-lasting way, if they are targeted at reducing inequal- ity, redistributing revenue, building social cohesion, and cultivating/ protecting the common good. 2. Competences, Education, Work. Learning and working: a case in point 2. Competences, Education, Work. Learning and working: a case in point An integration between the educational system and the world of work, based on the promotion of human life and real justice, must be considered essential for the development of future competences and professions, and for steering the education of young people and adults in a fair and supportive way (Bertagna 2011; Bocca 2000; Federighi 2011; Vischi 2014). The complex issue of employment calls for the transition from school and university to work to then be constantly improved. On the one hand, it is important to enhance policy and the educational offering in relation to the professional profiles and qualifications re- quired; on the other, we need to improve the acquisition of compe- tences that are adapted to the demands of the production and services system. Meanwhile, we must restore the confidence of many of the 300 PIERLUIGI MALAVASI young people who do not study and do not work, for whom person- al frustration leads to the mistrust of institutions and the lack of any sense of belonging, which is likely to result in insecurity, confusion and a marginalized experience of life. In Italy, the introduction of obligatory participation in the work- related learning programme in the last three years of the high school is a profound innovation and marks a real change. National Law no. 107/2015 encourages young people and schools to engage in work- based learning programmes. Learning and working, beyond the restrictions of complying with regulations and implementation chal- lenges, brings us closer to vital situations and contexts: corporations, associations, social voluntary work, and service learning, etc. Specifi- cally, it «enables students to develop confidence in terms of tasks, schedules, and ways of working in the professional word, as well as testing out their own competences (including social and emotion- al ones)» (Rosina 2015: 83). Our future prosperity depends on the young, as the European Commission points out in its report Youth on the Move, in support of the Europe 2020 Strategy. This is particularly relevant to Italy, which is one of the advanced economies most lack- ing in complete and suitable tools for including new generations in the enhancement and creation of wellbeing (European Commission 2011). 2. Competences, Education, Work. Learning and working: a case in point Young people who could potentially serve as a resource for development may become, paradoxically, a social cost, partly because of the absence of comprehensive, active work policies, incentives for enterprise, and sufficient investment in research, development, and innovation. If we do not take incisive action, such social disadvan- tages will become chronic. Indeed, authoritative international re- search institutes anticipate a potential lost generation (IMF 2015). The issue of employment must be integrated into a structured system of measures that coherently combine vocational guidance with educa- tion-to-work transition, «but also the achievement of autonomy and the construction of a solid basis for planning one’s own life. Policies, therefore, that are not only motivating but also enabling: tools that encourage autonomy […], reduce starting inequality and make young people more dynamic and responsible […] less vulnerable to becom- ing trapped in long-term unemployment or entirely precarious em- ployment (Rosina 2015: 98). A culture that is constantly considering new work-related forms and ethical content, within the changing historical and social context, con- stitutes an educational endeavour hinging on personal freedom and the equal dignity of the various professions, with important legal, eco- nomic, technological, religious, and media-related implications. This article is exploratory in nature, and its aim is not to examine the many issues and technicalities at play, nor to develop the ethical and moral INTERPRETATIONS. MARKET, WORK, TRAINING 301 dimension of work, the relevance of which is often overlooked. The issue of organizational wellbeing and the fostering of loyalty in pro- fessional relations should be carefully explored, along with means for reconciling family time and time at work, training for professional abnegation and overcoming doubts and resistance regarding invest- ing in the younger generation. Pedagogy, the science of education and training, understands change governance, in its multi-faceted complexity, to be an inex- haustible field of experience and processes upon which to reflect in terms of planning. Beyond and through particular conjunctures and events, the success of policies for socioeconomic growth and related ethical and moral enterprises to raise the quality of life for everyone on the planet, is closely linked with the ability to generate a creative momentum and enhance the talents of one and all. 2. Competences, Education, Work. Learning and working: a case in point The anthropologi- cal choice behind work-related education stems from a holistic view of humanity, which understands the education of the individual to mean promoting their dignity and responding to the human voca- tion for work; this is considered essential for development, against a backdrop of rapid change and the pervasive nature of mediatization throughout the world. g The role of a form of pedagogy open to multidisciplinary dialogue is: to evaluate the fusion between professional knowledge and busi- ness practices; to identify needs and provide vocational guidance; to develop theories and operative protocols to instruct and engage the community, taking a holistic view of humanity (Giuliodori, Malavasi 2016). The inequity of issues such as absolute poverty, hunger, and youth unemployment is related to environmental, social, and economic sustainability; it calls for the ‘deployment’ of the sciences and technol- ogies, imposes a participatory model for doing business, and calls for the contribution of religious values. g In the face of the dominant ‘market’ ideology centred on short- term profit, research and intervention on ways of ensuring that our schools, universities and businesses do not create inequality in society are essential. The importance now attributed to the human capital econo- my, amid much controversy, must be transformed into competences as a common good, competent planning (Birbes 2012) increased dignity and value, and opportunities for enterprise on an intergenerational scale. Competences are often represented in a functional way, as a sort of unit of certified educational capital, which can be exchanged on the global work market as a standardized commodity, with a ‘guaranteed origin’ to be recognized by potential interested parties for its declared value. This supposed transverse validity about the curriculum justifies the adoption of the term in close continuity with that of ‘certification’ (Guasti 2000). According to the usual interpretation, a certified skill is g In the face of the dominant ‘market’ ideology centred on short- term profit, research and intervention on ways of ensuring that our schools, universities and businesses do not create inequality in society are essential. The importance now attributed to the human capital econo- my, amid much controversy, must be transformed into competences as a common good, competent planning (Birbes 2012) increased dignity and value, and opportunities for enterprise on an intergenerational scale. 2. Competences, Education, Work. Learning and working: a case in point Competences are often represented in a functional way, as a sort of unit of certified educational capital, which can be exchanged on the global work market as a standardized commodity, with a ‘guaranteed origin’ to be recognized by potential interested parties for its declared value. This supposed transverse validity about the curriculum justifies the adoption of the term in close continuity with that of ‘certification’ (Guasti 2000). According to the usual interpretation, a certified skill is 302 PIERLUIGI MALAVASI now considered the necessary condition for an individual’s education- al and professional mobility. The rigour of certification procedures and the reliability of the results lead to the dynamics of interperson- al communication and inter-professional exchange. Above all, one needs to gather the coherent features between the two dimensions of competency, the internal interaction between knowledge and expe- rience, and external universal recognition, which reflect and justify each other, the one influencing the other. Competences are related to technological development and man- agement principles: flexibility and mobility within a company, as well as on an inter-company and inter-system level, surpass national bound- aries and constitute the necessary attributes in a context where the scale and quality of production is changing, and not only in negative conjunctures, but as a normal condition of market unpredictability. Linked to the faith in succeeding to create a system for rationalizing human capital centred on competences, is the by-now considerable credit attributed to standards, which ‘legitimize’ the prescriptive value of competences by internationalizing them (Guasti 2003). f p y g In many respects, the debate on competences affects the theory regarding work-related training and is one of its main heuristic cross- roads. Nevertheless, at the same time, we cannot overlook the criti- cism of a new formalism that could impact research programmes if it endorses a procedural culture based on certification, indicators, and standards. Beyond reductive semanticizing, B. Rey underlines the importance of second-level analysis, according to which, com- petences maintain an irreducible position, since they define the ca- pacity to choose, combine, and modify knowledge to respond to an unprecedented situation (Rey 2006). p ( y ) The epistemology of business practices has brought to light the fact that these have authentic, specific, theoretical properties. 2. Competences, Education, Work. Learning and working: a case in point Plac- ing the central position of the act of learning at the start of the pro- cess means not foregoing a technical and personal parameter with which to consider the credibility of the term competence in terms of its vast use in international regulations. Educational and economic models of human capital − or, as U. Margiotta theorizes in Italy, welfare delle capacitazioni (capacity-building welfare) (Margiotta 2011) − can recov- er, through a clear-sighted appraisal of the real condition of the skills market, a sphere of knowledge that cannot be reduced to traditional discipline-based systems (no less specific, such as business adminis- tration), compared to which they have sometimes been considered minor or simply applied versions. The discovery that every generation of entrepreneurs, teachers and students must make on the key role of practical knowledge, which is in many ways original both in terms of phylogenetic and socioge- INTERPRETATIONS. MARKET, WORK, TRAINING 303 netic reconstruction, is what provides the richness in combining the «curricular route with the construction of an individual’s identity». ii y Learning also happens through identification with qualified pro- fessionals and a certain degree of mimicry typical of non-didactic (i.e. practical/applied) learning. Skills, beyond the didactic mediations of teaching and the taxonomies of learning, belong to the world from which emerges: the profession. The proactive synergy between school, university and work, the alternation between skills-based and creative life experiences can cre- ate the future, subverting situations that appear hopeless, alongside realistic forecasts of decline. The education of individuals and com- munities in navigating towards a good life, with and for others in decent institutions is not dependent on the current situation, unchangeable as this may seem, and opposes the processes breaking down the hu- man experience and those of the contextual, associated paroxysmal diversifying of convictions and conventions. Building society in an intentionally participatory and creative way calls upon the educational sector or the entirety of essentially human experiences that define and form humanitas, laying down the undeni- able principles such as the pursuit of the common good, respect for life and moral responsibility in the practices of freedom, employabil- ity, and competences. Innovative Curricula and New Professions. Lectures towards the Job mar- ket and Education. Innovative Curricula and New Professions. Lectures towards the Job mar- ket and Education. References Alessandrini G. (ed.) 2007, Comunità di pratica e pedagogia del lavoro, Pensa MultiMedia, Lecce. Bertagna G. 2011, Lavoro e formazione dei giovani, La Scuola, Brescia. Birbes C. 2012, Progettare competente. Teorie, questioni educative, prospettive, Vita e Pensiero, Milano. Bocca G. 2000, Pedagogia della formazione, Guerini, Milano. Fabbri L. 2008, Cultura del lavoro e formazione universitaria, FrancoAngeli, Milano. Federighi P. 2010, Professioni educative e lavoro, «Pedagogia oggi», 1, 70-86. Federighi P. 2011, La ricerca evidence-based in educazione degli adulti, «Pedagogia oggi», 1-2, 112-120. Giuliodori C. and Malavasi P. (eds.) 2016, Ecologia integrale. Laudato sì. Ricerca, formazione, conversione, Vita e Pensiero, Milano.i Guasti L. 2000, Curricolo, competenze, significati, «Pedagogia e Vita», 4, 102-131. Guasti L. 2000, Curricolo, competenze, significati, «Pedagogia e Vita», 4, 102-131. Guasti L. 2003, Il sistema degli standard, «Pedagogia e Vita», 3, 139-149. Guasti L. 2000, Curricolo, competenze, significati, «Pedag Guasti L. 2003, Il sistema degli standard, «Pedagogia e Vita», 3, 139-149 Malavasi P. 2007, Pedagogia e formazione delle risorse umane, Vita e Pensiero, Milano. 304 PIERLUIGI MALAVASI Margiotta U. 2011, Per una nuova pedagogia dell’età adulta. Crisi del welfare e apprendimento adulto: un new deal per la ricerca in scienze della formazione, «Pedagogia oggi», 1-2, 67-79. g g gg Morin E. 1999, La Tête bien faite. Repenser la réforme, réformer la pensée, Seuil, Paris. Pope Francis 2015, Lettera enciclica Laudato sì sulla cura della casa comune. Pope Francis 2013, Esortazione apostolica Evangelii Gaudium. Rey B. 2006, Les compétences professionnelles et le curriculum: des réalités conciliables?, in Lenoir Y. and Bouillie-Oudot M.H. 2006, Savoirs professionels et curriculum de formation, Les Presses de l’Université de Laval, Saint-Nicolas (Québec), 83-108. Rosina A. 2015, NEET. Giovani che non studiano e non lavorano, Vita e Pensiero, Milano. Sen E. 1999, Development as freedom, Oxford University Press, New York.l Vischi A. 2011, Riflessione pedagogica e culture d’impresa. Tra responsabilità sociale e progettualità formativa, Vita e Pensiero, Milano. Vischi A. (ed.) 2014, Learning city, Human grids. Governance, processi formativi, conoscenza scientifica, Pensa MultiMedia, Lecce. * Sergio Bellantonio, PhD, Postdoctoral Research Fellow in General & Social Pedagogy, Department of Physical Education, University of Naples “Parthenope” Italy, Email: sergio.bellantonio@uniparthenope.it. Sergio Bellantonio (University of Naples Parthenope)* Sergio Bellantonio (University of Naples Parthenope)* Abstract: Education is a constantly ongoing process that allows individuals to interpret and transform reality. The variety of life contexts, formal, non-formal or informal, are explicitly or latently involved in this process and contribute de- cisively to building a personal knowledge and competence system at the basis of subjectivity. The aim of this paper is to highlight the role played by knowledge and competence arising from personal experience within profiles of professional competences, to implement possible reflective paths in higher education guid- ance processes. y g p g p y subjectivity. The aim of this paper is to highlight the role played by knowledge and competence arising from personal experience within profiles of professional competences, to implement possible reflective paths in higher education guid- ance processes. Keywords: personal knowledge and competences, higher education, subjectiv- ity, guidance. 1. Introduction Education is a process of acquiring a continually evolving form of one’s identity that allows individuals to transform and occupy the world they live in, through the construction and use of diverse forms of knowledge able to foster their adaptation to the environment (Cam- bi 2010). Such a process must embrace a multitude of living condi- tions and experiences, and constitute an important contribution to the construction of a system of personal knowledge and competences at the basis of subjectivity (de Mennato 2003). From this perspective, human knowledge must assume a particular, existential importance, in that it is characterized by its interpretation in the world, beginning from the variety and significance of individual experiences (Bruner 1992; Neisser 1981; Polanyi 1990). y ) Knowledge is ‘rooted’ in the environment, in a relationship of co-adaptation with the mind, which cognitively and emotionally organizes knowledge (Bateson 1976) both explicitly, through formal facts and in tacit, latent ways too (Bruner 1992). At this point it is well understood that knowledge is expressed by one’s own person- al way of living in the world, based on individual, autobiographical repertories that constitute a subjective key to access the real world. Consequently, each fragment of knowledge has no meaning in and 306 SERGIO BELLANTONIO of itself, but acquires it through the mental processes that connect new knowledge to that acquired previously (Neisser 1981), weaving a tight web between identity and alterity. In this way, human knowl- edge assumes a social value, in view of the circularity with which it constructs and shares itself with and between individuals; in other words, personal stories of education are realized in the light of other people’s lives and experiences. FOSTERING PERSONAL KNOWLEDGE AND COMPETENCES The paradigmatic crisis of postmodernity (Bauman 2002) has in- tensified the climate of widespread distrust towards formal education, which seems to have progressively lost the ability to foster in students the competences needed to plan for the future, from both an existential and a professional perspective, decreasing the employability of students at the end of their career. If a vision of work that is generally coher- ent and fairly stable with its own formal education, still very socially rooted, is less and less clear or at least defined as job placement, it fol- lows that formal education is gradually losing its appeal; this means that higher education is undergoing a lack of recognition of its value in use which, in the past, distinguished it from other forms of education, es- pecially since it was considered a possibility for individuals to improve or enhance their own living and working conditions (Morin 2000).i g g The widespread crisis in which many young adults find themselves is, therefore, a difficulty in overcoming the so called developmental tasks (Havighurst 1953), which largely concern the construction of a life project that precedes a progressive transition to those areas of training and working that characterize adult life (Boffo, Gioli 2016). All of this is related to a negative vision of change and an inability to imagine the future, creating forms of discomfort that prompts youngsters to assume a distrustful attitude of a certain disorientation (Cunti 2008; Cunti, Priore, Bellantonio 2015). i An extremely unnatural and artificial compartmentalization of knowledge, overly analytical and fragmented, coupled with a lack of effective employability of the higher education system, requires a systemic approach to the issue. From this point of view, if individual existences are increasingly discontinuous, in the sense that they tend to change rapidly, with individuals the first to adapt to the environ- ment (Bauman 2002, 2006), one wonders what kind of education is most suitable to overcome this impasse. 2. Processes of knowledge and development of subjectivity in higher education 2. Processes of knowledge and development of subjectivity in higher education With reference to the above, it is understood that students in- volved in higher education contexts enter the educational system ‘in their wholeness’, in that they bring with them personal knowl- edge and competences coloured by various personal epistemologies representing the way in which these relate to the system itself. Such settings, unlike other learning contexts, can create unique oppor- tunities for contact with a vast array of knowledge, which should, ideally, facilitate individual and social experiences of all-round and self-knowledge. However, too often, individuals are not completely aware of the deeper causes that motivate their lives and educational choices, and interpret study as being isolated from other experien- tial contexts. Explanations for such a division most likely hark back to the in- dividuals’ educational stories. A frequently found scholastic culture still concentrates excessively on the acquisition, retention, and rep- etition of content, while excluding the more intimate and personal components of learning, such as the emotional aspects. Consequently, these components are rarely recognized as the best way of supporting a meaningful learning in didactics, according to a constructivist per- spective of learning processes (Cunti 2014), a perspective now backed up by scientific research in didactics and pedagogy. p yi p g gy Knowledge and competences developed outside formal teach- ing settings are understood as needing to remain separate, a concept that seems completely unsustainable, especially since every form of knowledge shifts continuously between an ‘open system’ of individ- ual construction, and a ‘closed system’ of intersubjective and contex- tual construction (Varela, Thompson, Rosch 1992). This means, in other words, that every form of knowledge has at the same time a personal value, because it is filtered by subjective processes of decodi- fication, selection, and construction of reality, and a social value, since it assumes a significance in the context that produced it, which also makes it shareable and desirable to a greater or lesser extent (Bateson 1976; Maturana, Varela 1985). FOSTERING PERSONAL KNOWLEDGE AND COMPETENCES 307 2. The role of guidance processes in higher education Responding to the young generations’ needs for the future cannot merely dissolve into the simple offering of prospects and opportuni- ties, but requires these to be recognized as important chances by the individual. The necessity is to connect higher education educational paths with employability and life projects through guidance strategies that seek not only to undermine the widespread mistrust of the future, but, above all, to promote critical-reflective competences for students to imagine themselves as competent adults (Lo Presti 2015) and there- fore effectively support existential transition processes from higher education to working contexts (Boffo, Gioli 2016). 308 SERGIO BELLANTONIO The importance of guidance practices in higher education is now largely agreed upon by national and international governance poli- cies, at least in terms of intentionality expressed on a declarative level. As a result, the fragmentation and precariousness of individual lives have prompted an important reconsideration of operational guidance strategies, revised considering theoretical paradigms of a constructivist matrix (Guichard 2005, 2012; Savickas 2002, 2005, 2015). From this perspective, the construction of a professional career is closely related to individuals’ ability to adapt in a flexible manner to the flow of job events; in this sense, a current educational request seems to educate individuals to take reflexive attitudes that help them continuously define and re-define themselves in the workplace (Savickas 2015). In the dynamic construction of the Self, then, reflexivity and the exer- cise of metacognitive thought become a valid personal competence to be developed for the construction of a life project (Guichard, Pouy- aud 2015). Today, individuals are constantly involved in dealing with various aspects of their lives and, for this reason, it is no longer con- ceivable to think that professional development is a totally separate field from the personal sphere, also considering that increasingly fre- quently, individuals are engaged in dual careers that see them involved in the foreground on both personal and professional levels (Arthur, Hall, Lawrence 1989; Hall 1976). This theme is highly significant, due to the fact that nowadays, ‘existential bustles’ are increasingly frequent between the personal and professional spheres, which demands guidance strategies that urge the individual to deal with ever more frequent existential transitions. 2. The role of guidance processes in higher education Guiding the subject to a personal life project, therefore, means nurtur- ing a lifelong process of self-construction, emphasizing and articulat- ing the various roles individuals play in their existential complexity; in this sense, guidance becomes an educational strategy to build one’s own identity (Lo Presti 2015). In the end, accessing personal knowledge and competencies also means working on professional epistemologies; personality traits and implicit theories play a key role in defining personal knowledge and contribute, often in a tacit and latent form, to the construction of changing professional profiles that seem to be necessary in a historical period strongly marked by precariousness and liquidity (Savickas 2015). 3. Fostering Personal Knowledge and Competencies in Higher Education In the light of the above considerations, it is important to under- line the prominent role of personal knowledge and competencies de- rived from personal experience through higher education contexts. FOSTERING PERSONAL KNOWLEDGE AND COMPETENCES 309 From a review of the literature on scientific research engines and through the use of concepts and keywords referring to the themes of interest, it emerges that a certain awareness has developed: to pro- mote more sustainable employability a range of personal knowledge and competences is necessary which, if accompanied by knowing-how skills (Boyatzis 1982; Campbell et al. 1970; Jones, Lichtenstein 2000) can determine a more effective job placement. In this sense, personal knowledge and competences, individual skills and psychosocial re- sources make it possible to convert technical and specialist knowledge in a specific field of work into more efficient work practices (Allen, Remaekers, Van Deer Velden 2005; Boyatzis 1982, 1995, 2002, 2008; Spencer and Spencer 1993); beyond the specialist skills, therefore, those acquired in other life contexts can be defined as effective professional amplifiers (Allen, Remaekers, Van Der Velden 2005). Research undertaken in recent years (Boyatzis 2002, 2008; Spen- cer, Spencer 1993) has also highlighted the role of emotional intelli- gence (Goleman 1995; Goleman, Boyatzis, McKee 2002) in describing and predicting human behaviour in the professional field, too; from this point of view, this particular kind of intelligence is understood as a real competence capable of effectively managing conflicts in the workplace and actively tackling work-related stress (Ashkanasy, Daus 2002; Jordan et al. 2002; Humphrey 2002, 2006). It has also been considered that individuals with elevated levels of such intelligence are more able to handle their own and others’ emotions by develop- ing a quality of positive relationship that can expressly improve the quality of work (Wong, Law 2002). 3. Fostering Personal Knowledge and Competencies in Higher Education Some of the empirical evidence in certain scientific studies also asserts that personal knowledge and competences play a prominent role in improving the construct of professionalism both in terms of emotional (Goleman 1995) and rela- tional aspects (George 2000), significantly influencing the success and effectiveness of professional careers (Sturges, Simpson, Altman 2003).ii f p ( g p ) Despite the obvious benefits of much scientific evidence, higher education still seems reluctant to promote personal knowledge and competences; in this sense, university students are not yet accustomed to the development of metacognitive abilities through the exercise of critical-reflective thinking about themselves and their own education; in fact, higher education still fosters knowledge characterized by rep- etition in a passive and uncritical way (Busana, Banterle 2008). This is also due to the fact that many teachers consider the development of personal knowledge and competences the task of job placement offices or external experts from business contexts (Boyatzis, Stubbs, Taylor 2002); teaching programmes are still mostly structured in a traditional way, which seems in direct contrast with certain research data, ac- cording to which formal education that also considers the personal 310 SERGIO BELLANTONIO components of individuals is capable of educating individuals who can deal more effectively with their career path, making them more motivated and result-oriented, as well as able to work better in groups (Longenecker, Ariss 2002; Zimmerman-Oster, Burkhardt 1999). ( g ) The literature examined also emphasizes the importance of incor- porating the development of personal competences into the mission of higher education and the enhancement of students’ personal knowl- edge through implementation of curricular and extra-curricular pro- grammes (Astin, Astin 2000; Zimmerman-Oster, Burkhardt 1999). To this end, some authors (Freshwater, Stickey 2004) have suggested guidelines to foster knowledge and competences gained from personal experience in higher education programmes, in which they take on a prominent position in the exercise of critical-reflective thinking, the implementation of tutoring and mentoring activities, and the devel- opment of empathy and emotional intelligence. Only in recent years have higher education curricula welcomed this perspective (Dugan, Komives 2007); for this reason, some universities have introduced courses on ethics and skills assessment programmes. References Allen J., Ramaekers G. and Van der Velden R. 2005, Measuring Competencies of Higher Education Graduates, in D. J. Weerts and J. Vidal (eds.), Enhancing Alumni Research: European and American perspectives, Jossey-Bass, San Francisco. Arthur M.B., Hall D.T. and Lawrence B.S. 1989, Handbook of Career Theory, Cambridge University Press, New York. Ashkanasy N.M. and Daus C.S. 2002, Emotion in the Workplace: The New Challenge for Managers, «Academy of Management Executive», XVI (1), 76-86. Astin A.W. and Astin H.S. 2000, Leadership Reconsidered: Engaging Higher Education in Social Change, W.K. Kellogg Foundation, Battle Creek. Bateson G. 1976, Mente e Natura. Un’unità necessaria, Adelphi, Milano (orig. ed. 1949). Bauman Z. 2002, Modernità liquida, Laterza, Roma-Bari (orig. ed. 2000). Bauman Z. 2006, Vita Liquida, Laterza, Roma-Bari (orig. ed. 2005).f Boffo V. and Gioli G. 2016, Transitions to Work and Higher Education: Listening to What the Graduates Have to Say, in Pejatovic A., Egetenmeyer R., Slowey M. (eds.), Contribution of Research to Improvement of Adult Education Quality, Institute for Pedagogy and Andragogy, Belgrade, 9-28.f Boyatzis R.E. 1982, The Competent Manager: A Model for Effective Performance, John Wiley & Sons, Chichester. Boyatzis R.E. 1995, Cornerstones of Change: Building a Path for Self-Direct Learning, in Boyatzis R.E., Cowen S.C. and Kolb D.A. (eds.), Innovation in Professional Education: Steps on a Journey from Teaching to Learning, Jossey- Bass, San Francisco, 92-108. Boyatzis R.E. 2002, Unleashing the Power of Self-Directed Learning, in Sims R.R. (ed.), Changing the Way We Manage Change, Quorum Books, Westport. Boyatzis R.E. 2002, Unleashing the Power of Self-Directed Learning, in Sims R.R. 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Final remarks Although there have been some appreciable changes, the big- gest challenge remains to promote and enhance the role of personal knowledge and competences in university curricula, as a fundamen- tal part of the educational process (Boyatzis, Stubb, Taylor 2002). For this reason, it is necessary to guide subjects in their life cycle through innovative teaching methodologies that can strengthen the link be- tween the academic world and the workplace, and make learning a self-directed process (Boyatzis, Kolb 1995).l p y In the light of the literature, as well as a reflective and transforma- tive theoretical framework of learning (Mezirow 1991; Schön 1991), it could prove interesting to develop the phenomenon through quali- tative research methods (focus groups, semi-structured and in-depth interviews) with the aim of further revealing the significance of per- sonal experience in the construction of professional paths. A formal educational setting designed with such a framework implies directing individuals to enter into a close bond with the processes of knowledge from which an idea of themselves and the future can arise. There is then the need to implement educational practices that can commu- nicate personal knowledge with expanded experience, culture, for- mal education, and the concrete conditions that surround the living and educational environments (Lo Presti 2015). FOSTERING PERSONAL KNOWLEDGE AND COMPETENCES 311 References 2006, Abitare il disincanto. Una pedagogia per il postmoderno, Utet, Torino. Campbell J.P., Dunnette D.M., Lawler E.E. III and Weick K. E. Jr. 1970, Managerial Behaviour, Performance and Effectiveness, McGraw-Hill, New York. 312 SERGIO BELLANTONIO Cunti A. 2008, Aiutami a scegliere. Percorsi di orientamento per progettare e progettarsi, FrancoAngeli, Milano. g Cunti A. 2014, Formarsi alla cura riflessiva. Tra esistenza e professione, FrancoAngeli, Milano. 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FOSTERING PERSONAL KNOWLEDGE AND COMPETENCES 313 * Natascia Bobbo, PhD, Researcher in General, Social and Health Pedagogy, Department of FISPPA, University of Padua, Italy, Email: natascia.bobbo@unipd.it; Silvia Lazzaro, PhD, Health Professional Educator, Department of FISPPA, University of Padua, Italy, Email: lazzaro.silvia@gmail.com. FOSTERING PERSONAL KNOWLEDGE AND COMPETENCES Lo Presti F. 2015, Forming the Reflective Self for the Life Project. The Guidance Function of Education, «Educational Reflective Practices», FrancoAngeli, Milano. Longenecker C.O. and Ariss S.S. 2002, Creating Competitive Advantage Through Effective Management Education, «Journal of Management Development», 21, 640-654. Maturana H. and Varela F. 1985, Autopoiesi e cognizione. La realizzazione del vivente, Marsilio, Venezia (orig. ed. 1980).i Mezirow J. 2003, Apprendimento e trasformazione. Il significato dell’esperienza e il valore della riflessione nell’apprendimento degli adulti, Raffaello Cortina, Milano (orig. ed. 1991). Morin E. 2000, La testa ben fatta. Riforma dell’insegnamento e riforma del pensiero, Raffaello Cortina, Milano (orig. ed. 1999). Neisser U. 1981, Conoscenza e realtà. Un esame critico del cognitivismo, il Mulino, Bologna (orig. ed. 1976).ii Polanyi M. 1990, La conoscenza personale. Verso una filosofia post-critica, Rusconi, Milano (orig. ed. 1958). Savickas M.L. 2002, Career Construction. A Developmental Theory of Vocational Behavior, in D. Brown et al., Career Choice and Development, Jossey-Bass, San Francisco, 149-205. Savickas M.L. 2005, The Theory and Practice of Career Construction, in Brown S.D., Lent R.W. (eds.), Career Development and Counseling: Putting Theory and Research to Work, John Wiley & Sons, Hoboken, 42-70. Savickas M.L. 2015, Designing Projects for Career Construction, in Young R.A., Domene J., Valach L. (eds.), Counseling and Action, Springer, New York, 16-17.l Schön D.A. 1991, The Reflective Turn. Case Studies in and on Educational Practice, Teachers College Press, New York. g Spencer L.M. and Spencer S.M. 1993, Competence at Work: Models for Superior Performance, John Wiley & Sons, New York. Struges J., Simpson R. and Altman Y. 2003, Capitalizing on Learning: An Exploration of the MBA as a Vehicle for Developing Career Competencies, «International Journal of Training and Development», VII (1), 53-66. Varela F.J., Thompson E. and Rosch E. 1992, La via di mezzo della conoscenza. Le scienze cognitive alla prova dell’esperienza, Feltrinelli, Milano (orig. ed. 1991)f Wong C.S. and Law K.S. 2002, The Effects of Leader and Follower Emotional Intelligence on Performance and Attitude: An Exploratory Study, «The Leadership Quarterly», XIII (3), 243-274. Zimmerman-Oster K. and Burkhardt J.C. 1999, Leadership in the Making: Impact and Insight from Leadership Development Programs in US Colleges and Universities, W.K. Kellogg Foundation, Battle Creek. Natascia Bobbo (University of Padua), Silvia Lazzaro (University of Padua)* Natascia Bobbo (University of Padua), Silvia Lazzaro (University of Padua)* Abstract: Increasing life expectancy and the growing number of chronic diseases have changed the kind of patients who need to be assisted. This paper presents a qualitative study conducted with a group of nursing students near graduation, aimed at describing and discussing vocational preferences and desirable health- care settings for future employment. Keywords: nursing students, vocational calling, professional identity, qualita- tive research, patients’ needs. 1. Introduction The increase in life expectancy as well as the growing number of patients suffering from chronic diseases require the development of a specific nurse’s profile with clinical skills and vocational motivations suitable for this care target (Mazhindu et al. 2016). If, after gradua- tion, students acquire adequate theoretical and clinical knowledge as well as critical-thinking skills, they will be able to express an elevated level of professional performance (Rothwell, Sensenig 2002; Spencer, Spencer 1995; Marañón, Pera 2015; Ames 1992). Recent studies have underlined that a nurse’s vocational motiva- tion seems to be linked to a self-realization need expressed as a car- ing attitude towards fragile subjects (Mackintosh 2007; Miers et al. 2007; Williams et al. 1997).f Nevertheless, students have different caring targets in mind. In- deed, a significant percentage of them tend to focus their employment expectancies on specific clinical areas that are more preferred than others: critical rather than general medicine, paediatric rather than the elderly (Rognstad et al. 2004; Storen, Hanssen 2011). This could be due to students’ development of a professional over- view influenced by some trivialized and simplified patterns that might NATASCIA BOBBO, SILVIA LAZZARO 316 even involve the image of patients (perhaps influenced by unrealis- tic television series). These distortions are incoherent with the com- plexities of caring for ill children, and critical or terminal patients (Gurses et al. 2009; Aujoulat et al. 2006; Brewster 1982). On the other hand, these patterns seem inadequate to respond to the real needs of a healthcare that is more and more oriented to assisting an increas- ing number of chronic patients and, implicitly, to caring for them at home (Italian Law. 38/2010). ( ) This kind of motivational structure may subject nursing students to frustration in their vocational project; moreover, future nurses may be unable to meet the real needs of their patients. A frustrated subject may be potentially exposed to great psychophysical stress and, con- sequently, suffer from some secondary traumatic stress disorder that can elicit a shirking of professional responsibility, emotional distance, and the activation of defence mechanisms. These elements can all provoke professional burn-out (Schwam 1998; Kearney et al. 2009; Conrad, Kellar-Guenther 2006). ) The aim of the present study is to qualitatively describe vocational preferences and desirable future employment areas in a group of nurs- ing students near graduation. 1. Introduction The description will focus particularly on speculation concerning the coherence between students’ prefer- ences and the complexity of patients’ healthcare needs. The interviews were recorded in their entirety and transcribed. Data were analysed using Atlas.ti, 7.1.8. 2. Methods A qualitative study was conducted with a group of nursing students near graduation at Padua University. The study was conducted using a bottom-up approach, and within a phenomenological perspective (Reinharz 1983; Berger, Luckmann 1966). The researchers’ inten- tion was to go beyond a quantitative description of students’ choices to reach a hermeneutic comprehension regarding their desires and subjective perception. j p p Semi-structured interviews were realized individually with each student. The interview protocol was based on Happell’s paper (2000). During the interviews, the students’ personal data was col- lected, while the protocol included some specific questions useful to explore students’ motivations regarding their desire to become a nurse and their preferred clinical areas for future employment. Clinical areas proposed to the students were: Paediatrics, Critical/ A&E, Surgery, General Medicine, Geriatrics, Mental Health, and Home Care. The interviews were recorded in their entirety and transcribed. Data were analysed using Atlas.ti, 7.1.8. NURSING STUDENTS’ FUTURE EMPLOYMENT PREFERENCES 317 Results The study was conducted in September 2013. 37 students near graduation were involved (8 males and 29 females; the mean age (±DS) was: 25.6±5.9 years. Considering students’ reasons linked to the choice of this university course, test analyses led to the identifi cation of 10 diff erent motiva- tions. Some of them seemed similar for most of the subjects involved. Others, instead, were only mentioned by a few students (Fig. 1). Figure 1 – Motivational choices on Nursing Degree Courses. One of the most frequent motivations described concerns the need to fi nd a satisfying strategy to express solidarity. A substantial number of students talked about the desire to receive gratitude from patients and therefore to feel that they were useful. Frequently, students reported knowing other nurses, especially among their family members and friends. Another frequent motiva- tion that they referred to was having had experience of social volun- teering. This reason was often linked to a clear declaration of feeling pleasure when involved in a work activity with both technical and relational dimensions. In many cases, students referred to an innate vocation, without any possible rational explanation; a sort of desire (that seems to have emerged during childhood or adolescence) to work in a healthcare setting, especially in a hospital. g p y p Few students reported having chosen this Degree Course to have a guaranteed job, occasionally as a fresh start after a layoff in later life. Instead, not a few students admitted that their choice was induced by a failure to pass the test for a medical or physiotherapist’s degree. Nevertheless, in all these cases, they admitted their satisfaction con- cerning their Nursing Degree. 318 NATASCIA BOBBO, SILVIA LAZZARO Other motivations, reported by only one or two students, were linked to a passion for some bio-scientifi c subjects of the nursing de- gree curriculum, or the human body as an object of interest. g y j Regarding preferred clinical areas for future employment, the stu- dents were clearly infl uenced by the positive experiences that they had had during their internships. Students frequently referred to a particular emotional context or a skilful and charismatic clinical tu- tor making the diff erence. Other reasons were also identifi ed, distin- guished by preferred healthcare areas as follows. Five out of 37 students chose the Paediatrics Area: they expressed two preferences, for Paediatrics and Maternity units (Fig. 2). NURSING STUDENTS’ FUTURE EMPLOYMENT PREFERENCES Figure 2 – Preferences for Maternity and Paediatric areas: students’ explanations. Figure 2 – Preferences for Maternity and Paediatric areas: students’ explanations. Three students chose the maternity unit because of their desire to assist new mothers in learning to care for their baby, and because of a natural aptitude to socializing with other women. Lastly, because in this area there were no chronic patients. p Those who chose the Paediatric unit explained their choice sim- ply with the statement, «I love children, even better if new-born». Moreover, they discussed the complexity of caring for an ill child, since professionals are more emotionally engaged, this might con- tribute to letting them feel useful to young patients as well as their parents. There was another interesting result: the students who chose the Paediatric and Maternity units underlined that these areas are less exposed to the risk of having to cope with death, pain, or a therapy failure compared to other areas (e.g.: Geriatrics or General Medicine). p g Three units were chosen as being in the ‘critical area’: Coronary Intensive Unit, Intensive Care Unit, and A&E. This sector was cho- sen by many students: 11 out of 37 (Fig. 3). 319 NURSING STUDENTS’ FUTURE EMPLOYMENT PREFERENCES Figure 3 – Preferences for Critical areas: students’ explanations. Figure 3 – Preferences for Critical areas: students’ explanations. Regarding the reasons linked to this choice, the students reported that working in this sector represented a chance to cope with a vari- ety of situations, pathologies, and diff erent patients and, in addition, might be an opportunity to acquire (or reinforce) new knowledge and skills. They affi rmed that in this sector nurses could play an active role in managing the critical situation and working with the medical team. From the students’ point of view, these elements all seemed to contribute to a more detailed nurse profi le. pi Above all, in the intensive units, students mentioned the inclusive relationship they could establish with a sedated patient who, in this way, seemed to be utterly dependent on their care. The situation characterized by A&E, the chance to save patients’ lives and the adrenaline-fi lled condition deriving from this idea, clear- ly represented some salient motivations for these students. Five students chose the surgical area; the Surgical Ward and the Operating Theatre were the two units they pointed to (Fig. 4).i The fi rst motivation reported concerned the chance to obtain more information, knowledge, and skills linked to the acquisition of more responsibilities. Figure 5 – Preferences for Medical and Geriatric area: students’ explanations Figure 2 – Preferences for Maternity and Paediatric areas: students’ explanations. The students preferred the operating theatre where they could experience direct work on patients, linked to the chance to satisfy their interest in the human body (as a tangible object to be handled). In this healthcare setting the relationship with the doctors seems to have been perceived at the same level. 320 NATASCIA BOBBO, SILVIA LAZZARO Figure 4 – Preferences for Surgery area: students’ explanations. Certain stereotypes, derived from television medical dramas, emerged from the students’ accounts. Additionally, they stated that these areas let them avoid emotional involvement with the patients. Seven students chose General Medicine (5) and Geriatric Area (2); results are described together because they present some analo- gies. Many clinical units have been pointed out by students (Fig. 5). Figure 5 – Preferences for Medical and Geriatric area: students’ explanations. Students stated that they preferred these contexts because they of- fer many chances to establish a continuative, holistic, and deep re- lationship with the patient. This area was considered a good setting Students stated that they preferred these contexts because they of- fer many chances to establish a continuative, holistic, and deep re- lationship with the patient. This area was considered a good setting NURSING STUDENTS’ FUTURE EMPLOYMENT PREFERENCES 321 for initial employment, because it let new graduate nurses learn the basics of their job in a safe condition. Even in this area, nevertheless, one student reported fear of patients dying. Only one student chose the Home Care Area. His motivation was linked to the opportunity to establish with the patients and their fam- ily a caring alliance and a human relationship; this would let him be- come a reference point for them. In this way, he expected to receive signifi cant payback for his eff orts. Four students chose the oncological area. They indicated the On- cology, Chemotherapy, and Ambulatory Units, and the Hospice (Fig. 6). Figure 6 – Preferences for Oncology area: students’ explanations. Figure 6 – Preferences for Oncology area: students’ explanations. Figure 6 – Preferences for Oncology area: students explanations. g gy p These students told us about the opportunity to play − as a nurse − a complete role in terms of caring for a patient who needs human attention. Almost all said that taking care of a dying patient during his or her last days seemed to be a virtuous professional aim. One of them mentioned some personal grief that infl uenced his choice; this personal experience may have signifi cantly contributed to his future professional preferences. It is possible to refl ect on the extent of his elaboration of grief. These students told us about the opportunity to play − as a nurse − a complete role in terms of caring for a patient who needs human attention. Almost all said that taking care of a dying patient during his or her last days seemed to be a virtuous professional aim. One of them mentioned some personal grief that infl uenced his choice; this personal experience may have signifi cantly contributed to his future professional preferences. It is possible to refl ect on the extent of his elaboration of grief. g Students seemed to perceive the oncology patient as a complex per- son and, for this reason, someone who could stimulate their profes- sional learning. This complexity, indeed, and the great need for care of these patients let students imagine − or expect? − to receive sig- nifi cant feedback (in terms of self-esteem and self-effi cacy), not com- 322 NATASCIA BOBBO, SILVIA LAZZARO parable to other areas. Students involved in this study seemed not to be suffi ciently aware of the risk of emotional involvement. Very few refl ections on this theme were recorded. Four students chose the Mental Health Area. Everyone indicated the Dependency Service (Fig. 7). Figure 7 – Preferences for Mental Health area: students’ explanations. These students mentioned the operative and autonomous role that nurses can play in this context. Besides this, patients suff ering from drug addiction seemed to be particularly stimulating for these stu- dents, since they were described as complex and unpredictable but, at the same time, captivating and capable of great payback. Additionally, the continuity of the assistance allows nurses to take care of these pa- tients more through a human relationship than a technical approach. NURSING STUDENTS’ FUTURE EMPLOYMENT PREFERENCES NURSING STUDENTS’ FUTURE EMPLOYMENT PREFERENCES 323 their future job in the various healthcare areas. Firstly, a sort of in- stinctive love for children drove some students towards the mater- nity or paediatric sector and let us hypothesize a desire for maternage – ‘mothering’, rather than a real understanding of the complexity of paediatric patients (Happell 2000). i In other cases, choosing a specific sector seemed to be a way to avoid suffering, death, or patients’ pain: all these elements could com- promise the need for security and control that many of these stu- dents expressed directly or indirectly (Rosenbloom et al. 1999). On the other hand, those who chose the oncological context seemed to express a heroic will, but perhaps they were seeking a social payback that they perceived as necessary for their self-esteem. The security they mentioned in coping with the risk of human involvement in a patient’s history of pain, is indeed incoherent with their youth and the competences they may have acquired a few months before taking their degree. In fact, solidarity (like the main professional motiva- tions) appears linked to a major risk of exposure to burn-out (Join- son 1992; Crumpei, Dafinoiu 2012). pi Lastly, the students’ desire (frequently expressed) to work in a crit- ical setting was very meaningful: they wished for a job that would give them the chance to be, continually, in an adrenaline-filled con- dition, to help a critical patient utterly dependent on their skills. This dependence concerned both the patient’s therapeutic needs and (even more significantly for the students) his or her life. This attitude may have derived from a prolonged adolescent desire for power and con- trol (Barone 1990 Rosenbloom et al. 1999), alternatively, it could be an attention-seeking behaviour. Moreover, it could be coming from the current social and cultural context characterized by a hyper-stim- ulating environment which might induce them to need continuous stimuli at every moment of their life (even from a professional point of view) (Griffiths 2000). fi Considering the preferences and desires expressed by the students involved in this study, there is little space for the concrete needs of the patient. It is not simply arguing what level of awareness these students near graduation possess regarding patients’ human needs, or about their skills and competence (or limits) in caring for them (Cowin 2001). 3. Discussion and fi nal remarks The choices and preferences that moved these students near gradu- ation were coherent with other previous studies (Williams et al. 1997). They were keen (except for few cases) to stand caring for a patient, even if suff ering from a chronic condition. Despite this, the analysis of the students’ answers concerning the explanation of their future professional preferences, revealed some inappropriate − or unrealis- tic − ideas. Students seemed not to have a clear awareness regarding NURSING STUDENTS’ FUTURE EMPLOYMENT PREFERENCES i The results of the present study let us partially confirm our initial hypothesis; generally, it was indeed possible to identify in these stu- dents a limited consideration of the concrete opportunities for em- ployment and a poor awareness of the real complexity of both their job and patient’s needs. These distortions, within a few years, might expose them to some dangerous consequences concerning their own identity as well as their emotional wellbeing (Yoder 2010; Stamm 1999; Fingley 1995). This risk appears increased by a clear absence 324 NATASCIA BOBBO, SILVIA LAZZARO in their portfolio of competences in some meta-cognitive skills that could protect them from any disturbances or vicarious traumas (Yassen 1995). These are some serious educational gaps that must be bridged by some specific paths to help them comprehend and integrate in- to their vocational wishes a more realistic idea of patients, as well as a clear idea of the nursing profession (a difficult job, full of pro- fessional, emotional, and personal obstacles). The literature appears rich in suggestions regarding training strategies fit for this student target: there are some didactic strategies by which it appears possible to lead students to integrate their – prevalently − rational thinking with a more contemplative one (Heiddeger 1966; Hixon 1978). In this way, they would able to care for a patient while thinking about his or her wellbeing (which only the patient can describe − Mottura 1986), avoiding wasting their good intentions and vocational moti- vation on role profiles which, in the present healthcare systems, are less and less requested. NURSING STUDENTS’ FUTURE EMPLOYMENT PREFERENCES 325 Gurses A.P., Carayon P. and Wall M. 2009, Impact of performance obstacles on intensive care nurses’ workload, perceived quality and safety of care, and quality of working life, «Health Services Research», XLIV (2p1), 422-443. g f ( p ) Happell B. 1999, When I grow up I want to be a …? Where undergraduate student nurses want to work after graduation, «Journal of Advanced Nursing», XXIX (2), 499-505. Happell B. 2000, Love Is All You Need? Student nurses’ interest in working with children, «Journal for Specialists in Pediatric Nursing», V (4), 167-173. Heidegger M. 1966, Discourse on Thinking, Harper & Row, New York. gg g p Hixon L. 1978, Coming home. The experience of enlightenment in Sacred Tradition, Larsen, New York. Joinson C. 1992, Coping with compassion fatigue, «Nursing», XXII (4), 116-118. gg g p Hixon L. 1978, Coming home. The experience of enlightenment in Sacred Tradition, Larsen, New York. h f Larsen, New York. Joinson C. 1992, Coping with compassion fatigue, «Nursing», XXII (4), 116-118. , nson C. 1992, Coping with compassion fatigue, «Nursing», XXII (4), 116-118. Kearney M.K., Weininger R.B., Vachon M.L.S., Harrison R.L. and Mount B.M. 2009, Self-care of physicians caring for patients at the end of life, «Jama», CCCI (11), 1155-1164. Legge 15 marzo 2010, n. 38: Disposizioni per garantire l’accesso alle cure palliative e alla terapia del dolore, Gazzetta Ufficiale, 65. 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Reinharz S. 1983, Phenomenology as a dynamic process, «Phenomenology + Pedagogy», I (1), 77-79. Rognstad M.K., Aasland O. and Granum V. 2004, How do nursing students regard their future career? References Ames C. 1992, Achievement goals and the classroom motivational climate, «Student perceptions in the classroom», 327-348. Aujoulat I., Simonelli F. and Deccache A. 2006, Health promotion needs of children and adolescents in hospitals: a review, «Patient education and counseling», LXI (1), 23-32.i Barone P. 2009, Pedagogia dell’adolescenza, Guerini Scientifica, Milano. Berger Peter L. and Luckmann T. 1966, The social construction of reality: A treatise in the sociology of knowledge, First Anchor, Garden City. Brewster A.B. 1982, Chronically III Hospitalized Children’s Concepts of Their Illness, «Pediatrics», LXIX (3), 355-362. Conrad D. and Kellar-Guenther Y. 2006, Compassion fatigue, burnout, and compassion satisfaction among Colorado child protection workers, «Child abuse and neglect», XXX (10), 1071-1080. g Cowin L. 2001, Measuring nurses’ self-concept, «Western Journal of Nursing Research», XXIII (3), 313-325.i Crumpei I. and Dafinoiu I. 2012, The relation of clinical empathy to secondary traumatic stress, «Procedia-Social and Behavioral Sciences», 33, 438-442. Deci E.L. and Ryan R.M. 2008, Self-determination theory: A macro theory of human motivation, development, and health, «Canadian Psychology», XLIX (3), 182. Fingley C.R. (ed.) 1995, Compassion Fatigue. Coping with Secondary Traumatic Stress Disorder in those who treat the traumatized, Routledge, New York-London. Griffiths M. 2000, Does Internet and computer “addiction” exist? Some case study evidence, «CyberPsychology and Behavior», III (2), 211-218. NURSING STUDENTS’ FUTURE EMPLOYMENT PREFERENCES GUIDANCE MODELS AND PRACTICES ADOPTED INTERNATIONALLY TO PROMOTE THE EXPLORATION OF SKILLS RELATING TO THE EMPLOYABILITY OF STUDENTS WITH DISABILITIES. A FIRST META-ANALYSIS* Valentina Paola Cesarano (University of Naples Federico II), Marianna Capo (University of Naples Federico II), Maria Papathanasiou (University of Valentina Paola Cesarano (University of Naples Federico II), Marianna apo (University of Naples Federico II), Maria Papathanasiou (University of Naples Federico II), Maura Striano (University of Naples Federico II) Capo (University of Naples Federico II), Maria Papathanasiou (University of Naples Federico II), Maura Striano (University of Naples Federico II) Abstract: Introduction: Employability is defined as an interweaving of a person’s hu- man, social and psychological capital, mediated by situational variables, which allows individuals to enter the job market with a professional personal project (Grimaldi, Porcelli, Rossi 2014). Nowadays, young people enter the job market through long, precarious, and poorly contextualized paths, while the socialization processes be- come recursive, discontinuous, and fragmented (Lodigiani 2010). A key role can be played by guidance services, which can start at university, to meet the demands of the (many) young people who are discouraged and disillusioned to the point where they cannot even imagine a job while still at university. In the employability stakes, what is even more complex is the encounter between young people with disabilities and the world of work, due to the persistence of stereotypes and stigmas. Research questions: What are the intervention models and guidance practices adopted by uni- versity guidance services internationally to promote the exploration of skills relat- ing to the employability of students with disabilities? Objectives: To analyse the main intervention models and guidance practices adopted internationally to explore the skills associated with employability in students with disabilities. Methodology: It was decided to carry out a theoretical analysis of 20 scientific articles concerning the models and practices adopted to explore the competences relating to employability in certain university orientation services for students with disabilities in Italy, France, the UK, and the United States. NVivo software was used (Richards 1999) to system- atically explore the scientific literature. Preliminary Findings: A first scientific paper showed that, like in Italy and France, the «Competence Balance Sheet» (Ardouin 2010) is the guiding practice in the USA, while in the UK, it is the Career Guidance Approach (Reid, Scott 2010). In the literature, orientation models and practices are also closely linked to the various patterns of employability. NURSING STUDENTS’ FUTURE EMPLOYMENT PREFERENCES Career preferences in the post-modern society, «Nurse Education Today», XXIV (7), 493-500. Rosenbloom J., Pratt A. and Pearlman L.A. 1999, Helpers’ responses to trauma work: understanding and intervening in an organization, in Stamm B.H. (ed.) Secondary Traumatic Stress Disorder. Self-care and issues for clinicians, researchers and educators, Sidran Press, Baltimore-Maryland, 65-79. y Rothwell W.J. and Sensenig K.J. (ed.) 1999, The sourcebook for Self-Directed Learning, HRD Press, Amherst Massachusetts. Schwam K. 1998, The phenomenon of Compassion Fatigue in perioperative nursing, «AORN Journal», LXVIII (4), 642-648. 326 NATASCIA BOBBO, SILVIA LAZZARO Spencer L.M. and Spencer P.S.M. 2008, Competence at work models for superior performance, John Wiley & Sons. p f J y Stamm B. H. (ed.) 1999, Secondary Traumatic Stress Disorder. Self-care and issues for clinicians, researchers and educators, Sidran Press, Baltimore, Maryland. Støren I. and Hanssen I. 2011, Why do nurses choose to work in the perioperative field?, «AORN Journal», XCIV (6), 578-589. Williams B., Wertenberger D.H. and Gushuliak T. 1997, Why students choose nursing, «Journal of Nursing Education», XXXVI (7), 346-348. g J f g WHO and Consultation F.E., 2003, Diet, nutrition, and the prevention of chronic diseases, «World Health Organ Tech Rep Ser», 916, I-VIII. Yassen J. 1995, Preventing Secondary Traumatic Stress Disorder, in Fingley C.R. (ed.) Compassion Fatigue. Coping with Secondary Traumatic Stress Disorder in those who treat traumatized, Routledge, New York-London, 178-208. g Yoder E.A. 2010, Compassion fatigue in nurses, «Applied Nursing Research», XXIII (4), 191-197. ** Valentina Paola Cesarano, PhD student, Department of Humanistic Studies, University of Naples Federico II, Italy, Email: valentinapaola.cesarano@unina.it; Marianna Capo, PhD, SInAPSi Centre, University of Naples Federico II, Italy, Email: marianna.capo@ unina.it; Maria Papathanasiou, Volunteer researcher, Department of Humanistic Studies, University of Naples Federico II, Italy Email: mpapsal@gmail.com; Maura Striano, PhD, Professor of General and Social Pedagogy, Department of Humanistic Studies, University of Naples Federico II, Italy Email: maura.striano@unina.it. * Maria Papathanasoiu is the author of the Introduction; Marianna Capo is the author of the first and second paragraphs; Valentina Paola Cesarano is the author of the third, fourth and fifth paragraphs; Maura Striano is the author of the Final remarks. * Maria Papathanasoiu is the author of the Introduction; Marianna Capo is the author of the first and second paragraphs; Valentina Paola Cesarano is the author of the third, fourth and fifth paragraphs; Maura Striano is the author of the Final remarks. ** Valentina Paola Cesarano, PhD student, Department of Humanistic Studies, University of Naples Federico II, Italy, Email: valentinapaola.cesarano@unina.it; Marianna Capo, PhD, SInAPSi Centre, University of Naples Federico II, Italy, Email: marianna.capo@ unina.it; Maria Papathanasiou, Volunteer researcher, Department of Humanistic Studies, University of Naples Federico II, Italy Email: mpapsal@gmail.com; Maura Striano, PhD, Professor of General and Social Pedagogy, Department of Humanistic Studies, University of Naples Federico II, Italy Email: maura.striano@unina.it. GUIDANCE MODELS AND PRACTICES ADOPTED INTERNATIONALLY TO PROMOTE THE EXPLORATION OF SKILLS RELATING TO THE EMPLOYABILITY OF STUDENTS WITH DISABILITIES. A FIRST META-ANALYSIS* Final remarks: The im- plementation of guidance counseling paths aimed at exploring the skills associated with employability among all students and graduates is crucial to the completion of a viable strategic action in the University’s social function, as a part of new or- ganizational models that take the plurality of learning opportunities into account. Keywords: employability, orientation, disability. V.P. CESARANO, M. CAPO, M. PAPATHANASIOU, M. STRIANO 328 1. Introduction The theme of employability involves orientation, training, and work, as well as their various junctures. In the face of today’s com- plexity characterized by recursive, irregular, and fragmented recruit- ing and socialization processes (Lodigiani 2010), guidance services can play a leading role, which can begin at university, to meet the demands of the (many) young people who declare themselves dis- couraged and disillusioned to the point that they are already unable to imagine a job during their university studies (Grimaldi, Porcelli, Rossi 2014). In the context of employability, there is an even more difficult encounter, namely, between young people with disabilities and the world of work, a match that is still very difficult to imple- ment, and is sometimes even denied, since very often the persistence of stereotypes and stigmas, coupled with the lack of a genuine po- litical and systemic will in applying and enforcing the norms, often makes the world of work inaccessible, or disinclined to change in its organizational culture. It is important to bear in mind the functional and cognitive characteristics, individual goals, and potential, in the academic and professional spheres of undergraduate students with dis- abilities when they are transitioning to the adult and professional life. Indeed, Carter et al. (2012) already pointed out that disabled students usually complete their university course without the skills, abilities, and experience, and sometimes even without the support, that would allow them to have concrete job opportunities. It is therefore essen- tial to develop training and guidance paths at university geared to the employability of students with disabilities. d d h h d p y y To understand which orientation patterns and practices encour- age the exploration and advancement of employability and related competences of undergraduate students with disabilities in Italy and abroad, we chose to make a first meta-analysis of the Italian and in- ternational scientific literature on this subject, using NVivo software (Richards 1999), with the aim of systematically exploring a corpus of selected scientific material. An analysis was made of scientific litera- ture from Italy, France, the UK, and the USA, since there is a prolif- eration of studies and reflections in these contexts in relation to the subject in question. 1. Introduction Beginning from the Grounded Theory perspec- tive (Glaser, Strauss 1967), with the support of the NVivo Software, the collected articles were coded, and the following analysis catego- ries were formulated: • The construct of employability in Italy, France, the UK, and the USA l b l l h d h • Employability orientation practices in Italy, France, the UK, and the USA • Theoretical reference models based on orientation practices • The relationship between skills and employability PROMOTE THE EXPLORATION OF SKILLS RELATING 329 In the process of the contribution, the subdivision of these cat- egories will be intensified to achieve the enucleating of a core cat- egory from a qualitative meta-analysis, defined as ‘Orientation as an educational task’. 1.1 The employability construct: a brief theoretical excursus and the Italian INAPP proposal Back in the ’50s and ’60s, the term ‘employability’ had already ap- peared in the scientific literature. From a review by Cavenago and Martini (2012), it is apparent that the concepts of dichotomic em- ployability and socio-medical employability were in use in those years to distinguish between ‘manageable’ and ‘unmanageable’ peo- ple based on personal predisposition and aptitude, and physical and mental health. The increase in unemployment in the 1970s called for the introduction of the term workforce employment ability with which the alignment (or misalignment) between the knowledge and skills possessed by the individual was categorized with respect to market demand. In the 1980s, the term ‘employability’ was adopted by com- panies to identify the flexibility of the workforce, which at that time became an indispensable competence for addressing the demands of a brand-new job market. In political and institutional spheres, the European Employment Strategy and the various EC documents that have been around since the ’90s interpret employability as people’s ability to enter the job market. It was from the ’90s onwards that a proliferation of studies can be noticed on the issue of employability, seen as a multidimensional construct in which certain factors come together, both external ones related to the contexts of life and the job market, and the internal ones of individuals (Lefresne 1999; Forrier, Sels, 2003; Fugate, Kinicki, Ashforth 2004; Fugate, Kinicki 2008). 1. Introduction In constructing employability, attention has sometimes been given to more contextual/objective aspects (the job market situation, the characteristics of organizations/companies, the number of changes in work, etc.) and sometimes to more individual aspects (personal characteristics, individual adaptability, flexibility, etc.) until patterns developed that interwove both factors (Fugate, Kinicki, Ashforth 2004; McQuaid, Lindsay 2005; Cavenago, Magrin, Martini, Moni- celli 2012). Of particular interest is the definition of employability formulated by Grimaldi, Porcelli & Rossi (2014) as «the intertwin- ing of a person’s human, social, and psychological capital − mediated by situational variables − which allows individuals to venture into/ re-enter the job market with a professional personal project obeying the context» (p. 58). V.P. CESARANO, M. CAPO, M. PAPATHANASIOU, M. STRIANO 330 This definition reflects the employability model developed by IN- APP (National Institute for Public Policies Analysis), which considers individuals’ biographical and curriculum data, their context, and their environmental and life conditions. 2. The Italian context: some examples of experimentation and good practice guidance 2. The Italian context: some examples of experimentation and good practice guidance In Italy, the education of people with disabilities is based on a socio- bio-psycho-pedagogical approach that considers the cultural context, the physical and psychological wellbeing (but not only), and indi- vidual abilities. In the fields of accompanying study and orientation in higher education, the Italian system considers the relationship be- tween the student and the consultant who will be co-responsible not only for the development of the student’s university project but also their professional project. Particularly interesting in the Italian con- text is the approach and methodology of the Foro Italico University of Rome, which focuses on the orientation of undergraduate students with disabilities towards the job market. The Foro Italico University of Rome has an internship and job placement office where the pro- fessional experiences of the students during their Degree Course are collected, and advice is provided on the future possibilities of use. In general, the university offers two professional outlooks: specific train- ing (internships) and recognition of the work carried out. It is through the university’s relationship with employers (compa- nies, associations, etc.), regarding the availability and skills of the stu- dents concerned, that the internships become possible depending on the level of study. Meanwhile, the employer may also ask the univer- sity for a Trainee Profile that responds to their interests. Specifically, for disabled students, the internship and job placement office works closely with the Specialized Education support service. The main dif- ference from other students is that the personal data on students with disabilities and related information are available from the Specialized Educator’s Office. Overall, the students’ skills and specific needs are examined in accordance with their social and educational development. Decisions on issues concerning people with disabilities are taken in agreement with the Support Centre, the Managing Director, and the individual student. The theoretical model for accompanying students with disabilities on which the orientation practices of the Foro Italico University of Rome are based, was inspired by a pilot work developed by the Danish University of Aarhus. In the framework of the Europe- an Leonardo Project, Univers’emploi (2012), the four partner countries involved, France, Ireland, Italy, and Denmark, adopted this model. p Decisions on issues concerning people with disabilities are taken in agreement with the Support Centre, the Managing Director, and the individual student. 2. The Italian context: some examples of experimentation and good practice guidance The theoretical model for accompanying students with disabilities on which the orientation practices of the Foro Italico University of Rome are based, was inspired by a pilot work developed by the Danish University of Aarhus. In the framework of the Europe- an Leonardo Project, Univers’emploi (2012), the four partner countries involved, France, Ireland, Italy, and Denmark, adopted this model. 331 PROMOTE THE EXPLORATION OF SKILLS RELATING The basic idea of the model was the inclusion of people with different learning modes, both in the educational system and in society, with an emphasis on supporting people in their transition from university education to work. This model was based on four key concepts: edu- cation, inclusion, learning and life project. The theoretical reference substrate, based on the capacity model developed by Amartya Sen (1999) and Martha Nussbaum (2010) contains a very useful picture. This model, like the ICF (International Classification of Functioning, Disability, and Health Classification), focuses on people’s abilities − or rather, on their potential. The concept of ‘potential’ is grounded in the theory of experimental learning by Peter Jarvis (1995) and the philosophy of Martin Heidegger (1927) based on human development. Guidance practices inspired by this model relate to Career Counseling and Career Coaching, according to a humanistic-existential approach where subjects are questioned, and meaning is given to their transition to the job market (Univers’emploi 2012a). In general, disabled students have low expectations regarding their future in the job market. They therefore need to examine and clarify their motivations and potential in relation to the job market. Career Focus is an extension of the stu- dents’ field of opportunity in terms of access to professional realiza- tion. To achieve this, it is important for students to become aware of both their resources and potential, and the difficulties and challenges associated with future job placement. Consequently, the reflection must be on their disability status in relation to inclusion and exclu- sion processes, for example, to a future working environment. In this light, career counseling can therefore create a space for reflection on their professional project to activate a process of clarification and to be able to plan career goals (Univers’emploi 2012b). p g ( p ) In Italy, certain permanent orientation tools have been prepared for self-assessment by students with disabilities. 2. The Italian context: some examples of experimentation and good practice guidance It is the dimension of the meeting with the Other that subsequently justifies all the other aspects considered relevant to the work, such as the use of one’s own skills, prestige, group work, pro- fessional creativity, economic security, involvement, and commit- ment. This is a first element of divergence with respect to previous surveys of samples of normative associates attending the same uni- versity (Magnano, Paolillo 2008), the centrality accorded even be- fore their own self-realization (in terms of matching work to their own interests and capabilities), the ability to be of help to others. In addition, considering the characteristics of the group involved in the research, it was noted that the decision to face a university course in search of a job is a motivating factor in itself, implying, however, an important level of investment and interest in the future, and the will to be as independent as possible.f within the area of a person’s proximal development to realize their own personal, social, and professional identity. The results of research conducted in collaboration with the Chair of Psychology of Educa- tion and Professional Orientation and the Centre for Orientation and Training of the University of Catania (Magnano, Paolillo 2008) on a representative sample of the population of disabled students at the Centre for Active and Participating Integration of the University of Catania (CinAP), included an exploration of the following compo- nents: professional values; working styles and motivation; thoughts on the future − self-image; perceived self-efficacy; the relationship between all the above dimensions. The tools used for research pur- poses were taken from the Portfolio for Assessment, Treatment, and Integration of Disabilities − ASTRID-OR (Sores, Note 2007). To these was added an instrument for self-image capture from the Guid- ance Questionnaire of the University of Catania. The synthesis of the results of this research reminds us of a fundamental concept: the importance for those in a ‘peculiar’ state to find a dimension in their personal and professional realization, including the Other, which lets them establish a relationship where they feel useful to another person, thus being able to play, but also to develop and expand their skills and abilities. 2. The Italian context: some examples of experimentation and good practice guidance For example, the Univer- sity of Macerata (Formiconi, Nicolini, Regolo 2015) has developed an online platform that allows a disabled person to self-assess their knowledge, skills, strengths, and working conditions, to increase the specificity of the match between the specific professional profile of the candidate with disabilities and the various training and/or work fields, enhancing the characteristics and uniqueness of their compe- tences. The various professional profiles are then operationalized by articulating them into: knowledge, skills, needs in working conditions linked to disability, plus a profile of their own intellect. The value of the platform is the active role of self-assessor played by the candi- date in disability status, thereby becoming the key resource and not a person hired because of legislative obligation. In this sense, orien- tation assumes the value of a continuous and articulate process whose main purpose is the support of self-awareness and potential, acting V.P. CESARANO, M. CAPO, M. PAPATHANASIOU, M. STRIANO 332 within the area of a person’s proximal development to realize their own personal, social, and professional identity. The results of research conducted in collaboration with the Chair of Psychology of Educa- tion and Professional Orientation and the Centre for Orientation and Training of the University of Catania (Magnano, Paolillo 2008) on a representative sample of the population of disabled students at the Centre for Active and Participating Integration of the University of Catania (CinAP), included an exploration of the following compo- nents: professional values; working styles and motivation; thoughts on the future − self-image; perceived self-efficacy; the relationship between all the above dimensions. The tools used for research pur- poses were taken from the Portfolio for Assessment, Treatment, and Integration of Disabilities − ASTRID-OR (Sores, Note 2007). To these was added an instrument for self-image capture from the Guid- ance Questionnaire of the University of Catania. The synthesis of the results of this research reminds us of a fundamental concept: the importance for those in a ‘peculiar’ state to find a dimension in their personal and professional realization, including the Other, which lets them establish a relationship where they feel useful to another person, thus being able to play, but also to develop and expand their skills and abilities. 2. The Italian context: some examples of experimentation and good practice guidance It is the dimension of the meeting with the Other that subsequently justifies all the other aspects considered relevant to the work, such as the use of one’s own skills, prestige, group work, pro- fessional creativity, economic security, involvement, and commit- ment. This is a first element of divergence with respect to previous surveys of samples of normative associates attending the same uni- versity (Magnano, Paolillo 2008), the centrality accorded even be- fore their own self-realization (in terms of matching work to their own interests and capabilities), the ability to be of help to others. In addition, considering the characteristics of the group involved in the research, it was noted that the decision to face a university course in search of a job is a motivating factor in itself, implying, however, an important level of investment and interest in the future, and the will to be as independent as possible.f What emerges, therefore, is a second difference with respect to normative students: the extent to which independence is seen as a professional value for these subjects is at the basis of the scale of pri- orities (Magnano, Paolillo 2008). Qualitative data analysis showed the choice of the degree programme mattering more than the pro- fession to which it aspired, while the professional experiences already gained (for some) also determined the respondents’ decision. Thus, their targets remained strongly ‘desired’, as stages of a process aimed at obtaining their autonomy and independence to the greatest degree possible. For disabled students, the moment of impasse and greatest PROMOTE THE EXPLORATION OF SKILLS RELATING PROMOTE THE EXPLORATION OF SKILLS RELATING 333 pressure would seem to come later, when their training path has al- ready been ‘decided’ or is about to end, and the choices to be made concern their own, already concrete, professional future. From an op- erational standpoint, therefore, it is necessary to think of the initia- tives proposed by the departments as providing guidance to organize outbound paths for those students with ‘peculiar’ health conditions, who are in the process of graduating. This often means that they are working on scaling down the stereotypes or irrational ideas that are sometimes present and can impact their professional future, and above all their decision-making processes, ideas that still arouse some trep- idation about the consequences of their actions, at times impeding unrealistic mental openness in dealing with the uncertainty and de- cisions associated with a professional choice. Therefore, a key result for disabled university students is the adaptation of means and the de- signing of future goals for their life (Nota, Rossier 2015). In addition, the use of inclusive modalities and achievements in everyday life can make for an inclusive university experience, a model of participation to be carried with them through life and with which the university prepares its students (Getzel, Wehman 2005). 3. Guidance on work and employability in France: Mission Handicap from a constructivist perspective 3. Guidance on work and employability in France: Mission Handicap from a constructivist perspective In France, the accompaniment of disabled students is based on the medical-social approach. Taking care of students through the services is subject to prior recognition of the disability, which includes a med- ical assessment. In French universities, Mission Handicap is a point of reference for disabled students, a key pillar, and the place where pedagogical compensation and coordinated construction of the pro- fessional project (orientation, reorientation, internships, university/ work Alternance). The French universities’ approach places the student as the centre of the ‘accompaniment work’. Orientation is character- ized here by a constructivist approach that gives greater weight to the student’s relationship and life experience, and emphasizes problem- solving and an emphasis on student assessment with the delivery of a final response. This approach encourages individuals to tell their sto- ry, and to identify their own life and career themes. The consultant thus operates more like a biographer who interprets life rather than an accountant who coldly deals with passivity and activity. The goal of the orientation process is thus to promote empowerment, to en- able students to deal with transitions and to govern their own career path. This occurs to the extent that the accompanying practice calls for the development of social skills, adaptation, relationalism, mobi- V.P. CESARANO, M. CAPO, M. PAPATHANASIOU, M. STRIANO 334 lization, and participation in projects, facilitation of material, human and intellectual studies, all elements that are part of a global project. In other words, success in studies is a condition for professional success. (Univers’emploi 2012). The demand to accompany vocational training comes increasingly from the students themselves, when they present a problem and question their path. Whether they come to this point of reflection is either because they have been forced to quickly find a compulsory internship, they have to enter the world of work, or because they have been regularly questioned by Mission Handicap on issues relating to job placement and the idea that the road gradu- ally continues, to start verbalizing their professional expectations and begin the project. 3. Guidance on work and employability in France: Mission Handicap from a constructivist perspective Starting from the Mission Handicap model, the contribution of the Univers’emploi project methodology enabled inclu- sion of the activity in an integrated transition system focusing on the following dimensions: to foster students’ accompaniment in terms of their ‘becoming’ professional, in any case, an openness to the world of work; to measure the evolution of the course begun by the student; to formalize the skills gained after an internship or work experience (usually done during the summer holidays); a bonding between the accompanying, orientation and job placement services; implement- ing regulations between universities and companies involving issues relating to the employment of students with disabilities. Employ- ability in the French context is defined as the probability of retriev- ing or retaining a job. It depends on personal factors (aptitudes and abilities) and/or situational factors (job market, workplace accessi- bility and work methods, adaptability of workstations and organiza- tional schemes, professional constraints, etc.) (Busnel 2010: 17). To promote employability, on 22 July 2013, by law, France introduced education in entrepreneurship into Higher Education and Research. Several analysts see in the ‘Entrepreneurship University’ the future of the university alongside its new ‘third mission’, connected with the world of business (Vorley, Nelles 2008). 4. The Anglo-Saxon context: Career guidance services In British universities where there is an increase in students and graduates with disability status, career orientation services are provid- ed which provide information, counseling and guidance in an acces- sible way to all students. These practices are based on the theoretical model of educational and vocational guidance (Reid 2010) that ‘ed- ucates’ students to build and explore the narrative of their own vo- cations. Furthermore, in the designing of career guidance services, people with disabilities are also involved as key eyewitnesses in the PROMOTE THE EXPLORATION OF SKILLS RELATING 335 co-designing of orientation activities. Equally important is the rela- tionship with the career counsellor, who accompanies the student in pursuit of job opportunities and internships both during the univer- sity course and up to two years after graduation, as in the case of Bru- nel University. The university encourages feedback from all students on Career Guidance services. Some universities, such as Manchester Metropolitan University, have developed a peer mentoring system. This provides opportunities for disabled students to develop the so- cial skills required by the workforce and promotes career guidance services. The realization of Career Guidance services in Anglo-Sax- on contexts involves exploring the possible barriers to entry into the workplace in advance, including the problems experienced by stu- dents and graduates with disabilities. Loughborough University has set up a group of professional consultants in a career guidance service together with an employment centre intended to deal effectively with the problems of students with disabilities regarding job placement and to offer effective individualized solutions, as well as the pursuit of opportunities to do internships. For example, the University of Bradford provides all students with placement opportunities during the summer break. Students meet with a professional counsellor and together find suitable apprenticeship opportunities and before doing so, there is a preparatory phase to reflect on the planned tasks and activities. The consultants visit the host company and at the end of the internship experience a ‘reflexive folder’ is compiled in which the students report on the activities and the skills acquired that contrib- uted to the construction of their professional project. 4. The Anglo-Saxon context: Career guidance services In regard to the employability construct, the Anglo-Saxon literature tends to oppose a simplistic model of employability, that is, the model of the ‘magic wand’ − according to which a student is employable by virtue of his or her university education − a model of employability development that takes into account one side of the individual’s potential in terms of self-cultivation and, on the other, the actual employment the in- dividual has had, mediated by economic and social factors (Harvard 2010). There would then be factors linking these two dimensions of work experience, namely; the development of self-promotion and career management skills; the will to learn and reflect on acquired learning. Hence the need to create pedagogically-oriented spaces and places for reflection and orientation to employability. 4. The Anglo-Saxon context: Career guidance services In regard to the employability construct, the Anglo-Saxon literature tends to oppose a simplistic model of employability, that is, the model of the ‘magic wand’ − according to which a student is employable by virtue of his or her university education − a model of employability development that takes into account one side of the individual’s potential in terms of self-cultivation and, on the other, the actual employment the in- dividual has had, mediated by economic and social factors (Harvard 2010). There would then be factors linking these two dimensions of work experience, namely; the development of self-promotion and career management skills; the will to learn and reflect on acquired learning. Hence the need to create pedagogically-oriented spaces and places for reflection and orientation to employability. co-designing of orientation activities. Equally important is the rela- tionship with the career counsellor, who accompanies the student in pursuit of job opportunities and internships both during the univer- sity course and up to two years after graduation, as in the case of Bru- nel University. The university encourages feedback from all students on Career Guidance services. Some universities, such as Manchester Metropolitan University, have developed a peer mentoring system. This provides opportunities for disabled students to develop the so- cial skills required by the workforce and promotes career guidance services. The realization of Career Guidance services in Anglo-Sax- on contexts involves exploring the possible barriers to entry into the workplace in advance, including the problems experienced by stu- dents and graduates with disabilities. Loughborough University has set up a group of professional consultants in a career guidance service together with an employment centre intended to deal effectively with the problems of students with disabilities regarding job placement and to offer effective individualized solutions, as well as the pursuit of opportunities to do internships. For example, the University of Bradford provides all students with placement opportunities during the summer break. Students meet with a professional counsellor and together find suitable apprenticeship opportunities and before doing so, there is a preparatory phase to reflect on the planned tasks and activities. The consultants visit the host company and at the end of the internship experience a ‘reflexive folder’ is compiled in which the students report on the activities and the skills acquired that contrib- uted to the construction of their professional project. 5. The American context: skills-based guidance services American universities offer their students with disabilities Career Counseling founded on a work-based learning model to help them V.P. CESARANO, M. CAPO, M. PAPATHANASIOU, M. STRIANO 336 make decisions about their careers. Through interaction between work and study experiences, students can improve their academic knowl- edge, personal development, and professional preparation (Brand, Valent, Danielson 2013). The purpose of this service is to promote ‘career readability’, namely career readiness in the achievement and demonstration of skills that prepare graduates for a successful transition to the world of work (National Association of Colleges and Employ- ers 2015). American literature highlights the importance of explora- tion and orientation skills, particularly for disabled students, in cases where they are considered incapable of working autonomously and this has a negative impact on their rates of employment (US Bureau of Labor Statistics 2014). Employers cite employability skills as being the most important (Hart Research Associates 2015; British Council 2011). Lack of employability skills can contribute to a ‘talent short- age’ (Manpower Group 2012). Demonstration of employability skills is correlated with top hiring rates, job success, and profits (Lippman, Ryberg, Carney, Moore 2015). The Washington Department of Edu- cation has elaborated a model of employability skills that could play a crucial role in university transition programmes for disabled students, for success in the job market. The framework for occupational skills consists of three principal areas of competence:ff • Effective relationships that can be translated into effective relationship skills, i.e., those skills that help individuals interact effectively with clients, colleagues, and employers. In this area they associate inter- personal skills and personal qualities. • Workplace skills that can be translated into skills in the workplace, i.e., those skills that enable successful performance of work assignments. This category includes resource management skills, information usage, communication skills, systematic thinking, and technological skills. • Applied Knowledge, i.e., integration of the knowledge acquired in the academic context and technical skills put into practice in the workplace. This category covers academic knowledge applied to the working context and critical thinking. 6. Final remarks: The relationship between skills and employability for orientation as educational work 6. Final remarks: The relationship between skills and employability for orientation as educational work Despite the criticalities associated with the employment of uni- versity students in a disability state, our meta-analysis shows that the scientific literature highlights the fundamental role played by vo- cational guidance services to support the transition to the world of work through various instruments from Career Guidance to Career Counseling. However, in the international context, some of these PROMOTE THE EXPLORATION OF SKILLS RELATING 337 guidance instruments involve theoretical efforts to build specific em- ployability models and related skills. It is possible to see how skills as the orchestration and mobility of knowledge and competences are transferred to various contexts and their elaboration contributes to the definition of employability and makes it possible. Nevertheless, it is impossible to establish a universal set of skills for employability, not only because these skills vary according to the job market seg- ments and the stratification of roles, but also because the pluralism of the value scales for the quality of work is related to competences in different socio-cultural contexts. Meanwhile, in their diversity, these models emphasize both individual factors and abilities as well as external factors related to the socio-economic reality of the world of work. In addition to this, there is a need to carry out accompany- ing practices for the transition to the world of work of all students, characterized not only by a ‘technocratic’ orientation involving the compilation of psycho-attitudinal tests to be cross-referenced with professional profiles; the ability to apply curriculum vitae and mo- tivational writing strategies, targeted research techniques, and self- marketing practices. In line with this, the use of the NVivo software has enabled the core category ‘Orientation as an educational task’ to enucleate orientation as a process that people put into practice to guide their relationships with training and work through develop- ment, in the lifelong and life-wide dimensions of Competency 3B recognizable as the ability to define and make plans for life and per- sonal projects. We could consider this competence as ‘reflective’ since it supports individuals in interpreting their life giving it meaning and significance (OECD 2005). Consequently, as highlighted by Grimaldi, Rossi, and Porcelli (2015), self-orientation skills (thinking and intentionally choosing one’s own future), as well as those of design and self-design (life de- sign) allow individuals to become employed, that is, to enter the job market with a professional personal project in tune with the context. 6. Final remarks: The relationship between skills and employability for orientation as educational work Moreover, it is vital to value both the relationship with the working world from the viewpoint of Business − University Cooperation, and the promotion of support for human and social development in the sense understood in the Capability approach. This approach aims to restore people’s dignity through the centrality of the human being as a set of individual aptitudes made up of opportunities, abilities, and their interaction with access to resources. Martha Nussbaum (2010) has drawn up a list of basic capabilities that are the same for all hu- mans, trying to overcome the distinction between ‘normal’ people and people with disabilities, giving everyone the same rights. If, then, someone – whether disabled or not − cannot perform one of these key functions at the appropriate threshold level, society must create V.P. CESARANO, M. CAPO, M. PAPATHANASIOU, M. 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For academic reasons, it should be noted that Marianna Capo is the author of the third and fourth paragraphs, Valentina Cesarano of the second paragraph, Maria Papathanasiou of paragraph 2.1 and Maura Striano is the author of the conclusions. ** Valentina Paola Cesarano, PhD student, Department of Humanistic Studies, University Federico II of Naples, Italy, Email: valentinapaola.cesarano@unina.it; Marianna Capo, PhD, SInAPSi, University Federico II of Naples, Italy. Email: marianna.capo@ unina.it; Maria Papathanasiou, Volunteer researcher, Department of Humanistic Studies, University Federico II of Naples, Italy. Email: mpapsal@gmail.com; Maura Striano, PhD, Ordinary Professor of General and Social Pedagogy, Department of Humanistic Studies, University Federico II of Naples, Italy. Email: maura.striano@unina.it. 1 [Author’s note] The work described in the article was carried out by Dr. Marianna Capo, PhD in Psychological and Pedagogical Science, and Carolina Galdo, Psychologist. Among the most recent publications of Dr. Capo, the following should be mentioned here: Capo 2016c and 2016d; Capo, Navarra 2016; Capo 2017. y p y @ 1 [Author’s note] The work described in the article was carried out by Dr. Marianna Capo, PhD in Psychological and Pedagogical Science, and Carolina Galdo, Psychologist. Among the most recent publications of Dr. Capo, the following should be mentioned here: Capo 2016c and 2016d; Capo, Navarra 2016; Capo 2017. TELLING TRANSVERSAL COMPETENCES… TO BE PROFESSIONALLY PROMOTED* Marianna Capo (University of Naples Federico II), Valentina Paola Cesarano (University of Naples Federico II), Maria Papathanasiou (University of Naples Federico II), Maura Striano (University of Naples Federico II) Marianna Capo (University of Naples Federico II), Valentina Paola Cesarano (University of Naples Federico II), Maria Papathanasiou (University of Naples Federico II), Maura Striano (University of Naples Federico II) Marianna Capo (University of Naples Federico II), Valentina Paola Cesarano (University of Naples Federico II), Maria Papathanasiou (University of Naples Federico II), Maura Striano (University of Naples Federico II)** Abstract: This article introduces experimental reflection on the experiences at an ‘Employability Skills’ laboratory1 of a group of young volunteers from the National Civilian Service under the ‘Support and Inclusion’ project of the Employment Promotion Section (SPO in Italian) of the University of Naples Federico II SInAPSi Centre. Young volunteers were included as unstructured support figures in activities that sought mainly to assist and serve students with disabilities. More specifically, these activities included: accompaniment and support during lessons; digitization of teaching material; providing support for the assorted services offered by the University Centre; general training implemented by AMESCI staff; specific training implemented by SInAPSi operatives. The experiences, which were accomplished in cooperation with the Europe 2020 programme, included the creation of an integrated system to recognize and validate formal, non-formal, and informal skills, as a tool to promote youth employment (Striano, Capobianco 2016). Keywords: employability, narration, skills, storytelling, social services. * The contribution is the result of a collective work. For academic reasons, it should be noted that Marianna Capo is the author of the third and fourth paragraphs, Valentina Cesarano of the second paragraph, Maria Papathanasiou of paragraph 2.1 and Maura Striano is the author of the conclusions. ** Valentina Paola Cesarano, PhD student, Department of Humanistic Studies University Federico II of Naples, Italy, Email: valentinapaola.cesarano@unina.it; Marianna Capo, PhD, SInAPSi, University Federico II of Naples, Italy. Email: marianna.capo@ unina.it; Maria Papathanasiou, Volunteer researcher, Department of Humanistic Studies University Federico II of Naples, Italy. Email: mpapsal@gmail.com; Maura Striano, PhD Ordinary Professor of General and Social Pedagogy, Department of Humanistic Studies University Federico II of Naples, Italy. Email: maura.striano@unina.it. 1. The synergy between the SInAPSi Centre and National Civilian Service 1. The synergy between the SInAPSi Centre and National Civilian Service Italian National Civilian Service (SCN in Italian) is an opportu- nity for young people between the ages of 18 and 28, regardless of gender, to spend twelve months of their lives supporting community service commitments, social cohesion values, and realizing everyone’s wellbeing, collectively and individually. M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. STRIANO 342 As ratified by the National Civilian Service Act (Law 64/2001, Article 1), the goal is to: • foster implementation of the constitutional principles of social ser- vice (Article 1, para. b); • foster implementation of the constitutional principles of social ser- vice (Article 1, para. b); • promote solidarity and cooperation at national and international levels, above all with regard to the protection of social rights, ser- vices for the people, and the bringing of peace to communities (Article 1, para. c); ( p ) • participate in the safeguarding and protection of the nation’s heri- tage […] (Article 1, para. d); and finally, ( p ) • participate in the safeguarding and protection of the nation’s heri-i • contribute to the civic, social, cultural, and professional education of young people through activities that may also be carried out at authorities and administrations operating abroad (Art.1, para. e). As stated above, civilian service is to be considered an active citizen- ship experience that guarantees the young volunteers both human and professional growth. Moreover, for the entire duration of the project, it allows them to devote time to themselves and others, thus enrich- ing their education from various perspectives: personal, professional, cultural, social, and civic. In this way, young people reap the opportu- nity to become citizens who are participants and protagonists of their own future and that of others (Muroni 2017). All made possible by the heterogeneity and multiplicity in the sectors of intervention the SCN addresses, i.e.: education; cultural promotion; the protection and safe- guarding of historical-artistic, cultural, environmental, and forestry heritage; civil protection and assistance. The civilian service volunteer experience as job creation in an organized context is conceived as an occasion for knowledge and training, especially and above all of the professional variety (Capo et al. 2015) whose outcome and failures are rarely formalized and validated through instruments and tools spe- cifically tailored to the user’s profile. 1. The synergy between the SInAPSi Centre and National Civilian Service For this reason, it was suggested that the training of volunteers should not only include a specific sec- tion to assess inbound and outbound cross-skills, in order to provide volunteers with an opportunity to discover knowledge and acquired skills as well as measuring the occupational levels possessed. The in- troduction of a functional narrative instrument through the dedicated use of multimedia technologies was also suggested, with the aiming of promoting and evaluating the volunteers’ employability levels. p g g p y y The long-term objective is the development of a Balance of Com- petence tool that allows operationalization of the construction of em- ployability and exploration of the related competences according to the model prepared by INAPP (previously ISFOL 2016), which, starting from this experimental encounter, can be systematized, and extended to other types of user (university students, graduates, research fellowships, TELLING TRANSVERSAL COMPETENCES 343 research doctors). From the perspective of further education, orientation is a necessary interface to ensure continuity and significance for train- ing processes, facilitating the recognition of a personal and professional development project consistent with the expectations, needs, resources, and potential of individuals, in relation to their life contexts (Striano 2004; Savickas et al. 2010). The Occupational Promotion Service (SPO) plays a strategic role in enhancing and promoting integrated actions of guidance, motivation, and support to civilian service volunteers in de- veloping their training and work scheme through analysis of intersecting skills possessed by the following aims and objectives, i.e., it proposes to: • help individuals become aware of their own transverse and pro- fessional skills; • help reveal often latent knowledge, aptitudes, skills, interests, and aspirations that define them as particular individuals;i • maintain the definition of a professional project consistent with the skills developed in the various training paths (formal, non-formal and informal) and with personal expectations and also aims to: • promote ability in self-assessment and activation;i • help define and valorize individuals’ strengths and room for possible improvement;i • know themselves and their potential (with specific reference to the potential of internal employability); • develop interests and motivation. This is therefore a way to intensify volunteers’ training and pro- fessional pathways by means of a narrative description of the skills and knowledge gained during a work experience placement. 1. The synergy between the SInAPSi Centre and National Civilian Service From this standpoint, the training proposal is an intervention that allows volunteers to acquire explicit awareness of their personal and profes- sional identity, an indispensable outlook to successfully address their own educational and professional pathways (De Mennato 2006; Cunti 2008; Lo Presti 2009). 2. The participants in the training experience and the methodological proposal The reference sample consisted of 40 volunteers from the National Civilian Service (27 females and 13 males) of whom 5 had high school qualifications, 5 were university graduates (seeking employment) and 30 were undergraduates from the following university courses: Law (4); Engineering (5); Psychological Science (9); Social Service (4); Ge- g g y (4); Engineering (5); Psychological Science (9); Social Service (4); Ge- M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. STRIANO 344 ology (1); Languages (3); Biology (3); Biotechnology (1). The proposed promotion of employability for volunteers at the University SInAPSi Centre was based on the use of diversified tools, and used a method- ology founded on the narrative approach of Life Design Counseling (or Narrative Career Counseling) (Savickas et al. 2010; Savickas 2005, 2012; Savickas, Porfeli 2012). As Savickas points out, this is a frank at- tempt to bring meaning and shape to our future. In addition, the abil- ity to narrate leads clients to better understand the driving motives of their own existence, their vocational personality, and their personal adaptability resources (Savickas 2005). Alongside this approach, the Balance of Competence was adopted according to the French model (Le Boterf 2000; Lemoine 2002; Lévy Leboyer, 1993; Aubret 2009), which requires self-assessment to activate the re-appropriation and enhance- ment of individuals’ competences. This investigation was structured in 4 phases, for a total of 6 encounters, involving the following steps: • reception (plenary session); • exploration (group and individual mode); p (g p ) • in-depth analysis (group and individual mode); • conclusion (group mode). The operational proposal for civilian service volunteers focuses on studies and research that support the use of narrative/autobiographi- cal instruments that ‘tell the story of one’s life’. This can encourage greater awareness, revealing to the subject unimagined possibilities for change, and thus activating a power of existential and design re- configuration. Indeed, the narrative is presented as a ‘sense organizer’, since it restores a sense of direction to heterogeneous and compos- ite subjective experiences (Delory Momberger 2015; Formenti 1998; Pineau 2000). 2. The participants in the training experience and the methodological proposal Furthermore, the operational proposal refers to the outcomes of the European KVALUES project, revised within the Biographical Self Labs project: Objective Portfolio Worker, funded by the Puglia Region (Bando Giovani Idee 2012) (De Carlo 2014), considering the plurality of approaches and instruments for validation of learning, collected, and distributed by CEDEFOP in the European Inventory on Validation of Non-Formal and Informal Learning (Brussels 2005- 2011) and ISFOL (2014). 2.1 The promotion path to employability and the autobiographical Self Indeed, in recent years, a series of narrative orientation practices have spread as the most suitable for exploring oneself, but also for knowing and relating to others and to contexts; in fact, «there are narratives that can stimulate narration on oneself, fundamental narrative, and necessary for a guidance path […]. Then the confrontation appears, where the listening to the narratives of others, the identification of the narratives of others on us, constructs the collection of narratives» (Batini 2005: 49). Parker too argues that behind any autobiography there is always a person who gives importance between one’s own self and the designability of one’s own self. This reconstruction is intended as a journey through which subjects dis- cover and re-elaborate experiences, grasping not only objectives and orientation, but also the ability to re-invent themselves, recognizing a set of elements to lay the foundations of their own development strategy on. The specific autobiographical dimension of the evalua- tion allows the individuals who use it to generate, through a narrative exposition, self-defensive cognitive processes that clarify the signifi- cance of the personal cognitive-emotional paths of experience and create a ‘broader’ sense of self, embracing the past, the present and the future. In this sense, the focus of the entire evaluation is to out- line precisely the Autobiographical Self, so dear to Damasio (2000), who, in The Feeling of What Happens, describes the Extended Self, also defined as the Autobiographical Self, present within the extend- ed consciousness as an elaboration that can provide individuals with a developed sense of self that considers the past and the anticipated future. For Damasio «here and now is still present, but it is accom- panied by the past […] and, equally important, it is accompanied by the foreseen future» (Damasio 1999: 237). The evaluation of compe- tences is, in fact, a retrospective of the past, which individuals form, and which makes them ‘competent’ in return, to re-read their past in the present while creating a large opening towards their future: all this autobiographical reconstruction work of experiences is part of a pedagogical framework in which training is understood as a complex, dynamic, active, continuous, and multidimensional process, attentive to relationships, inter- and intro-subjectivity. 2.1 The promotion path to employability and the autobiographical Self The training process to promote employability (including the Bal- ance of Competence) is a qualitative/quantitative tool that can prompt autobiographical reconstruction, that is not merely a description of the past anchored to an ephemeral dimension, but a dynamic connection 345 TELLING TRANSVERSAL COMPETENCES between one’s own self and the designability of one’s own self. This reconstruction is intended as a journey through which subjects dis- cover and re-elaborate experiences, grasping not only objectives and orientation, but also the ability to re-invent themselves, recognizing a set of elements to lay the foundations of their own development strategy on. The specific autobiographical dimension of the evalua- tion allows the individuals who use it to generate, through a narrative exposition, self-defensive cognitive processes that clarify the signifi- cance of the personal cognitive-emotional paths of experience and create a ‘broader’ sense of self, embracing the past, the present and the future. In this sense, the focus of the entire evaluation is to out- line precisely the Autobiographical Self, so dear to Damasio (2000), who, in The Feeling of What Happens, describes the Extended Self, also defined as the Autobiographical Self, present within the extend- ed consciousness as an elaboration that can provide individuals with a developed sense of self that considers the past and the anticipated future. For Damasio «here and now is still present, but it is accom- panied by the past […] and, equally important, it is accompanied by the foreseen future» (Damasio 1999: 237). The evaluation of compe- tences is, in fact, a retrospective of the past, which individuals form, and which makes them ‘competent’ in return, to re-read their past in the present while creating a large opening towards their future: all this autobiographical reconstruction work of experiences is part of a pedagogical framework in which training is understood as a complex, dynamic, active, continuous, and multidimensional process, attentive to relationships, inter- and intro-subjectivity. The immense impor- tance attributed today to the subjective dimension of the balance of competences rather than to the objective one, concerns the use of all those narrative practices that represent privileged opportunities to en- able individuals to become better acquainted with themselves, their desires, their own projects, their own successes and failures, but also their own expectations and concerns, and, above all, those skills pos- sessed or implemented. 2.1 The promotion path to employability and the autobiographical Self The immense impor- tance attributed today to the subjective dimension of the balance of competences rather than to the objective one, concerns the use of all those narrative practices that represent privileged opportunities to en- able individuals to become better acquainted with themselves, their desires, their own projects, their own successes and failures, but also their own expectations and concerns, and, above all, those skills pos- sessed or implemented. Indeed, in recent years, a series of narrative orientation practices have spread as the most suitable for exploring oneself, but also for knowing and relating to others and to contexts; in fact, «there are narratives that can stimulate narration on oneself, fundamental narrative, and necessary for a guidance path […]. Then the confrontation appears, where the listening to the narratives of others, the identification of the narratives of others on us, constructs the collection of narratives» (Batini 2005: 49). Parker too argues that behind any autobiography there is always a person who gives importance and priorities to certain matters over others. […] These are the essential ‘goods’ with which the subject lives, which determine choices and ac- M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. STRIANO 346 tions based on a particular ethic. […] Such goods […] inevitably form the stories the subject talks about when planning the future, reconstructing his or her past or present (Parker 2007: 1). For this reason, the skills budget must be a sharing space ‘where the subject feels free’ to name these essential goods. A space of sharing that can reflect the subject, helping him to fully realize his or her cognitive and emotional skills. And is «the interweaving between the human, so- cial, and psychological wealth of a person − mediated by situational variables − that allows subjects to re-enter the job market with a pro- fessional personal project in tune with the context» (Grimaldi, Porcelli, Rossi 2014: 58). Through this training to promote employability, indi- viduals can connect, integrate, and articulate their resources in the light of a reflexivity that becomes an articulate analysis which then makes it possible to recognize the motivations that have guided their actions and also the skills and abilities gained in other experiences. 3. Variation in educational training for SCN volunteers operating at the SInAPSi Centre 3. Variation in educational training for SCN volunteers operating at the SInAPSi Centre The course is structured in two modules, Inbound (BdC1) and Outbound (BdC2), each divided into different training days where individual work moments alternate with activities in groups and/or subgroups. The work is based mainly on the direct experience that participants have through exercises, games, moments for reflection, focus groups, and brainstorming. Each volunteer is invited to collect all the material produced in a personal ‘archive’, a personal ‘Travel Di- ary’ representing the memory of their experience. Below we illustrate the three steps of the incoming route and the related tools used. The first phase is devoted to the reception, the creation of a collaborative environment, and the formation of the group. The group instrument is assumed as a context suitable to accommodate the «stresses of a plu- rality of models, each of which bear a selective enhancement of one or more structural dimensions of competence» (Galdo, in Striano, Capobianco 2016: 187). Through reciprocal presentation and inter- active exchange, the relationship between the participants begins: the main dimensions the work emphasizes are motivation and relation- al attitudes. At this stage of the work, the participants are also given a ‘Travel Diary’, a folder to collect the tools used, which represents the memory of each participant’s route. At this point, the contract or training agreement has also been clarified, which is where the presentation of the content of the training (inbound and outbound modules) is also planned. The second exploration phase is aimed at TELLING TRANSVERSAL COMPETENCES 347 subjective exploration through the distribution of a self-assessment questionnaire on cross-skills, and the AVO Giovani (ISFOL 2016) to assess internal employability potential. The third step is «to present an autobiographical narrative instrument, the My Skills Patchwork, an instrument designed to ‘activate personal promotion’ with the aim of supporting volunteers in reconstructing and presenting their personal history and training with particular attention to the various formal, non-formal, and informal contexts» (Capo, in Striano, Capo 2015: 142). This session is therefore key in «activating a subjective recon- naissance and reflection on personal training experiences, but also in interpreting students’ expectations and aspirations» (Capo 2015: 142). 3. Variation in educational training for SCN volunteers operating at the SInAPSi Centre At this stage, some time is also dedicated to the clarification of moti- vations, expectations, and general reflections: participants are offered the opportunity to reveal their personal motivations and expectations on the path they are facing through guided reflection. The ultimate step follows the final phase during which the skills profile returns in a paper version (Fig. 1), elaborated both by an analysis of My skills Patchwork and by the questionnaire on the self-evaluation of skills (quantitative analysis). From the analysis of the training experiences in the various contexts (formal, non-formal, and informal), initial and non-professional experiences, but above all through analysis of the questionnaire, «it becomes possible to identify for each student those skills that we could identify and define as “weak” and “strong”, while photographing the set of core competences possessed by the subject» (Striano, Capobianco 2016: 145) as illustrated in the following chart: Figure 1 – Graphic representation of the individual profile of cross-competences. Figure 1 – Graphic representation of the individual profile of cross-competences. 348 M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. STRIANO The reception phase of the incoming module (T0) provides an introductory moment during which the goals and stages of the en- tire evaluation path are presented to the volunteers. At this initial stage, and at the opening of subsequent workshops, the group is of- fered the use of the photo set ‘Inside the photo’ created and developed by ISFOL (2006). After specifying that this is a game of imagina- tion where there is no right or wrong answer, the trainer asks each volunteer to choose one or more photographs (up to three), among those available, on the basis of which the person presents him- or herself and explains the expectations nurtured by the SCN expe- rience. Volunteers point to a Post-it note positioned next to each selected image, with the size and characteristics identified. Then, positioned in a circle, they are invited to present themselves based on the reflections triggered. The introduction of such a procedure is particularly effective in facilitating exchange between the various members of the group and the establishment of a positive climate, essential for carrying out subsequent experiential activities. Within this game of imagination, the descriptive features of stimuli can be elaborated by the participants in a subjective manner. 3. Variation in educational training for SCN volunteers operating at the SInAPSi Centre Each volun- teer tends to attribute the pictures in the photos, their expectations and feelings, to the experience, as well as their own personal char- acteristics, skills and competences: the images become the starting point to narrate their own personal and professional experiences, fa- cilitating the emergence and sharing of thoughts and emotions. Af- ter the presentation ends, the activity continues with a later stage of work, to be held this time in a group. In the absence of the trainer, the participants will arrange the photos, individually selected, on a single noticeboard: the task is to choose, from images and individ- ual representations, a name and/or an allegorical figure to represent the group’s identity. The goal is to work on defining the identity of the volunteers’ group and to bring out its motivations and expec- tations. The next exploration phase is dedicated to the distribution of the My Skills Patchwork tool which each volunteer will have to deal with individually (Tab. 1). This is a narrative instrument divided into two thematic sections of in-depth analysis: 1) ‘My Educational Experiences’, with a focus on: a) the context’s learning experiences (formal, non-formal, and informal); b) percep- tion with respect to the educational implications of the experiences. 1) ‘My Educational Experiences’, with a focus on: a) the context’s learning experiences (formal, non-formal, and informal); b) percep- tion with respect to the educational implications of the experiences. p p p 2) ‘My Portrait’ with a focus on: a) personal events considered par- ticularly significant for professional growth; b) attitudes; c) the rela- tionship with the professional context (Capo 2016b). Below is a chart of the sections the narrative instrument is divid- ed into. Below is a chart of the sections the narrative instrument is divid- ed into. 349 TELLING TRANSVERSAL COMPETENCES Table 1 – Diagram depicting the sections of My Skills Patchwork. Therefore, the biographies/fi ction sheet, My Skills Patchwork, for volunteers on the course is a useful tool to facilitate the approach to and comparison with diff erent subjective dimensions such as self- image, interests, motivations, and attitudes. From this point of view, the subjects, with the trainer as a facilitator, can identify their skills more clearly, positively surpass critical situations, and, as far as the ‘biographical wealth’ is concerned, gain and increase their skills, abilities, and knowledge (Malrieu 2003; Guichard 2005; Rossi, Fab- bri 2005; Delory Momberger 2013; Capo 2016a). 3. Variation in educational training for SCN volunteers operating at the SInAPSi Centre Beginning from 2016, the Occupational Health Services (SPO) section started collaborating with INAPP (previously ISFOL) and allowed the BdC orientation path specialist to administer a pilot ex- periment, anchored to the employability construct for a new audi- ence of users, or young volunteers from the National Civilian Service (SNC). The AVO Giovani questionnaire (ISFOL 2016) identifies the employability profile (Fig. 2) of a subject based on some of the per- sonal resources available that permits recognition of internal em- ployability potential. The AVO Giovani questionnaire is subdivided into several sections which allows the investigation and elaboration of those aspects that have a major impact in determining subjects’ employability (Grimaldi, Porcelli, Rossi 2014), e.g.: t ki bilit Subsequently, in the phase to intensify the evaluation, the vol- unteers are provided with a self-assessment questionnaire on cross- sectoral skills based on the key competences of the European Qualification Framework (2008), and Life Skills (OMS 1999, 2004). These skills are considered fundamental, especially in the field of work and in other aspects of life in general. In addition to these competences, as indicated by the EQF, the questionnaire examines Life Skills, a range of basic cognitive, emotional, and relational skills that enable individuals to work with both individual and social com- petence. Of the ten Life skills, eight were taken into consideration, since problem-solving and effective communication, which are two Life Skills, were already part of the EQF’s competences. The ques- tionnaire, consisting of 46 items with Likert-type scale responses (from 1 = nothing to 5 = very much), allows the subjects to express and attribute their value to the skills they are examining. This compi- lation formula is a training highly focused on the subject, since it uses a methodology aimed at the investigation and promotion of a subject’s personal resources (Capo 2016a; Capo 2016b; Capo, Na- varra 2016). Subjects’, by expressing their own opinion with respect to the characteristics of a given competence (i.e. indicating what is consistent with a mark from a minimum of 1 to a maximum of 5) reflects and gains greater self-awareness (Maiocchi, Porcelli 2007). 3. Variation in educational training for SCN volunteers operating at the SInAPSi Centre It is therefore «a process that facilitates the recognition of personal abilities and com- petences for the subject, meaning the latter “not as static”, given components but rather as “emerging” dimensions, or distinctive el- ements of a subjective dynamic and refl ective reconstruction» (Capo 2016b: 4). The subject is promoted through the act of narrating and self-telling, therefore, the account does not favour access to a story that is already there, instead, the narration represents the space-time through which subjects create their own story and establish them- selves as the subject of this story (Delory Momberger 2004; Guichard 2004). From this standpoint, the life story represents a «performa- tive act» (Pineau 2000). M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. STRIANO 350 Subsequently, in the phase to intensify the evaluation, the vol- unteers are provided with a self-assessment questionnaire on cross- sectoral skills based on the key competences of the European Qualification Framework (2008), and Life Skills (OMS 1999, 2004). These skills are considered fundamental, especially in the field of work and in other aspects of life in general. In addition to these competences, as indicated by the EQF, the questionnaire examines Life Skills, a range of basic cognitive, emotional, and relational skills that enable individuals to work with both individual and social com- petence. Of the ten Life skills, eight were taken into consideration, since problem-solving and effective communication, which are two Life Skills, were already part of the EQF’s competences. The ques- tionnaire, consisting of 46 items with Likert-type scale responses (from 1 = nothing to 5 = very much), allows the subjects to express and attribute their value to the skills they are examining. This compi- lation formula is a training highly focused on the subject, since it uses a methodology aimed at the investigation and promotion of a subject’s personal resources (Capo 2016a; Capo 2016b; Capo, Na- varra 2016). Subjects’, by expressing their own opinion with respect to the characteristics of a given competence (i.e. indicating what is consistent with a mark from a minimum of 1 to a maximum of 5) reflects and gains greater self-awareness (Maiocchi, Porcelli 2007). 3. Variation in educational training for SCN volunteers operating at the SInAPSi Centre B i i f 2016 h O i l H l h S i (SPO) Beginning from 2016, the Occupational Health Services (SPO) section started collaborating with INAPP (previously ISFOL) and allowed the BdC orientation path specialist to administer a pilot ex- periment, anchored to the employability construct for a new audi- ence of users, or young volunteers from the National Civilian Service (SNC). The AVO Giovani questionnaire (ISFOL 2016) identifies the employability profile (Fig. 2) of a subject based on some of the per- sonal resources available that permits recognition of internal em- ployability potential. The AVO Giovani questionnaire is subdivided into several sections which allows the investigation and elaboration of those aspects that have a major impact in determining subjects’ employability (Grimaldi, Porcelli, Rossi 2014), e.g.: • networking ability; • knowledge and enjoyment of local services; • perception of the job market; • endurance − coping;fi • perceived self-efficacy; • adaptability; • locus of control 351 TELLING TRANSVERSAL COMPETENCES Figure 2 – Employability profile. [AVO Giovani questionnaire, 2016] As can be seen from Figure 2, the calculation of the questionnaire scores allowed us to obtain both a score for each measurement scale, and an internal employability potential index. At the in-depth analy- sis phase, which follows the concluding one, feedback is given to each volunteer, in a paper version, as well as an individual skills map devel- oped from the questionnaire analysis and the employee employability profile. Through recognition of professional and firsthand experienc- es, personal events, representation of the current state of volunteer- ing, knowledge, and personal resources, and above all through the compilation and analysis of the questionnaires, it becomes possible to identify the ‘strong’ skills that represent the personal assets of the core competences possessed by the volunteer, as well as the ‘weak’ ones, i.e., those skills present at a medium to low levels. During feed- back in a group context, from an individual evaluation, subjects are offered the opportunity to talk about their employability profile and the skills highlighted by the course. 351 TELLING TRANSVERSAL COMPETENCES Figure 2 – Employability profile. [AVO Giovani questionnaire, 2016] Figure 2 – Employability profile. [AVO Giovani questionnaire, 2016] As can be seen from Figure 2, the calculation of the questionnaire scores allowed us to obtain both a score for each measurement scale, and an internal employability potential index. 3. Variation in educational training for SCN volunteers operating at the SInAPSi Centre At the in-depth analy- sis phase, which follows the concluding one, feedback is given to each volunteer, in a paper version, as well as an individual skills map devel- oped from the questionnaire analysis and the employee employability profile. Through recognition of professional and firsthand experienc- es, personal events, representation of the current state of volunteer- ing, knowledge, and personal resources, and above all through the compilation and analysis of the questionnaires, it becomes possible to identify the ‘strong’ skills that represent the personal assets of the core competences possessed by the volunteer, as well as the ‘weak’ ones, i.e., those skills present at a medium to low levels. During feed- back in a group context, from an individual evaluation, subjects are offered the opportunity to talk about their employability profile and the skills highlighted by the course. 4. Reflection on the data obtained during the training course 4. Reflection on the data obtained during the training course As part of the 2017-2018 year, the set of 40 young people from the National Civilian Service was divided and organized into two M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. STRIANO 352 groups: Group 1 and Group 2. Compared to the ‘Inside the Pho- to’ activity proposed during the reception phase, the first BdC1 group proceeded to implement their commitment by first select- ing the photos chosen by its individual members. Subsequently, a second skimming allowed the removal of a further series of pho- tographs. Once the most important choices had been made, mem- bers thought about applying the images, leaving a space to fit the chosen phrase into. p As can be seen from Fig. 3, the title of the poster is the result of the observation and analysis of the participants in the training course. The Civilian Service Project collects boys and girls of different ag- es, culture, profession, and education. In this diversity, it is possible to find a common element which needs to be emphasized: the emo- tion of working together with a shared purpose. This is how the title ‘The Rainbow of Identities’ was born. The term ‘identity’ was chosen deliberately to indicate what goes beyond the skills or competences that emerge in a given context. In this sense, there is a need to re- fer to the concept of identity spoken of by Erickson (1982), namely, the individual’s awareness of a constant and continuous sense of self within time, and also the recognition by others of such qualities of an individual’s self. Figure 3 – The outcome of the ‘Inside the Photo’ activity carried out by the 1st BdC1 group. Figure 3 – The outcome of the ‘Inside the Photo’ activity carried out by the 1st BdC1 group. 353 TELLING TRANSVERSAL COMPETENCES Figure 4 – The outcome of the ‘Inside the Photo’ activity carried out by the 2nd BdC1 group. Figure 4 – The outcome of the ‘Inside the Photo’ activity carried out by the 2nd BdC1 group. The identity dimension, not yet well defined, of the ‘tribe’ (group) and the travel metaphor, found space in the work of the second sub- group (Fig. 4) which was to support the recovery of a temporal and emotional dimension of the newly formed training experience, en- abling an explanation of motivations and expectations. The pivotal point of the image configuration proposed by the group was the fire around which the ‘tribe’ gathered, after numerous experiences (rep- resented by the horizon and the sea behind the tribe). Fire becomes the element of a design thinking: the tribe is reunited, determined to enhance its ability to be, and to improve its knowhow − the ‘tribe’ wants to learn. The tribe is therefore in the game. The goal, not yet visible, has to do with intentional change (Boyatzis, McKee 2006), with both personal and group growth. It strongly emerges in the discussion when each member of the ‘tribe’ brings out any desires and dreams related to the goal yet struggling to define him- or her- self around a clear personal and professional project. In this case, the group becomes a privileged place to learn, slowly coming to define itself as a space of self-awareness, as a reflection on its designing ex- periences in a design perspective. The group context becomes an op- portunity to establish relationships and a place to achieve an active confrontation with oneself and with others, with multitude points of The identity dimension, not yet well defined, of the ‘tribe’ (group) and the travel metaphor, found space in the work of the second sub- group (Fig. 4) which was to support the recovery of a temporal and emotional dimension of the newly formed training experience, en- abling an explanation of motivations and expectations. The pivotal point of the image configuration proposed by the group was the fire around which the ‘tribe’ gathered, after numerous experiences (rep- resented by the horizon and the sea behind the tribe). Fire becomes the element of a design thinking: the tribe is reunited, determined to enhance its ability to be, and to improve its knowhow − the ‘tribe’ wants to learn. The tribe is therefore in the game. The goal, not yet visible, has to do with intentional change (Boyatzis, McKee 2006), with both personal and group growth. It strongly emerges in the discussion when each member of the ‘tribe’ brings out any desires and dreams related to the goal yet struggling to define him- or her- self around a clear personal and professional project. In this case, the group becomes a privileged place to learn, slowly coming to define itself as a space of self-awareness, as a reflection on its designing ex- periences in a design perspective. The group context becomes an op- portunity to establish relationships and a place to achieve an active confrontation with oneself and with others, with multitude points of The identity dimension, not yet well defined, of the ‘tribe’ (group) and the travel metaphor, found space in the work of the second sub- group (Fig. 4) which was to support the recovery of a temporal and emotional dimension of the newly formed training experience, en- abling an explanation of motivations and expectations. The pivotal point of the image configuration proposed by the group was the fire around which the ‘tribe’ gathered, after numerous experiences (rep- resented by the horizon and the sea behind the tribe). Fire becomes the element of a design thinking: the tribe is reunited, determined to enhance its ability to be, and to improve its knowhow − the ‘tribe’ wants to learn. The tribe is therefore in the game. The goal, not yet visible, has to do with intentional change (Boyatzis, McKee 2006), with both personal and group growth. It strongly emerges in the discussion when each member of the ‘tribe’ brings out any desires and dreams related to the goal yet struggling to define him- or her- self around a clear personal and professional project. In this case, the group becomes a privileged place to learn, slowly coming to define itself as a space of self-awareness, as a reflection on its designing ex- periences in a design perspective. The group context becomes an op- portunity to establish relationships and a place to achieve an active confrontation with oneself and with others, with multitude points of M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. STRIANO 354 view, motivations, and attitudes and «yet just by listening to such a wealth of visions, a small (but significant) change [can be inaugurat- ed]» (Formenti 2017: 75). Figure 5 – Graphic representation of the ‘parent’ and ‘child’ nodes found during the analysis with NVivo 11 Pro software. TELLING TRANSVERSAL COMPETENCES From the graph above, it emerges that the motives which stimulated the volunteers who chose to try National Civilian Service, and in par- ticular those that led them to choose the SInAPSi Centre project were: • Personal values and inclinations: 20% of the volunteers stated that they chose to do this experience because it seemed to be in line with their values, ideals, and interests. For example, in the narratives stimulated by the My Patchwork skills some responded: «I chose this project because it complied with my personal inclinations and val- ues»; or, «I have always tried before to help others, but the SInAPSi centre allowed me to develop this inclination through volunteering, acquiring new skills at the same time […] it has turned out to an op- portunity that has enriched me significantly»; «I chose to do civilian service in this area because it was adapted to my personal disposition»; • Consistency with a study course: 16% of the volunteers claimed to have chosen to undertake National Civilian Service, and in particu- lar to have become involved in this project in the ‘Assistance’ field, because it was in continuity with the course of studies they were attending or had already completed. Some of them stated: «I want- ed to do an activity that was close to my study course»; or, «I was interested in having experience in a context where the knowledge and skills I had acquired during my studies were involved»; «The SInAPSi Centre project caught my attention because it belonged to a field related to my social studies»; • Training experience: 15% of the sample stated that they wanted to undertake National Civilian Service in order to enjoy a training experience that could serve them at multiple levels (personal, pro- fessional, social, civic, etc.) and that would accompany them for the rest of their life as a pleasant reminder of their youth. The collection of ‘biographical’ materials produced during the training has made it necessary for a qualitative analysis of the collected narrative material from the compilation of the My Skills Patchwork instrument using specific software: NVivo 11 Pro (Non-numerical Unstructured Data Indexing, Searching and Theorizing Vivo). This is a programme to analyse texts, images, and multimedia, and is part of the Computer Assisted Qualitative Data Analysis (CAQDAS) programme (Pacifico, Coppola 2010; Coppola 2011). Primarily, we identified the ‘parent’ node, which is a princi- pal category, and then their respective ‘child’ nodes, which are sub- categories. Below we show a graphic representation (Fig. 5) of the dimension explored with the respective ‘child’ nodes that emerged: Figure 5 – Graphic representation of the ‘parent’ and ‘child’ nodes found during the analysis with NVivo 11 Pro software. The next step shows the compilation of charts related to the di- mension of motivation, as core-categories in the speech produced by volunteers through the support of the narrative-autobiographical in- struments (Fig. 6). Figure 6 Graphic representation of the motivations that led volunteers to Figure 6 – Graphic representation of the motivations that led volunteers to choose to try SNC. 0% 5% 10% 15% 20% 25% Figure. 6 Graphic representation of the motivations that led volunteers to choose to try SNC Figure 6 – Graphic representation of the motivations that led volunteers to choose to try SNC. 0% 5% 10% 15% 20% 25% Figure. 6 Graphic representation of the motivations that led volunteers to choose to try SNC 355 TELLING TRANSVERSAL COMPETENCES TELLING TRANSVERSAL COMPETENCES Some volunteers stated in this regard: «I wanted to have a new experience, but also one that was profitable»; «I participated in the civilian service call out of an economic issue, […] I had just lost my job and then I decided to take this road because […] it gave me the guarantee of a monthly reimbursement of expenses»; g y • Economic reward: 9% of the volunteers stated that amongst the moti- vations that led them to choose National Civilian Service was to ben- efit from the monthly payment of €433.80, which could be useful not only to cover their expenses but also to save small sums of money to invest in other areas of their lives. Some volunteers stated in this regard: «I wanted to have a new experience, but also one that was profitable»; I i i d i h i ili i ll f i i [ ] «I wanted to have a new experience, but also one that was profitable»; «I participated in the civilian service call out of an economic issue, […] I had just lost my job and then I decided to take this road because […] it gave me the guarantee of a monthly reimbursement of expenses»; g g y p ; • Familiarity with the university: 7% of the sample involved in this study stated that they chose to do National Civilian Service while taking part in the project proposed by the SInAPSi Centre, because the latter was part of an already familiar context, namely, the uni- versity where they were studying or had already studied (Univer- sity of Naples “Federico II”). Some volunteers stated in this regard: «Being in service at the SInAPSi Centre was convenient for me, since it was on the university campus that hosted my study course»; «I chose the SInAPSi Centre project because, being located at my university centre, it gave me a sense of security and protection»; y g y p • Number of vacant posts: 6% of the volunteers stated that they chose to do National Civilian Service because the latter, unlike most other Calls, made 40 positions available to candidates. This strikingly in- creased the chances of being selected and winning the contest. TELLING TRANSVERSAL COMPETENCES Some vol- unteers said, «I wanted to live a new experience, but at the same time one that was highly educative, learning directly in the field»; «I chose this project because it promoted interesting activities, able to enrich my collection of theoretical and practical knowledge»; «In my eyes, the civilian service experience was an opportunity for personal, professional, and civic growth»; p p g Transition to the working world: 11% of the volunteers involved in the research said they had completed this National Civilian Service experience because it was midway between university and work. In this way, young volunteers, still students, or who had just com- pleted their university and/or higher education, were given a ‘taste’ of what the working world entails and requires, namely: respect for rules, responsibility, interpersonal confrontation, etc.). In this regard, the volunteers stated: «Volunteering for me represented an oppor- tunity to have an experience that was a bridge between study and work»; «I wanted to do a volunteer experience that I could add to M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. STRIANO 356 my curriculum at the same time, […] have an experience that would give me the opportunity to discover new awarenesses and abilities, working in a relatively secure context»; • Becoming involved: 9% of the volunteers claimed they had chosen to do National Civilian Service because it offered them the opportunity to test themselves as people «learning how to handle new situations and thus assuming tasks that require a sense of responsibility, the man- agement of emotions or stress, etc.»; or to kick-start their life situation, which had stalled for various reasons. Others asserted: «I wanted to have an experience which I would get by myself»; «I wanted to get into the game of life, to discover a different world from mine, and then to mea- sure and get to know better my own abilities, talents and resources»; g y • Economic reward: 9% of the volunteers stated that amongst the moti- vations that led them to choose National Civilian Service was to ben- efit from the monthly payment of €433.80, which could be useful not only to cover their expenses but also to save small sums of money to invest in other areas of their lives. TELLING TRANSVERSAL COMPETENCES In fact, the volunteers said: «I honestly chose this project because it made 40 positions available and I was more likely to become a part of it»; «I chose this project because it is one of the few at a national level that offers such a high number of places»; «I thought: the project seems interesting to me, there are several places, I want to try it!»; g p y • Through word of mouth: 5% of the volunteers claimed to have become aware of the existence of National Civilian Service and the project in question through word of mouth, or through stories of positive experi- ences among their friends, former volunteers, or those in service at the 357 TELLING TRANSVERSAL COMPETENCES TELLING TRANSVERSAL COMPETENCES STRIANO 358 The graph (Fig. 7) was shown to the group as a component to ini- tiate reflection on the results of the self-assessment questionnaire to provide them with the opportunity to share impressions, comments, and thoughts. The ability to Collaborate and Participate is anchored to the discussion on university experiences where, according to most participants, it seems that it was the privileged context of develop- ment and enhancement for that competence. Collaborating and Par- ticipating is mainly related to the pursuit of study activities during university years, but respectively important are the learning experi- ences in non-formal and informal learning contexts (such as sports, associations, or group travel). Probably these experiences have also ‘experienced and/or well-trained’ volunteers to interact with others, understanding differing points of view, enabling them to recognize the value of diversity and to collaborate with others. Moreover, the competence to Act Autonomously and responsibly, closely related to Collaborate and Participate, was evaluated by the young volun- teers as a strong point, the subjects themselves declaring that, at the appropriate time, they can enforce their rights and needs within the various contexts of reference, while recognizing those of others, as well as their own and others’ responsibilities. Certainly, personal responsibility depends on an ability to design, that is, to complete a project without the support of an adult, assuming responsibility for the consequences of one’s own actions. Equally, on medium to high values, the focus was on acquiring and Interpreting Information: the young volunteers valued being able to critically capture and inter- pret all information received, assessing its reliability and usefulness, distinguishing facts from opinions. It seems that the competence to critically evaluate the flow of information is closely linked to the competence of Planning, that is, the ability to use knowledge learned to achieve meaningful and realistic goals. Designing, therefore, re- quires the ability to identify priorities, evaluate existing constraints and capabilities, define action strategies, and subsequently test their outcome. This demonstrates how the volunteers were prepared to actively participate in the various experiences and to design, or at least identify and grasp, elements that enabled them to plan and de- sign future actions. Among the Life Skills, therefore, if Empathy, Emotion Management, and Interpersonal Relationships which were all at an average level, are excluded, young volunteers demonstrat- ed reduced Self-awareness. TELLING TRANSVERSAL COMPETENCES University Centre. Some volunteers said: «I was convinced about par- ticipating in this project because people who already knew the SInAPSi Centre talked about it in a very positive way»; «I became aware of the project through a professor who had contacts with the SInAPSi Cen- tre»; «I chose the SInAPSi Centre thanks to a person I knew, who had worked with professionals collaborating with the centre». University Centre. Some volunteers said: «I was convinced about par- ticipating in this project because people who already knew the SInAPSi Centre talked about it in a very positive way»; «I became aware of the project through a professor who had contacts with the SInAPSi Cen- tre»; «I chose the SInAPSi Centre thanks to a person I knew, who had worked with professionals collaborating with the centre». The outlining of a mapping of skills, both through the BdC ques- tionnaire and on the basis of the survey and reconstruction of each person’s training past, off ered volunteers the opportunity to focus on the skills they had acquired during the various training experiences. The following graph (Fig. 7) shows the aggregate results of the self- assessment questionnaire of cross-skills of the volunteers at time T0 (BdC entrance module). In general, the self-evaluation carried out by the volunteers on the dimension of the analysis proposed by the questionnaire, shows scores are mostly medium-high, which is, most probably, a consequence of the sample’s characteristics. In fact, the group mostly consisted of young graduates (LM) or others who had already had initial work experience. As can be seen, Creative Think- ing, Problem-Solving, and Self-Awareness are, however, the skills that volunteers allocated rather low scores. At an intermediate level, we fi nd the skills: Communicating (in the macro-category relationship with reality), Emotion Management, Interpersonal Relationship Manage- ment, Planning, Critical Thinking (in the Life Skills category), Iden- tifying Links and Relationships − category ‘relationship with reality’); Learning to Learn (in the macro-category ‘building the self’). Among the skills identifi ed as ‘strong’ because they re-enter an elevated level of competence, are: Collaborate and Participate; Act Autonomously; which are followed by Empathy; Time Management, Interpretation of Information and Planning (of the macro-category ‘building the self’). Figure 7 – Summary of the average cross-sectional skills of groups 1 and 2. ure 7 – Summary of the average cross-sectional skills of groups 1 and 2. M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. TELLING TRANSVERSAL COMPETENCES If the latter competence which is closely related to problem-solving and creative thinking, also showed very low scores, this confirms that the volunteers were not always prop- erly accustomed to recognizing their strengths and weaknesses and, consequently, translating the cognitive elements into new perspec- ti f h i th f t 359 TELLING TRANSVERSAL COMPETENCES The following graph (Fig. 8) shows the total responses provided for the individual scales of the AVO questionnaire (ISFOL 2016). The following graph (Fig. 8) shows the total responses provided for the individual scales of the AVO questionnaire (ISFOL 2016). Figure 8 – Graph of the resources involved in calculating the A and B Groups’ Volatility Index of volunteers. This instrument, as specifi ed previously, was administered at time T0, before the start of any training activity (general and specifi c) en- visaged by the civilian service project. Once the inbound training activities (BdC1) began for each volunteer, the individual result re- port was returned, where both the scores for individual scales and the concise employability index could be displayed in the form of a total score and graph. Re-establishing individual profi les was used within the group to initiate refl ection on the dimensions involved in computing the synthetic index. In response to clarifi cation by vol- unteers, manual calculations for scoring each measurement scale was considered (professional adaptability, coping, self-effi cacy perceived in job searches, perception of the job market, social networks, and per- ceived support). Overall, the data for the entire volunteer group had a medium-high synthetic index of 18.55. Volunteers were perceived as adaptable and learning-oriented. They said themselves that they used the most active ways to deal with challenging times, supported by an above average self-esteem. The average scores pertaining to the self-effi cacy scale found in job searches were above average: the views of the eff ectiveness of volunteers for the various activities which they could undertake to enquire about a job are therefore positive. Among the factors related to this scale, frustration tolerance was one that av- eraged the lowest scores: volunteers did not seem to perceive their ability to tolerate and manage any kind of diffi culties or possible fail- ures in job searches. TELLING TRANSVERSAL COMPETENCES The coping scale, as can be seen from the graph, produced two distinct scores in relation to the two specifi c dimen- Figure 8 – Graph of the resources involved in calculating the A and B Groups’ Volatility Index of volunteers. This instrument, as specifi ed previously, was administered at time T0, before the start of any training activity (general and specifi c) en- visaged by the civilian service project. Once the inbound training activities (BdC1) began for each volunteer, the individual result re- port was returned, where both the scores for individual scales and the concise employability index could be displayed in the form of a total score and graph. Re-establishing individual profi les was used within the group to initiate refl ection on the dimensions involved in computing the synthetic index. In response to clarifi cation by vol- unteers, manual calculations for scoring each measurement scale was considered (professional adaptability, coping, self-effi cacy perceived in job searches, perception of the job market, social networks, and per- ceived support). Overall, the data for the entire volunteer group had a medium-high synthetic index of 18.55. Volunteers were perceived as adaptable and learning-oriented. They said themselves that they used the most active ways to deal with challenging times, supported by an above average self-esteem. The average scores pertaining to the self-effi cacy scale found in job searches were above average: the views of the eff ectiveness of volunteers for the various activities which they could undertake to enquire about a job are therefore positive. Among the factors related to this scale, frustration tolerance was one that av- eraged the lowest scores: volunteers did not seem to perceive their ability to tolerate and manage any kind of diffi culties or possible fail- ures in job searches. The coping scale, as can be seen from the graph, produced two distinct scores in relation to the two specifi c dimen- M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. STRIANO 360 sions, namely, Analysis and Assessment of the situation and Evasion- Avoidance. The latter refers to the amount of passive coping whose score is understood to be inversely proportional to a calculation of the synthetic employability index. The data also shows a rather confus- ing, mostly static, and insecure perception of the job market as well as the presence of inadequate social networks that guarantee worth- while support in the search for employment. TELLING TRANSVERSAL COMPETENCES Overall, a perception of the job market was documented that was not very welcoming nor supportive of concern, anger, and resignation, and which influenced the proactivity and thereafter the potential of employability. Recent studies (Gilardi, Guglielmetti 2015) relate the perception of employ- ment opportunities and the feeling of self-efficacy to the perception of individuals’ employability; pointing out that feeling the job mar- ket hostile can affect both the sentiment of perceived employability, and the active search for work. 4. Final remarks The formative training presented in this contribution is intended to be a guidance experience for young volunteers of the National Ci- vilian Service (SNC) in the hope of becoming an effective and trans- ferable model for further orientation contexts in the transition to the professional world from a point of view of self-empowerment and the promotion of sustainable employability. Volunteering is constitu- tionally a privileged place for learning. In the Volunteering Charter of Values, adopted in 2001 in the Attitudes and Roles section, it is written that: «Volunteers are committed to training with perseverance and seriousness, aware of the responsibilities they are taking on, espe- cially towards the direct targets of their interventions. They receive from their organisation the necessary support and training necessary for their growth and for implementing the tasks they are in charge of» (Volun- teering Charter of Values 2001, Article 14).i This summarizes the twofold nature of volunteering, firstly as a ve- hicle for personal growth and secondly for social consistency. Training outcomes, in terms of skills acquired during volunteering, deserve to be recognized, certified, and valued in the same way as in other ar- eas of school, university, and workplace training: with their respon- sibility volunteers produce ‘strategic skills’ for themselves and for the company they are committed to. The aim of the laboratory for the promotion of employability is to comprehend and realize the need to update the vocational, organizational, and professional components of volunteering, pursuing the growth of volunteers by accompanying and promoting employment opportunities that can increasingly satis- TELLING TRANSVERSAL COMPETENCES 361 fy their needs. Cross-skills constitute a framework that every subject should cultivate in a particular way since they are characterized by a high degree of transferability to different contexts and demands. They are, in other words, a resource of fundamentally subjective means to achieve the social and ethical goals of volunteering. Aubret J. 2009, Sens et pratiques de l’accompagnement des adultes dans le démarches de validation de l’expérience, «Recherche et formation», 62, 25-38. 4. Final remarks The main goal of the formative action of the outgoing Laboratory Module (BdC2) is to provide transitional orientation activities aimed at promoting awareness among young volunteers for their work and training ca- reer and, in particular, promoting in volunteers «a positive self-eval- uation of the self-concept, [and therefore] a feeling of having control over the events of one’s life, solving problems in a creative way, […] applying decision-making skills to the various aspects of one’s own life, and making decisions in a careful and rational manner» (Di Fabio 2014: 100). Formative training for volunteers within the University SInAPSi Centre seems to pedagogically reflect on their skills set by making explicit the abilities, knowledge, and resources that would otherwise remain implicit. To ‘educate’ young volunteers is, in the first place, as Striano maintains, «to think that it can be seen, in the educational function, as a conscious thinking of someone’s Self, or else [of that thought that is capable to inaugurate a tale, recognizing the value of experiences], a thought that formulates hypotheses and tends to an incessant search for meaning» (Striano 1999: 47). Second- ly, from the point of the AVO questionnaire, it is about encouraging young people to reflect on their employability potentials, and on the various dimensions (and related sub-dimensions) that the question- naire looks at, such as: professional adaptability, coping, self-efficacy perceived in job searches, etc., in a society like today’s, in which self- redesign, grounded on an awareness of the skills acquired in various formal, non-formal, and informal contexts, the accomplishment of one’s own personal life project becomes the winning card. The very experience of volunteering and its educational implications thus be- come a fundamental element, not left to the mercy of oblivion, but reflexively ‘embedded’ in the puzzle of one’s own personal and pro- fessional realization, considering the skills and knowledge acquired before the beginning of volunteering as a prerequisite for giving meaning to the experience itself. fy their needs. Cross-skills constitute a framework that every subject should cultivate in a particular way since they are characterized by a high degree of transferability to different contexts and demands. They are, in other words, a resource of fundamentally subjective means to achieve the social and ethical goals of volunteering. 4. Final remarks The main goal of the formative action of the outgoing Laboratory Module (BdC2) is to provide transitional orientation activities aimed at promoting awareness among young volunteers for their work and training ca- reer and, in particular, promoting in volunteers «a positive self-eval- uation of the self-concept, [and therefore] a feeling of having control over the events of one’s life, solving problems in a creative way, […] applying decision-making skills to the various aspects of one’s own life, and making decisions in a careful and rational manner» (Di Fabio 2014: 100). Formative training for volunteers within the University SInAPSi Centre seems to pedagogically reflect on their skills set by making explicit the abilities, knowledge, and resources that would otherwise remain implicit. To ‘educate’ young volunteers is, in the first place, as Striano maintains, «to think that it can be seen, in the educational function, as a conscious thinking of someone’s Self, or else [of that thought that is capable to inaugurate a tale, recognizing the value of experiences], a thought that formulates hypotheses and tends to an incessant search for meaning» (Striano 1999: 47). Second- ly, from the point of the AVO questionnaire, it is about encouraging young people to reflect on their employability potentials, and on the various dimensions (and related sub-dimensions) that the question- naire looks at, such as: professional adaptability, coping, self-efficacy perceived in job searches, etc., in a society like today’s, in which self- redesign, grounded on an awareness of the skills acquired in various formal, non-formal, and informal contexts, the accomplishment of one’s own personal life project becomes the winning card. The very experience of volunteering and its educational implications thus be- come a fundamental element, not left to the mercy of oblivion, but reflexively ‘embedded’ in the puzzle of one’s own personal and pro- fessional realization, considering the skills and knowledge acquired before the beginning of volunteering as a prerequisite for giving meaning to the experience itself. References M. CAPO, V.P. CESARANO, M.PAPATHANASIOU, M. STRIANO 362 Batini F. (a cura di) 2005, Manuale per orientatori. Metodi e scenari per l’empowerment personale e professionale, Erickson, Trento. p p p f Boyatzis R. and McKee A. 2006, Intentional Change, «Journal of Organizational Excellence», XXV (3), 49-60. Capo M. 2016a, Le memorie professionali degli operatori dei nidi d’infanzia: uno sguardo biografico ai saperi in formazione professionale. 4. 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Alessandra Priore (University of Naples Parthenope)* Abstract: The aim of this paper is to see what happens when reflective practice is used in a guidance intervention made with a group. Fifty university students took part in 3 focus group sessions during which they could discuss the issue of guidance and the choices and construction of a life plan. The results show a clear perception of wellness and usefulness in the educational experience, based on the possibility of knowing oneself, comparing oneself with others, activating systematic reflection on oneself and promoting change. Keywords: guidance, focus group, reflexivity, higher education. * Alessandra Priore, PhD, Postdoc Researcher, Department of Motor Sciences and Health, University of Naples, Italy, Email: alessandra_priore@yahoo.it. THE GROUP AS AN EDUCATIONAL RESOURCE IN GUIDANCE PROCESSES Alessandra Priore (University of Naples Parthenope)* 1. Group guidance The construction of a life plan involves reflective processes that support the individual in exploring the perceived possibilities and in the critical connection between internal instances − needs, wishes, as- pirations − and the demands that life contexts impose (Cunti, Priore, Bellantonio 2015). Today, thinking and designing the future means risking, exploring the unknown, putting up with uncertainty, but also desiring, hoping, and choosing; all prerogatives that make the future a human time (Morace 2013), therefore, a time to educate, turning to the subjects that are projected towards it. Experimenting and cul- tivating choice in training venues allows individuals to prepare them- selves in the presence of the need to have to suddenly choose, and in the most critical transition phases, where they can feel disarmed and occasionally exploited. The educational task, then, comes to develop learning paths that promote a capacity to manage the Self in relation to change (Cunti 2015), to provide resources that enable students to choose properly. In fact, individual choices need to be the object of a reflection process (Schön 1987) that supports critical learning and cultural education. On the application side, this involves the use of techniques and instruments that assume a guiding value by making students aware of themselves through a continuous construction and re-construction of their personal story (Savickas et al. 2010), which may allow them to congruently connect personal features and their educational and professional choices (Lo Presti 2010; Loiodice 2009; Cunti 2008; Domenici 2003; Pombeni 1996). 366 ALESSANDRA PRIORE The aim of this paper is to investigate what happens when this re- flective exercise finds space and is cultivated in a guidance intervention carried out in a group context. The guidance practice, arranged in this way, attributes centrality to the subject through a work of Self on Self (Foucault 1985) that is mediated by others and is therefore based on the idea that such a work can be better valued by the individual when supported by others. 1. Group guidance In other words, the key guidance capabil- ity, i.e., the critical management of Self, is considered to be valued and integrated by individuals when they are supported by other listeners and interlocutors, when they are involved in a discussion, when they reflect themselves in others to know themselves better; indeed, the actual potentialities of an individual can be traced starting from the perspective of others: personal identity is built through a construc- tive discussion with the experience of others, but only when the in- timate dimension exceeds the social one. From an educational point of view, this implies setting out conditions and strategies to ensure that the internal aspects, who we are, how we represent ourselves, what we think, who we wish to become, meet the external aspects, the whole group, its participants, their experience, but above all, the opportunity to swap experiences on common ground. From this perspective, the group, with its relational and interactive dimension, may thus prefigure itself as a precious resource to trigger that critical reflexive leap (Guichard, Pouyaud 2015) useful, if not es- sential, to produce alternative ideas and possible solutions (Krueger, Casey 2000; Duggleby 2005). The sense of plurality that charac- terizes everyday life is thus reproduced inside the group in its wide range of perspectives, aspects that make it productive and functional; in the words of Usher and Edwards (2005), the efficacy of guidance consists in eliciting one’s self as an object of knowledge, but situated inside an actors’ network. In a pedagogical perspective of guidance, the group (Di Nubila 2008; Venza 2007) is thus interpreted as an educational setting in which to construct meaning about experiences that belong to the in- dividual members, and to lay the foundations of personal life plans; conflict, but also the mutual care that characterizes the creation of a group and the deployment of their work, will be the humus of that meaningful and potentially transformative learning (Mezirow 2003) so necessary for individual development. y p The methodological choice of group guidance therefore includes the perspective that the group represents a positive setting, facilitat- ing the activation of processes such as decentralization, comparison, cognitive conflict, and hence the development of transverse skills (Ladogana, Cardone, Mansolillo 2015). 1. Group guidance Should we like to view the group as a way to cope with critical issues, we can identify in its ho- THE GROUP AS AN EDUCATIONAL RESOURCE IN GUIDANCE PROCESSES 367 mogeneity (Pombeni, D’Angelo 1994), at least in terms of the needs shared by the participants, an element of efficacy in achieving com- mon goals. Although the uniqueness of individual experiences distin- guishes and offers different points of view and perspectives of meaning to various issues, the group remains centred on a common task that identifies and makes the various participants resemble one another. In fact, this mechanism facilitates the opportunity to feel closer to one’s own views, positions, and ideas that would never be considered alone. The group thus becomes an instrument that can activate criti- cal-divergent thinking, a learning opportunity that finds its founda- tion in experiential and relational aspects rather than in intellectual knowledge, as so often happens. g pp Such a guidance experiment implies a twofold function: on the one hand, it activates critical exercises addressed to self-management in choice systems, on the other, it is proposed as a privileged context of socio-emotional and relational experimentation in which to locate and verify operating modes which, in the working context, are con- sidered functional and productive; suffice to think of team-working in which cooperation (Kagan 2000; Kaye 1992) is based on skill in managing differences and conflicts. g gfl The group, as a mental space for discussing subjective experiences, offers the chance to increase the ability to find connections between themes and experiences, between stories and meanings, but also the forms of adaptation between internal and external dimensions. The complex dynamics that generically characterize each group, but above all the specific guidance aim that is discussed, depend especially on the role played by the moderator. The latter has the complex task of catalysing the group’s performance by working on transformative thoughts through feedback, reflecting but also emancipating; assem- bling, reorganizing, translating, reviving, and interpreting the par- ticipants’ interventions. These represent some basic steps that lead a working group to generate critical-reflective thought (Schön 1987). 2. Educational research with University Students Fifty university students (divided into four groups), enrolled in various active three-year Degree Courses at the universities involved in the national research project, took part in 3 focus group sessions (Merton, Kendal 1946) during which participants could discuss and exchange views on the guidance issue. In this research phase, it was decided to involve the users of uni- versity guidance services, i.e. students, with the aim of achieving the following research objectives: identifying their guidance needs; the 368 ALESSANDRA PRIORE individual and deeper aspects underlying their choices and life plan- ning; possible guidance strategies to respond to these. individual and deeper aspects underlying their choices and life plan- ning; possible guidance strategies to respond to these. However, when it came to the educational objectives, the focus group was assembled with the aim of supporting the participants in acquiring greater awareness regarding the choices and construction of a life plan, by galvanizing critical-reflexive thought. The structure of the focus group sessions was inspired by the way Savickas and Hartung (2012) organized the production of the career story according to the famous formula ‘Telling-Hearing-Enacting My Story’, which in the case of this research was adapted to the edu- cational story which becomes a life plan. y p The themes discussed during the 3 sessions related to the mean- ing in life, also through narrative education and the presence of sig- nificant institutional figures or family/friends, choices, transitions, and changes, of the questions; «Who am I?» and «Who will I be- come?», starting from the way each subject was located on his or her own educational path; the calling, how, whether, and what aspects and factors would make it changeable. Such themes supported the participants in reflecting together on the sense of university guid- ance, their deeper guidance needs, personal learning experiences, and identifying potential solutions and strategies through which the university as an institution could support their educational paths with specific guidance.l The reflective instruments used during the focus sessions were narration, metaphor, and brainstorming. All the data gathered, the methodological choices, and the results will be collected in subsequent publications; here we have chosen to investigate aspects of evaluating the path, and possible changes expe- rienced by the students. To this end, the participants were given an evaluation questionnaire centred on organizational elements, content, and method, and the student’s personal change. 2. Educational research with University Students The questionnaire consisted of a closed-response part that con- tained a series of statements to which the student was asked to in- dicate their level of agreement on a 5-point Likert-like scale (Not at All, Somewhat, Moderately, Quite a Bit, Very Much) or dichotomously (Yes, No); with an open narrative part that asked the students to dis- cuss more deeply the evaluation of the path they were participating in. As a last point, the students were asked to fill out the question- naire anonymously to avoid only desirable answers. Turning, instead, to a general evaluation of the course the stu- dents were attending, there was a clear sense of wellbeing and of the usefulness of the educational experience: in fact, all the students said that they ‘felt good’, considered participation in the project useful, and would recommend it to other students. When the students were THE GROUP AS AN EDUCATIONAL RESOURCE IN GUIDANCE PROCESSES 369 asked to explain the reasons behind their evaluation of the course as something useful for themselves, they referred to dimensions such as: asked to explain the reasons behind their evaluation of the course as something useful for themselves, they referred to dimensions such as: g y • self-knowledge («it allows you to evaluate aspects of yourself to which little importance is given»; «it encourages you to discover your iden- tity»; «so I can know myself better»); • comparison with others («you can listen to the experiences of others»; «you compare yourself with other people’s stories»);lll • reflexivity as systematic reflection («it helps you to reflect on your- self»; «there’s a need to reflect on yourself»; «it’s useful to find new ideas to think about»); • planning («it helps you make choices about your educational path»; «useful for understanding the goals you want to achieve»; «useful for clarifying your ideas»); • change («useful for understanding where to start to promote posi- tive change»). The reasons why the students would recommend that others par- ticipate in projects of this type were along the same lines. 2. Educational research with University Students The cat- egories that emerged in this regard highlighted the possibility of: • understanding the value of guidance («it makes you understand the usefulness of guidance, which is often undervalued»; «I would recom- mend it to confused university students because it helps you to think about yourself and to understand»); • in-depth self-analysis («we don’t always have the chance to analyse ourselves deeply»; «useful for personal reflection»); • comparing oneself with others («good experiences for sharing opin- ions and reflections»; «a way to get to know other people»; «helps you understand the lives of others»); ) • a stimulating experience («it was an interesting and fun experience»; «I had fun»; «very useful and stimulating»). The students’ description of their experience was summarized in the following categories: The students’ description of their experience was summarized in the following categories: • path of awareness («a positive experience that leads to greater awareness of yourself and your choices»; «it helps us dig deeply into ourselves»); • development path of people and their potential («it helps you improve yourself and understand how to do it»; «addressing important issues for the development of people and their potential»); • active participation course («a course in which you analyse issues to find solutions to university and guidance problems»; «a good way to feel active in the university»; «discussion of some concepts to under- stand university guidance and make it more effective»);l y gf • a reflective course («it helps you pause a moment to think about your- self»; «it’s a very reflective course»). 370 ALESSANDRA PRIORE At the end of the sessions the students were asked if they had sugges- tions for improving the guidance path they had followed together, but only 39% proposed improvements, which can be categorized as follows: • organizational proposals (avoid meetings during exams; schedule meetings at the beginning and end of the academic year; plan more meetings); • methodological proposals (list the topics before the meeting and give students time for pre-reflection; explain the goals better; discuss more issues). • methodological proposals (list the topics before the meeting and give students time for pre-reflection; explain the goals better; discuss more issues). 2. Educational research with University Students As for the involvement of the students during the focus groups, the responses revealed that about 66% said they felt ‘very much’ in- volved and about 34% ‘quite a bit’ involved; while to the question «Did something change in you?» 65% of the students said Yes and, for example, justified their answers in this way: Previously, the question «Who am I?» was obvious and superficial. Now it has a deeper meaning, and, for this reason, I no longer know how to respond. I am more convinced about myself as a person and student because I have understood more. I felt useful in raising the awareness of the institutions to address the problem proactively. I felt useful in raising the awareness of the institutions to address the problem proactively. The importance of guidance. The importance of guidance. I feel more aware of past choices and the author of future ones. I feel more aware of past choices and the author of future ones. From now on, I will no longer make too-hasty choices. From now on, I will no longer make too-hasty choices. I have a clearer picture for orienting myself in the future and new insights. THE GROUP AS AN EDUCATIONAL RESOURCE IN GUIDANCE PROCESSES THE GROUP AS AN EDUCATIONAL RESOURCE IN GUIDANCE PROCESSES 371 The theme of the choices and the recursive transitions (Boffo, Gioli 2016; Guichard, Di Fabio 2010) appears redundant in the participants’ narratives, and is described as an element that pervasively character- izes their life paths, in that, although, on the one hand, it represents an opportunity for personal growth and the education of the person, on the other, it seems to be experienced in negative terms, i.e., as a weight to carry on the shoulders and a moment of difficulty to cope with. It is often associated with a sense of obligation, conditioning by others, renunciation, and especially the absence of those fundamen- tal aspects – knowledge, passions, interests – starting points to make decisions that consider both rational and emotional-affective aspects. In other words, choices assume profound meanings that transcend a simple selection from among different alternatives and therefore re- quire greater attention from educational institutions. In fact, although change and transition now represent a structural feature of a person’s entire life cycle, those described in the narratives seemed to possess a potential criticality precisely due to the need to make choices that evidently also involved the resolution of conflicts with themselves and their surroundings. So, what seemed to have, at least in earlier decades, an evolutive potential for emancipatory change (transition as an exclusively positive event), is currently also charac- terized by a regressive character, procrastination, and/or stagnation. It is therefore necessary to rethink traditional guidance practices, in- tended in a diagnostic sense and focusing on linear paths of life, to try to respond to subjects’ need to reflect and know themselves, al- lowing for the dimensions of plurality and flexibility. In this framework, group guidance could represent a valid pro- posal, an opportunity to experiment and consciously cultivate choices within a cooperative learning process in which everyone leaves and takes something from comparison with others. Group guidance means focusing on an individual’s psycho-social resources, recognizing that each transition, decision, and project belongs to the individual, but is inevitably negotiated with others. The passage we wish to highlight is one which, from guidance based on an individualized approach, then tends to the construction of knowledge based on interpersonal, social, and cultural factors. 3. Final remarks Considering the recent theoretical models that have emerged in the literature (Savickas 2015, 2005, 2002; Guichard 2012, 2010, 2005), but also starting from the simple point of view of emerging adults engaged in constructing their own educational and professional path, guidance means educating people to become more flexible and criti- cal observers of themselves and their personal story. In this process, group work is set up as an educational instrument and a condition which, through comparison with others, allows people to intercept and recognize with greater clarity deep aspects of themselves. The passage from ‘us’ to ‘me’ and vice versa, typical of group dynamics (Bion 1961; Lewin 1948), and the result of proceeding through re- flection, identification, projections and spacings that fuel the change of perspective on oneself and on the world and, as a result, open the construction of new meanings to attribute to experience. THE GROUP AS AN EDUCATIONAL RESOURCE IN GUIDANCE PROCESSES The efficacy of the proposal is traceable in the perceived change, reported by the students, which, although not generalizable, finds in the pos- sibility of implementability and transferability in other contexts, a future prospect of research and education. The key element report- ed by the students is that they were fazed and surprised by how the stories and their meanings, nowadays given as certain, through the group took on a new light, and might steer their ideas on the future in a different direction. 372 ALESSANDRA PRIORE To conclude, the function of the group in guidance interventions is to re-create, in a protected mode, an «episode of social life» (Ush- er, Edwards 2005: 407), and to emphasize the network and the re- lationship between the actors, hence, the production of interrelated and dialogic knowledge. THE GROUP AS AN EDUCATIONAL RESOURCE IN GUIDANCE PROCESSES 373 Krueger R.A. and Casey M.A. 2000, Focus groups: A practical guide for applied researchers (3rd ed.), Sage, Thousand Oaks. ( ) g Kaye A. 1992, Learning together apart, in Kaye A.R. (ed.), Collaborative learning through computer conferencing, Springer-Verlag, Berlin, 117-136. 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THE GROUP AS AN EDUCATIONAL RESOURCE IN GUIDANCE PROCESSES * Marco Schiavetta: paragraphs 1 and 3; Sonia Startari: Introduction, paragraph 2 and reflective final remarks. ** Marco Schiavetta, PhD Student, Department of Educational Sciences, University of Genoa, Italy: marco.schiavetta.348@psypec.it; Sonia Startari, PhD, Research Fellow at Department of Educational Sciences, University of Genoa, Italy: sonia.startari@unige.it. 1 As stated by Decree 13/13 provided for the creation of a national system of com- petence certification Cedefop: European guidelines for validating non-formal and informal learning (2015). CAREER CALLING: LIGHTS AND SHADOWS* Marco Schiavetta (University of Genoa), Sonia Startari (University of Genoa)** abstract: In this moment the policies on lifelong guidance, recognition of learn- ing and skills certification seem more working on who should do what and not on how make. It would be desirable to create links between the various provid- ers of training, education and business, allowing for a reframing of orientation processes and of identification and certification of skills. All these practices can reduce distances for those who are likely to remain at the margins of education, training and work (Neet and over fifty). Keywords: lifelong learning, skills, orientation, guidance, identity, carrier. THE GROUP AS AN EDUCATIONAL RESOURCE IN GUIDANCE PROCESSES 2007, Dinamiche di gruppo e tecniche di gruppo nel lavoro educativo e formativo, FrancoAngeli, Milano. 1. Introduction This paper aims to develop a reflection on how the standards in Lifelong Learning, Lifelong Guidance, the Certification of Compe- tences, and the operating systems implemented by various institutions can represent a true resource for the needs of students and profes- sionals. As a result of repeated calls by the European Union, Italian Law 92/2012 Article 4 (paragraphs 51 to 68) equated formal learning with non-formal and informal learning and hence the different ways of acquiring professional and technical skills in all processes and at each stage of training, orientation, and work of specific competence1. It is our belief that lifelong learning and the various contexts (formal and non-existent) today represent the fundamental element of the re- sources whereby people cope with the needs of late modernity. Extreme- ly complex systems, where the autonomy of individual action increases, leave the student or worker the responsibility for building and growing their own knowledge base, skills, motivation, and propensity to action that blend in the term ‘competence’ (Beck, Gernsheim 1990; Lupton 2003). According to the writers, to disentangle the current debate, it is worthwhile speculating whether the current certification and guidance procedures really do allow students and workers in limbo MARCO SCHIAVETTA, SONIA STARTARI 376 between a ‘mechanical’ model and a model based on free self-entre- preneurship, to develop awareness and the relevant resources. The risk is for those who fail to keep up with the processes of training and acquiring new skills that a flexible and knowledge-centred orga- nization of work requires. Recognition of lifelong guidance, valida- tion of previous learning, and competence certification could be the means to guide the structuring and restructuring of professionalism, which, in turn, requires a process of re-socialization (Berger, Luck- mann 1995), thus redistributing values relating to the reality of the job market and education. 1. Introduction To date, however, there are still cultural barriers in the formalization of guidance procedures, recognition of learning, and skills certification, such as:i gi • the legal value of qualifications obtained through formal educa- tion and training paths; • the traditional weaknesses of further education and adult educa- tion and training, which in Italy are not as widespread and con- solidated as in other European countries;i • the absence of an explicit national system of qualifications and competences and auxiliary services for choice and lifelong guid- ance services; • the multitude of institutions involved in this issue at national, re- gional, and local levels (ISFOL 2012). Within this problematic context, the process of recognition, vali- dation and certification of skills acquired in formal, informal, and non-formal contexts can be significant in terms of both professional and personal development. Acquiring awareness of their skills can be an opportunity for individuals to redesign their career, but also to consider entering a path of education and training and, more gen- erally, it provides an opportunity for critical−propositive reflection on their past to design their own future. In the light of continuous changes in the forms of work and professionalism required, lifelong guidance can become a tool for continuous growth and development. It is also a process of learning intended as change, an ability to play, to choose, to take responsibility for ourselves; a process by which one becomes skilled, capitalizing not only on past experience, but all the experience of the very processes of orientation, identification, vali- dation, and certification. The Age of Uncertainty: Orientation and Life Design in the 21st Century was the title of the 16th SIO Conference (Italian Orientation Society), 2. Lifelong guidance today The Age of Uncertainty: Orientation and Life Design in the 21st Century was the title of the 16th SIO Conference (Italian Orientation Society), CAREER CALLING: LIGHTS AND SHADOWS 377 and it can be argued that this represents a social snapshot of today’s advanced demands, including orientation. The concept of ‘Life De- sign’2 is based on an ability to consciously design one’s own life (fol- lowing an individual’s real and profound needs) within a biocentric and holistic evolutionary model. To identify the needs to be met in a lifelong guidance perspective, studying work transitions allows us to investigate mechanisms to structure the opportunities and inequali- ties systems and their degree of permeability (Schizzerotto 2002). y g p y ( ) While, on the one hand, this approach has enabled one side to un- derstand the degree of societal openness or closeness, on the other, it has overlooked some peculiar configurations and mobility features. Reflection has focused primarily on all the ascending and descend- ing, inter- and intra-generational movements, seen as an indicator of the real change in life and affiliation opportunities. However, it has not focused on changes associated with horizontal displacements in social space (Bison 2002; Cobalti 1995); and while great emphasis has been placed on structural and context variables, the interest in subjective lives and the sense of the individual’s own work career has been studied little and left in the background. g Hence, our proposal is to widen knowledge of mobility dynamics by studying individual displacements along the horizontal axis and among working positions of different areas of use in which elements of regularity and predictability are induced by the action of social au- tomation, and attention is focused on the micro-social dimension of the passages previously indicated (subjective lives and the sense that individuals attribute to their work and training career)3. This could be achieved by focusing on the supply side of the job and seeking more precisely to look at aspects that have already been investigated but can provide other valuable information, such as motivations, ex- pectations, and individual preferences that generate and guide change. 2 Life Design (or Vocational Design) is based on the use of a narrative approach (Life Design Counselling, or Narrative Career Counselling). 3 Interesting in this line of research is the quantitative study on job-to-job mobil- ity, co-funded by the MIUR within the Prin Cofin 2005, as part of an inter-university survey of mobility and local job market transitions (Colasanto, Zucchetti 2008). 2. Lifelong guidance today p p g g g The purpose of lifelong guidance is to understand how the traits of uncertainty, volatility, and fragmentation typical of the current so- cial context (Bauman 2002; Giaccardi, Magatti 2003) affect work and employment transitions (including career choices made by university students) and to redefine not so much and not only the linearity and consequentiality (Fullin 2004; Sennet 1999), but also the intrinsic mean- ing. In particular, within a cultural and occupational universe whose traits are difficult to fix and where the contingency of the choices made MARCO SCHIAVETTA, SONIA STARTARI 378 is more than stability and long-term commitment (Bauman 2004; Sen- net 2006), all of this can contribute to generating and self-feeding a weakening, standardization, and a crumbling of social relationships. The most consistent hypothesis is that work forces are devoid of the meaning of a career as a social institution − or as a system of dis- ciplinary rules of substantial weight in individual and collective life (Castel 1995) − while, instead, individual protagonism becomes prev- alent in the form of a constant tension to achieve what makes more immediate sense, with personal experience and the construction of one’s identity thereby resulting in a narrowing of the temporal per- spective and an acceleration of subjective time (Aubert, Haroche 2013). p j ( ) Today, we no longer have the expectation of a better life for our children, on the contrary, we fear that their lives will become more difficult. If we wish to avoid this worsening, we must move at a fast- er pace, increasing our efforts, and innovating even more every year. The current crisis in the Eurozone is a practical demonstration; po- litical actions do not tend to create a better society, they do not create expectations, but simply focus on overcoming the crises by adopting the quickest workable solutions in order to avoid a worst-case scenario. q The linear social time ruled by our clocks has been replaced by a ‘timeless time’, a ceaseless flood of de-territorialized flows (capital, goods, people, ideas, as well as diseases and risks) that are emerging worldwide, giving rise to the phenomenon of de-synchronization of living places (Rosa 2012), and a consequent increase in uncertainties, inequalities, and discrimination (Piketty 2013). 4 See, for example, the Dublin Descriptors, a general outline of learning expecta- tions and skills for each of the closing titles of each ‘Bologna’ cycle, that is, the typical achievements of students who have obtained a qualification after successfully complet- ing a study cycle. These include elements of which there is no formal evaluation if not tacitly through examinations and positive evaluations of internships. ‘Knowledge and Understanding’. ‘Applied Knowledge and Understanding’ (applying knowledge and un- derstanding). ‘Making Judgments’. ‘Communication Skills’. ‘Learning Skills’. 3. Recognition of learning and certification of skills Legislative Decree 13/2013 provides a definition of competence that does not neglect the learning contexts: «Proven ability to use in a work, study, and professional development context, a structured set of knowledge and skills acquired in formal learning contexts, whether non-formal or informal». It is also explicit when it says that in the context of public education, training, work, competitiveness, active citizenship and welfare policies, the Republic promotes lifelong learn- ing as a person’s right, and ensures equal opportunities for the recogni- tion and valorization of competences., however acquired, in accordance with individual aptitudes and choices and from a personal, civic, social, and employment perspective. It focuses on people’s acquisition of knowledge and skills through- out their life, in their study and work lives, ensuring a transparent, CAREER CALLING: LIGHTS AND SHADOWS CAREER CALLING: LIGHTS AND SHADOWS 379 pertinent recognition system. These skills become an indispensable element for interventions aimed at professional development and job placement processes, which include the various training providers, employment services, schools, universities, and enterprises intercon- nected in the creation of institutional strategies and actions which seek to overcome a concept of accompanying and episodic orienta- tion, and which intend to create opportunities with specialized ac- tion plans that surpass the idea of welfare. p p From this perspective, the methodologies, tools, and techniques adopted by the competent entities and their actual functionality and usefulness regarding recipients’ real needs come into play. It is per- haps necessary to be careful not to fall into an «educator’s perspective» (Cepollaro 2008: 127), that does not surmount the possible contrast between personal goals and the means at hand and the actual op- portunities to achieve those goals. The challenge is how to work on the ‘weak link’ that exists between formal, informal and non-formal learning supporting the identification of skills. g pp gi We believe that validating and certifying are not one single process. Underlying these reflections is the belief that before you can evalu- ate and then certify the acquisition of a series of skills, it is necessary to make the skills possessed by the subjects visible, and subsequently recognize them as examples of knowledge or stable knowhow ac- quired, and subsequently certified. At a national level, it remains nec- essary to distinguish between the validation of previous learning and competence certification. These two paths are linked, but different. fi pi pf Among the most important difficulties remaining is the confusion in certifying skills with the recognition of non-formal and informal learning. As already mentioned, competence has come to the fore in terms of implementing specific actions in contexts. Professional repertoires are split into skills and knowledge, which contribute to producing competence and the level of autonomy with which ac- tions are carried out. The critical issue here is to unite the informal and non-formal learning acquired in the professional background of a person with transverse skills, which are not found in the region- al repertoires of professional figures, and have not even really been evaluated in formal paths4. CAREER CALLING: LIGHTS AND SHADOWS 380 MARCO SCHIAVETTA, SONIA STARTARI In implementing recognition and certification processes, it is equal- ly important to recognize the role of the accompanier, recalling that, in selectively reconstructing their biography in terms of training expe- riences or otherwise, candidates mature awareness of the professional applicability of these experiences, and focus on the profile proposed for certification. The accompanier plays a crucial role in recruiting candidates for the initial stages of education, further education, pro- fessional and personal experiences. The selection of experiences, carried out with the aim of facilitat- ing the recognition and worth/marketability of the experiences that subjects have developed in different contexts, should be fostered, in our opinion, through assisted self-assessment of skills with an ap- proach that recalls the output−links−resources model, aiming to ag- gregate appropriate skills to a specific role that should be explicitly expressed in: • output production processes with all the technical-realizational problems that have different degrees of predictability; • connections with clients and internal or external suppliers with the relational difficulties that these involve; fi • resources which, under their own responsibility, raise many dif- ficulties associated with management dynamics. The accompaniment should:i • define the objective of the entire recognition and certification path that can be identified:ii –– in the certification of competences for the recognition of qualifi- cations within the regional Directory of Professions system. The development of this goal may also include new profiles if they are not already present in the Directory; y p y –– in previous learning consistently with the content of training and/ or education paths that may allow for the acquisition of a formal diploma/qualification.i p qi • identify the most significant experiences and, for each experience identified, analyse the entire process of each macro, thus finding the relevant skills. • find continuous training paths in line with the goal. The whole accompanying phase is complex for both the accompa- nier and the candidate, since in the process of conceptualizing pro- fessional and personal biographies, all experiences seem valid, and it is not always possible to identify those that are genuinely marketable. The greatest risk is to fall into the trap of stating the possession of skills through an account of what the person did and to merely ex- hibit documentation that ‘certifies’ these. 5 Lifedeep Learning is a third dimension that has only recently begun to be debated. It relates to beliefs, values and life orientations. 6 On 19 February 2014, the MIUR published its National Guidelines for Lifelong Guidance, in which life-long orientation is recognized as the right of every person and exercised in different and specific forms and needs, contexts and situations (Lisbon 2010, Europe 2020). CAREER CALLING: LIGHTS AND SHADOWS However, we need to be CAREER CALLING: LIGHTS AND SHADOWS 381 very aware that this is a reflective narrative (Reggio, Righetti 2013) which shows the ability to reason on what has been done as well as the resources that have been mobilized to achieve the result or de- liver the performance. l This reflection is not always easy for potential candidates for cer- tification, and therefore it requires the support of a qualified guide. Moreover, it is indispensable to think about how to redesign study courses at all levels in a lifelong learning perspective. This means thinking about what skills learning should be offered, which deliv- ery method is most cohesive with the local territory and job market, and how to create validation and certification services and proce- dures that will be recognized by all education and training agencies. 5 Lifedeep Learning is a third dimension that has only recently begun to be debated. It relates to beliefs, values and life orientations. 6 On 19 February 2014, the MIUR published its National Guidelines for Lifelong d h h l f l d h h f d 4. The impact of orientation and lifelong learning on career paths Lifelong, lifewide and lifedeep5 are increasingly invaluable con- ditions for the effective exercise of the right to active citizenship. These variables, which are present to different degrees as resources in individuals, now more than in the past represent guarantees to pro- tect oneself from the multiple risks of marginalization or exclusion from the workplace6. This is the set that constitutes the underlying conditions from which mobility paths develop. Such paths are char- acterized by a degree of complexity and articulation of trajectories and are not necessarily driven by a search for material aspects, such as hierarchical progression and authority, and pay rises (which are among the aspects of gratification), but are instead aimed at achiev- ing a gratifying personal condition, defined by the level of initiative and autonomy exercised; by the correspondence of the job carried out with personal interests, expectations, and ways of being subjec- tive; by the possibility of satisfying integration between the personal and professional spheres (Bovone 1984; La Rosa, Meda 1998; Lewis, Rapoport, Gambles 2003). p p ) Today more than ever, Career Calling represents a challenge that requires us to reflect on how to launch a professional vocation; its dimensions and relationships with studies, motivation, satisfaction in life and work; the professional identity, and the career choices made MARCO SCHIAVETTA, SONIA STARTARI 382 by students (Fournier et al. 2016: 93-143) and adult/lifelong learn- ing. To deal with this task, three different key interpretations can be used. The first is based on reflections on career theory, above all, the approach where cultural patterns and norms of use depart from tra- ditional models towards a new, multifaceted, versatile, indeterminate, and self-taught business idea (Arthur, Rousseau 1996; Hall 1976). This perspective signposts increased opportunities for movement beyond organizational boundaries, between and within employment sectors, through a series of episodic, often unplanned jobs7, in particularly dynamic employment markets, especially for the most qualified pro- fessional profiles, but also for intermediate figures. These opportu- nities are organized in a design plan whose stages are characterized by lifelong learning and the acquisition of those multiple skills that this learning can help to build. In fact, there is a high probability of further inequalities,8 particularly on the road to success, since this is measured by the ability to ensure meaning and continuity through- out one’s life story (Arthur, Khapova, Wilderom, 2005). 7 Due also to changing market conditions and organizational structures, the re- sponsibility for deciding the ways and direction of such a career path falls entirely on individual subjects, who, in the absence of a reliable guide provided by the firm, under- take to collect information, skills and knowledge that will enable them to orient their subsequent choices. 8 On the one hand, thanks to the human and social capital possessed, they can play the protagonist and thus exploit the opportunities of realization that unfold. On the other, there are those who find themselves on a shaky unyielding path, made up of continuous moves, within a precariousness that they cannot abandon and which causes the lack of available resources. 9 The basis for individual success and social recognition is, in this cultural land- scape, risk predisposition, and an ability to adapt and be flexible; Thanks to these qualities, individuals succeed in nurturing an identity in progress, supported through the social networks they have created and the unceasing experiences that ensue in their life story. 4. The impact of orientation and lifelong learning on career paths y ( p ) The second follows the approach of the life course, which identi- fies itself as a combination of a series of events, transitions and paths, the practice by which individuals produce and reproduce their iden- tity, each time reaffirming themselves. The course of life and the working careers in it are interpreted as a cumulative result of the multiple career lines which make up an individual life story, and the trajectories of the ‘significant other’, including partners, family members or, more generally, members of one’s own social networks. These are, therefore, the results of experiences and change, whose outcomes can be both unexpected and foreseeable (Saraceno 2001; Schizzerotto 2002). These, however, seem to continue to follow a series of institutional references − typical of modern society: work, the family − whose relevance and normative nature seem almost unaltered (Kohli 2007). The third perspective is enclosed in the culture of the project, whose principles have been described by Boltanski (2005), pointing out how in the features that distinguish the ‘new spirit of capitalism’, CAREER CALLING: LIGHTS AND SHADOWS 383 of priority is the value assigned to the activity which, past an original identification with the work for the market, is reinterpreted as the in- volvement of the subject in changing and heterogeneous episodes and projects that are not confined to the employment dimension alone. These projects, by their very nature, have a limited timeframe; once completed, they require the ability and will to be questioned again, along with a promptness to change the scope of action, meaning, and relational values. Here, there is a very serious risk for individuals, namely, the loss of their roots as a result of the frantic success of the various projects, something which can create a deep sense of anxiety, fuelled by the fear that the plurality and complexity of the projects undertaken may actually prevent one from making sense of one’s life9. 5. Final remarks 5. Final remarks The development and implementation of lifelong guidance mod- els, the recognition of learning, and the identification and certifica- tion of competences, depend on how institutions face the challenges of European and national legislation. In current practice, the fo- cus seems more on who should do what and not on how, why this should be done, and what the effect will be on recipients of this new right to competence and recognition of learning matured in their lives. It would be desirable to find a common ground that cre- ates links between the various providers of training, education, and business, allowing for a reframing of orientation processes and the identification and certification of skills. This ought to represent a common system of development and inclusion for those who have failed to build a linear career in the name of flexibility, limiting themselves to experiencing several iotas of professionalism. Lifelong guidance, the recognition of learning, and skills certification can shorten the distances for those who are likely to remain at the mar- gins of education, training, and work. Operators should be careful not to remain involved in bureaucratic and documentary schemes that cannot create added value, but could produce symbolic and stigmatizing hierarchies for those who obtain a degree through a recognized traditional course. 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(a cura di) 2013, L’esperienza valida, Carocci, Roma. Rosa H. 2012, Beschleunigung-Die Veränderung Zeitstrukturen der in der Moderne, Reggio P. and Righetti E. (a cura di) 2013, Lesperienza valida, Carocci, Roma. Rosa H. 2012, Beschleunigung-Die Veränderung Zeitstrukturen der in der Moderne, Columbia University Press. * Massimiliano Costa, Associate Professor of General Pedagogy, Cà Foscari University, Venice, Italy. Email. Email: maxcosta@unive.it; Andrea Strano, Research Assistant at International Research Centre for Education and Advanced Studies - CISRE, Cà Foscari University, Venice, Italy. Email: andrea.strano@unive.it. Massimiliano Costa (Ca’ Foscari University of Venice), Andrea Strano (Ca’ Foscari University of Venice)* Abstract: Work personalization within cognitive capitalism (Alessandrini 2013) demands universities to certify competences that can promote new forms of em- ployability (Boffo, Federighi, Torlone 2015), connected to global network in- novation (Steiner et al. 2013). Personal entrepreneurship becomes the heuristic promoter of career changes (Federighi, Torlone 2013). This enables individu- als to ask new questions, to provide innovative solutions, and to create endeav- ours that can extend the current limits of knowledge, or define new ones (Costa 2014). Methodology: The research analysed variables and, by doing so, strength- ened entrepreneurial competence (Morselli, Costa 2015; Costa, Strano 2016) from an enabling perspective, involving more than one hundred people going through a career change (Sen 2000). Based on a mixed method (Ponce, Pagán- Maldonado 2015) the research was developed over four phases: 1) Self-assessment (Pittaway, Edwards 2012) of entrepreneurial attitudes; 2) Recognition (Federighi 2014) of emerging competences from global innovation networks; 3) Reflective thinking (Mortari 2003) and switching in terms of awareness (Mezirow 2003); 4) Capability (Sen 2006) of opportunities and resources for the instigation of en- trepreneurial action (Costa Strano 2016). Results: The data observed show that during career changes the development of competences is positively linked to age, education, and experience, above all when combined with enabling pro- cesses. As for entrepreneurship, training contexts in the service industry prove strategic, even if still too focused solely on the technical-informative dimension. Furthermore, the results show that entrepreneurial training (Pittaway, Edwards 2012) becomes competence that can promote action starting from resources/op- portunities in various career changes (Costa 2014). Keywords: entrepreneurship education, capability approach, agency, university education, work, education policy. 1. New forms of work representation within work transitions Nowadays, work is being traversed by profound transformations fol- lowing changes in demographic factors and production systems, along with digital transformations that require new skills to operate as effi- ciently as possible (Federighi 2014) within these new innovative con- texts (Costa 2013). Novel technological and production processes (smart economy, open innovation, Industry 4.0, Shared Economy) have trans- formed (Alessandrini 2016) the inner performance value of skills into MASSIMILIANO COSTA, ANDREA STRANO 390 ‘Ability/Competence to Act’ (agency) thus relaunching a new training and social deal (Learnfare) in which the educational system is the starting point to empower development based on people’s talents (Costa 2016). Consequently, the intention to strengthen our students’ employability brings new responsibilities (Ajello, Morselli 2016) to Universities, since social inclusion and citizenship are now related to employability (Boh- linger et al. 2015). Hence the need to identify new opportunities for our students along with new environments of activation which mean work- based learning. These processes can then support those progressions of consequential transition (Federighi, Torlone 2013) which imply an iden- tity change: the feeling of turning into a new person thanks to the spread of knowledge. When managing these collateral transitions1 (Engeström 1996), the young student will have the sensation of being uncertain, of being continuously on the move, and of experiencing new identities and new relationships. It follows that the university, within the current para- digm of cognitive capitalism, cannot address only technical and economic environment demands. In conjunction with this, it needs to promote the generation of a transformative identity among its students: a mix of abili- ties to seek, give meaning, and share. The link between transition and employability is no longer driven by a simple functional and productive logic, but is characterized by a stochastic, unforeseen process, capable of crossing the boundaries of action between university and the job mar- ket, mediating between personal and collective senses. 1 According to Engeström (1996), knowledge acquisition is the outcome of an ac- tive and constructive process, a path based on a strategy, on a form of control of the wide process of the codification, transformation, and storage of information. 2. From competence to enablement: the value of agency Work transitions in the post-Fordism period have been described as a protean career (Hall, Mirvis 1994), and a boundaryless career (Arthur, Rousseau 1996). The adaptable career (Protean) is a theoretical model which describes the role of individuals in a self-directed running of their own career. Those who implement an adaptable career have a strong lo- cus of internal control. They firmly believe in their ability to control and govern events and are individuals with strong adaptive skills, able to manage processes of professional and organizational learning. Capability Activation (Sen 2006) in support of employability, thus be- comes an expression and result of a reflective and recursive investigation process, able to engage peculiarity in the variety of contexts and systems of actions in which students will be non-passive performers of their own BOOSTING ENTREPRENEURSHIP CAPABILITY IN WORK TRANSITIONS 391 transition, leveraging their own inclinations (aptitudes, motivations and desires) and talents. This is why students’ employability value is now the result of social awareness, participation, responsibility, and an ability to think: the basis of the processes of action. Agency not only expresses the mere possession of practical knowledge − to master different situ- ations − but embodies the aptitude of willingness to perform an intel- lectual activity that begins from action. Furthermore, agency expresses the leaning to learn when at work, by combining thought and action, thanks to a flexibility that becomes a cognitive plasticity which supports individual capabilities (Sen 2000). Overcoming the perspective of neo- liberal culture, flattened by the functional need of a university caged in by the job market, it is obligatory to recover a new vision hinged on the generative and creative freedom of the student when it comes to learn- ing in the workplace. Enabling agency is linked to students’ ability to use different resources, preferences, aptitudes and values, to design their own future (Morselli, Costa, Margiotta 2014). Consequently, the development of youth entrepreneurship – as an enabling tool – becomes fundamental within educational systems. The role of education in promoting entrepreneurial skills (an entrepreneurial mind-set) has become increasingly significant in recent European strate- gies for employment and the strengthening of qualifications (EUCIS-Ill 2013). 2. From competence to enablement: the value of agency The term ‘entrepreneurship education’ has been defined as a set of knowledge, skills and aptitudes aimed at making individuals capable of ‘turning ideas into actions’, considering the possibilities and relationships in which they are involved at work but also in social contexts (hence the more appropriate term of ‘intrapreneurship’). A spirit of inventiveness, a proactive attitude, creative thinking, entrepreneurship (European Com- mission 2006), ability to take and manage risk are the core of those in- tangible strategic skills (Loiodice 2009) that activate students’ enabling agency, beyond the value of each individual skill acquired (Costa 2014). Entrepreneurship has been defined as one of the eight key European competences (European Commission 2006), so important that the En- trepreneurship Action Plan 2020 (European Commission 2013) indi- cates education and training as supporting axes to fund entrepreneurial development. Furthermore, most recent European documents reiterate the need to make education and training systems a growth laboratory for the entrepreneurial mindset of youth (European Commission 2016). E hi Ed i (M l ib 2010) i E i i Entrepreneurship Education (Mwasalwiba 2010) in Europe is main- ly compared, even if in a restricted way, with the simple technicalities that are the basis of ‘doing business’. However, talking about ‘Entrepre- neurship’ (Morselli, Costa 2015; Costa, Strano 2016), could be the way to overturn this logic, re-thinking entrepreneurship not as a mandato- ry skill to find or perform a job, but as a rich ability to realize an indi- vidual’s own development. Educating with this perspective, therefore, MASSIMILIANO COSTA, ANDREA STRANO 392 goes beyond the technical and economic outlook and becomes an act- ing aptitude that qualifies transformative thinking (Mezirow 2003). En- trepreneurship competence (Bohlinger et al. 2015) becomes the ability to combine and re-think those scenarios and new possibilities that arise from the social and working context experienced by students during their lives. From this point of view, therefore, educating in terms of entre- preneurship means generating in students an authentic entrepreneurship agency (Costa, Strano 2016) within the perspective of a lifelong learning culture, increasing the levels of social awareness, participation, account- ability, and thinking ability (Siebert, Walsh 2013). 2 The concept of Eutagogia exceeds that of andragogy since it broadens self-directed learning practices, through which young people and adults actively develop necessary learning skills to meet their needs (Canning 2010). 3 These skills are not technical skills, but intangible and strategical (Loiodice 2009), able to cross different areas of activities and subsequently support the acquisition of further technical skills. g ( g ) 3 These skills are not technical skills, but intangible and strategical (Loiodice 2009), able to cross different areas of activities and subsequently support the acquisition of further technical skills. 2 The concept of Eutagogia exceeds that of andragogy since it broadens self-directed learning practices, through which young people and adults actively develop necessary learning skills to meet their needs (Canning 2010). 4 Each skill was associated with five referential competences, which develop on progressive levels in terms of increasing degree of autonomy and responsibility (Tessaro 2011). These references help in defining the concept of competence. 3. The research: context, phases, and methodologies This research has been included in the framework of the overall ac- tion that C.I.S.R.E. (International Research Centre for Education and Advanced Studies) of Ca’ Foscari University of Venice has been pursu- ing on entrepreneurship education over the last years. The experimental action took place in 2015-2016 during some courses on entrepreneur- ship held at a Training and Entrepreneurial Development Centre in the Triveneto region, which saw the participation of several organizations, universities, institutions, and social partners. The main aim of the research was to recognize and clarify the cor- relation between entrepreneurship agency and professional (and person- al) youth development, trying to rethink adequate areas within tertiary education that could transmit the entrepreneurship value, better linking the university world to the job market and enhancing the heutagogic di- mension (Canning 2010)2 of the enabling process. This would empower young people’s freedom of choice, the ability to manage their own lives according to the future they have planned, valuing the experience with a critical approach and as an opportunity to create an alienation between past experiences and future scenarios. p p The concept of entrepreneurship education led the research to de- velop its own training tool to enrich the concept of entrepreneurship (Strano 2015, 2017). This tool consists of four ‘activation areas’ and sixteen ‘enabling skills’3, which can subsequently allow the adoption BOOSTING ENTREPRENEURSHIP CAPABILITY IN WORK TRANSITIONS 4. The research: results and initial findings The survey results show that skills development and the strength of entrepreneurship during work transitions are positively related to age, education level, and experience (both work and personal experiences and social life), above all when the experience is related to enablement processes, i.e., processes based on critical thinking, context analysis and alternative options analysis (Sen 2000), to achieve personal goals. The impact of education and, even more, of tertiary education on entrepreneurship development was identified through a series of results (even though these same results, not always so clear, opened up the long- standing issue of a tertiary education that needs to improve its orienta- tion to the entrepreneurship concept):f • The QACEN survey protocol showed a clear difference in certain dimensions concerning the level of social participation and personal activation between those who had taken a Degree and those who had not: graduates showed very high values in all the dimensions inves- tigated, while non-graduates showed very low values. g g y • Observing the GET2 Test, which analysed entrepreneurial dispositions through five large axes (need for self-realization, need for autonomy, creativity, aptitude for risk, locus of internal control), those with a Degree showed significantly higher values than those without one. y, p , ), Degree showed significantly higher values than those without one. BOOSTING ENTREPRENEURSHIP CAPABILITY IN WORK TRANSITIONS 393 of specific operating choices (Sen 2000, 2006)4. The four ‘activation areas’ were placed at the capability level (Sen 2000) namely: the Iden- tity Area (Demetrio 2003; Mezirow 2003), the Practice Area (Costa 2013; 2016), the Relational-Intersubjective Area (Alessandrini 2012; Rossi 2011) and the Organizational-Institutional Area (Nussbaum 2012; Margiotta 2012). Using this training instrument, the experimental research analysed several variables (context and development), and thereby strengthened the entrepreneurship competence (Morselli, Costa 2015; Costa, Strano 2016) from an enabling perspective (Sen 2000; Nussbaum 2012), involving more than one hundred people who were going through a work transi- tion. Founding the research on mixed methods (Ponce, Pagán-Maldo- nado 2015), the process covered four major phases: ) p j p 1) Self-assessment (Pittaway, Edwards 2012; Di Francesco 2015) of en- trepreneurial aptitudes; p j p 1) Self-assessment (Pittaway, Edwards 2012; Di Francesco 2015) of en- trepreneurial aptitudes; 2) Recognition (Tessaro 2011; Federighi 2014) of emerging skills;l 3) Reflective thinking (Mortari 2003) and awareness transformations (Mezirow 2003); ( ) 4) Capability (Sen 2000, 2006; Nussbaum 2012) of opportunities and resources for entrepreneurship activation (Costa, Strano 2016). The succession of these four phases furthered the entrepreneurial agency for the individuals involved thanks to the interaction of two im- portant pedagogical concepts: self-awareness-vision and enabling-capa- bility. They gave birth to «a recursive construction movement, through a complex mixture of actions and tools that continuously [returned] on individual’s agent function, outspreading the recognition of abilities and possibilities and, therefore, deploying the activating and enabling action» (Costa, Strano 2016). The tools chosen and elaborated to work within these pedagogical meanings were: p g g g a) GET2 test (General Enterprising Tendency 2 Test), of the University of Durham, oriented to predictivity, which investigates individuals’ potential entrepreneurial inclinations; b) The double-moment (survey and individual interviews) of QACEN (self-assessment questionnaire on entrepreneurship skills), designed by the research group; able to affect reflection and awareness processes, by carrying out a context analysis and revealing the consequent pro- cess of activation-enablement. 394 MASSIMILIANO COSTA, ANDREA STRANO Ajello A. and Morselli D. 2016, Assessing the sense of initiative and entrepreneurship in vocational students using the European Qualifications Framework, «Education+ Training», LVIII (7-8), 797-814. 4. The research: results and initial findings g gi y g • Returning to the surveys carried out through the QACEN survey protocol, we would like to briefly convey these additional data: –– observing the impact of different life contexts on entrepreneurship development, the contexts that had greater weight were profes- sional and family ones, while scholastic and university experiences were less relevant; • Returning to the surveys carried out through the QACEN survey protocol, we would like to briefly convey these additional data:f –– observing the impact of different life contexts on entrepreneurship development, the contexts that had greater weight were profes- sional and family ones, while scholastic and university experiences were less relevant; –– thinking about the skills of the training instrument, the graduates reported higher values in individual competences linked to cre- ative, innovative, and relational processes;i –– the radar chart, which plotted several profiles based on the levels of development of the sixteen training instrument skills, although reporting a general superiority of the graduates compared to the non-graduates in all four ‘areas of activation’ (Identity, Practice, Relational-Intersubjective, Organizational-Institutional), at the same time clearly highlighted that between these two categories there was not such a significant surplus in several competences, especially in those of the Practice and Organizational-Institu- tional areas; –– with respect to the competence of the training instrument, compe- tences of the Organizational-Institutional area stood out with the lowest development values, both for graduates and non-graduates. BOOSTING ENTREPRENEURSHIP CAPABILITY IN WORK TRANSITIONS BOOSTING ENTREPRENEURSHIP CAPABILITY IN WORK TRANSITIONS 395 5. Final remarks 5. Final remarks Work personalization within Cognitive capitalism requires univer- sities (Bohlinger et al. 2015) to categorize the skills needed to promote new forms of employability (Boffo, Federighi, Torlone 2015) in line with today’s timeframes and innovative contexts (Steiner et al. 2013). The con- cept of employability competency (Alessandrini 2013) exceeds the purely performative dimension and becomes an ability to act: any student, be- fore being competent to produce, must be competent to act and this is why universities play such a key role in empowering an entrepreneurial aptitude (Costa 2016). University education must know how to support these transformations, promoting in students (through teaching, men- toring and guidance) the ability to create problems and solve them, to solicit the certain from the uncertain, to cover different paths and to try out new solutions in their work transitions (Costa 2013). Research has shown that educating towards an entrepreneurial mind- set does not imply sticking to the linearity of economic theories and knowledge: universities should integrate competency and enablement, interpreting skills development as the functional element for agency en- hancement. Furthermore, this kind of education should know how to: «Formulate new educational models focused on an individual’s agency; re-thinking the paradigm of competencies relating to the concept of enablement; designing formative pathways that value individual, social and contextual complexities; focusing on work as a transformative pro- cess able to leverage the entrepreneurship agency» (Costa, Strano 2016). g p p g y ( ) Framing entrepreneurship development within this paradigm (Valerio, Parton, Robb 2014) requires the efforts of all university stakeholders to support and generate this precise, expansive variety of learning (Engeström 2001), the result of an intentional cooperation towards the co-generation that underlies the boundary zone (Konkola, Tuomi-Gröhn, Lambert, Lud- vigsen 2007). In summary, it could be said that leading the sense-making of work transitions recombines the sense of employability competency, focusing the power of choice as a key expression of an individual’s devel- opment (Sen 2006). Borrowing the words of Adriano Olivetti, from his book L’industria nell’ordine della comunità (Olivetti 1952: 21): «The new econ- omy we are imagining contributes to material progress and accompanies the individual while they perfect their personality and vocations». References References MASSIMILIANO COSTA, ANDREA STRANO 396 Alessandrini G. 2016, Smart Working. Nuove skill e competenze, Pensa MultiMedia, Lecce.i Alessandrini G. 2012, Le sfide dell’educazione oggi. Nuovi habitat tecnologici, reti e comunità, Pensa MultiMedia, Lecce. Alessandrini G. 2013, La formazione al centro dello sviluppo umano. Crescita, lavoro, innovazione, Giuffrè, Milano. 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Morselli D., Costa M. and Margiotta U. 2014, Entrepreneurship education based on the change Laboratory, «International Journal of Management Education», 30, 1-16. Morselli D. and Costa M. 2015, Il Laboratorio imprenditoriale per la formazione degli insegnanti all’imprenditività, RicercAzione, VII (2), 111-124.l Mortari L. 2003, Apprendere dall’esperienza. Il pensare riflessivo nella formazione, Carocci, Roma. Mwasalwiba E.S. 2010, Entrepreneurship education: A review of its objectives, teaching methods, and Impact Indicators, «Education+ Training», LII (1), 20-47. Nussbaum M. 2012, Creare capacità, il Mulino, Bologna. Olivetti A. 1952, L’industria nell’ordine della comunità, Edizioni di comunità, Ivrea. Pittaway L. and Edwards C. 2012, Assessment: Examining practice in entrepreneurship education, «Education+ Training», LIV (8-9), 778-800. Ponce O. and Pagán-Maldonado N. 2015, A mixed methods research in education: Capturing the complexity of the profession, «International Journal of Educational Excellence», I (1), 111-135. Rossi B. 2011, L’organizzazione educativa. La formazione nei luoghi di lavoro, Carocci, Roma. Sen A.K. 2000, Lo sviluppo è libertà, Mondadori, Milano. Sen A.K. 2006, Scelta, benessere, equità, il Mulino, Bologna.l Siebert S. and Walsh A. 2013, Reflection in work-based learning: Self-regulation or self-liberation?, «Teaching in Higher Education», XVIII (2), 167-178. Steiner L., Sundstrom A.C. and Sammalisto K. 2013, An analytical model for university identity and reputation strategy work, «Higher Education», LXV (4), 401-415. Strano A. 2015, Capacitare entrepreneurship per l’attivazione professionale dei giovani, «Formazione&Insegnamento», XIII (1), 109-116. MASSIMILIANO COSTA, ANDREA STRANO 398 Strano A. 2017, Capacitare l’imprenditività. Maria Luisa Iavarone (University of Naples Parthenope), Fausta Sabatano (University of Naples Parthenope) Abstract: This essay is an element of dialogue between educational practices acquired in territorial education contexts and the University. In particular, start- ing from the 10-year long experience consolidated in three educational centres operating in border areas of the Province of Naples, a series of ‘key competenc- es’ have been highlighted that are indispensable to the containment of social risk disadvantage in an inclusion (Bertolini 1977; Freire 2004; Rossi 2014; Sabatano 2015a, 2015b) and well-being project (Iavarone 2007, 2009) from an educational point of view. Such competencies have become subject of a ‘participatory didactic planning’ between expert educators working in these contexts and a university course on ‘Pedagogy of relationships’ within the Department of Motor Science and Well-Being at the University of Naples Parthenope. The participatory plan- ning practice has set the most ambitious goal of achieving a ‘system methodol- ogy’ to be used in the curriculum-design of the university courses in order to make the academic education offer a proper link element between the educa- tional demand of young people, the demand for professional skills in the territory and the emerging social needs in order to improve employability processes. The main results that this experience has highlighted can be deducted from the stu- dent’s satisfaction survey, as well as from the data collected and processed by the University Assessment Team, in the Department’s Joint Commission Reports, which show a clear and overall improvement of the communication processes between non-academic institutions collaborating with the University for the conduct of internships, training sessions and placement-targeted activities. The empirical evidence and the positive results obtained provide substantial comfort in considering that the experience gained can be a ‘good practice’ to be included in the didactic planning process of the courses, even in relation to the need to improve the educational and didactic offer with reference to the new quality as- surance parameters (QA) for the periodic accreditation of the CdS according to the AVA-ANVUR legislation in force. KEYWORDS: non-academic skills, employability, social educators Maria Luisa Iavarone, PhD, Full Professor of General and Social Pedagogy, University of Naples “Parthenope”, Italy. Email: marialuisa.iavarone@unipartheno- pe.it; Fausta Sabatano, PhD, Post-doc Researcher in General and Social Pedagogy, Department of Physical Education, University of Naples “Parthenope”, Italy. Email: fausta.sabatano@uniparthenope.it. * This essay, although prepared by the two authors in its overall content, shall be attributed as follows: first paragraph to Maria Luisa Iavarone, while second paragraph to Fausta Sabatano. BOOSTING ENTREPRENEURSHIP CAPABILITY IN WORK TRANSITIONS Agency tras-formativa per lo sviluppo e l’innovazione [unpublished PhD thesis], Università Ca’ Foscari di Venezia, Venezia. Tessaro F. 2011, Il formarsi della competenza. Riflessioni per un modello di sviluppo della padronanza, «Quaderni di Orientamento», XX (38), 24-40. Valerio A., Parton B. and Robb A. 2014, Entrepreneurship education and training programs around the world: dimensions for success, World Bank, Washington. KEYWORDS: non-academic skills, employability, social educators TRANSFER OF NON-ACADEMIC SKILLS IN ACADEMIC CONTEXT: TOWARDS A SUSTAINABLE EMPLOYABILITY* Maria Luisa Iavarone (University of Naples Parthenope), Fausta Sabatano (University of Naples Parthenope)** * This essay, although prepared by the two authors in its overall content, shall be attributed as follows: first paragraph to Maria Luisa Iavarone, while second paragraph to Fausta Sabatano. ** Maria Luisa Iavarone, PhD, Full Professor of General and Social Pedagogy, University of Naples “Parthenope”, Italy. Email: marialuisa.iavarone@unipartheno- pe.it; Fausta Sabatano, PhD, Post-doc Researcher in General and Social Pedagogy, Department of Physical Education, University of Naples “Parthenope”, Italy. Email: fausta.sabatano@uniparthenope.it. 1. Participatory didactic planning for sustainable employment Acquiring useful skills to improve employment is a crux in the contemporary socio-economic and productive debate which, how- * This essay, although prepared by the two authors in its overall content, shall be attributed as follows: first paragraph to Maria Luisa Iavarone, while second paragraph to Fausta Sabatano. MARIA LUISA IAVARONE, FAUSTA SABATANO 400 ever, should be primarily set in the context of education and training systems, first of all by means of a better focus on the curricula-design of the study courses, in order to make the educational offer a prop- er link between the demand of young people, the demand for skills coming from the labour market and the emerging socio-economic needs in the territory useful to improve the productive osmosis, con- dition which is indispensable for real employment.ffi p p y The awareness that a more effective and efficient reform of the training and didactic systems is the best prerequisite for training pro- fessionals who are more suited to the needs of the labour market is an inevitable challenge and, moreover, extremely problematic, given the intrinsic complexity of the training-employment relation, as it involves not only the training systems sensu stricto, but also the terri- tory, politics and local economy in its relationship system. Consistent with this analysis, the project – to which this essay refers – intended to make a righteous liaison between the academic world and the labour market, by means of a ‘participatory didactic planning’ experience between a university course and a social edu- cational enterprise that offers employment for educators qualified in inclusion processes of subjects with social risk. Specifically, the proj- ect, which took place at the University of Naples Parthenope, con- sisted in the experimentation of a teaching module called Pedagogy of the relationship of a Master’s Degree in Motor Science for Prevention and Well-Being, which was planned in a participatory manner between the Professor, the person who teaches the courses, and the Scientific Director of the Educational Centre. The idea underlying the experimentation and shared among the partners of the project was to start from destructuring the idea of ‘ac- ademic training’, traditionally understood as knowledge container, and to opt for a learning model based on the confrontation with ‘re- al professional environments’ through in-depth study of observation and reflection models in local contexts. TRANSFER OF NON-ACADEMIC SKILLS IN ACADEMIC CONTEXT TRANSFER OF NON-ACADEMIC SKILLS IN ACADEMIC CONTEXT 401 tives and the European directives on lifelong learning and higher edu- cation related to the “descriptors of Dublin” EHEA (European Higher Education Area) and to expected learning according to EQF (European Qualification Framework). fi ) In the second place, it turned out to be essential to pay close atten- tion to the need to create connections between formal and informal learning: a lot of literature emphasizes the utility of implementing crossover learning, that is fertile connections between the quality of knowledge coming from different contexts, in order to enhance the incisiveness of the experience through non-traditional learning meth- ods originating in the work places and in social and professional net- works. For this purpose, participatory planning has included seminars integrated with the presence of privileged witnesses from working contexts to assess the way methodologies and instruments learned in the classroom can have a real meaning and utility in the professional context of destination.i The ultimate purpose of the project was to create significant learn- ing environments and experiences on a pathway that is not always linear, and in which, however, the personal and self-conceptual di- mension of each trained individual is enhanced in order to allow the possibility of experiencing himself/herself, creating knowledge. Certainly, this way of rethinking education, especially in the aca- demic context, puts methods, practices and even institutions in cri- sis because participatory planning indispensably places the theme of training for employability in the centre. This makes that a recent de- bate (Dunia 2017) refer to five key aspects: • empowerment of the skills and young people; • empowerment of the skills and young people; p y g p • engagement of the territory and citizens; • endorsement of politics; • enforcement of regulations; With regard to the empowerment of skills for youth employability, it is necessary to refer to those which are useful and on which, there- fore, the educational pathways should be focused. This field covers in particular strategic or meta-competence skills that each individu- al can use to redefine their knowledge according to the needs of the personal and professional context, with particular attention to the ability to consciously use social media and the network. 1. Participatory didactic planning for sustainable employment Such analysis methodologies have been particularly used for studying contexts, related problems and needs of the ultimate recipients of educational work (minors at risk) in order to find strategies and interventions that are profession- ally appropriate and consistent with the local market demand and in tune with the academic curriculum chosen. This option has come forward to an education clearly understood not as a closed sector but as a personal and professional development environment meant for intercepting employability through a better understanding of the de- velopment processes of the territory and its communities, also in terms of social inclusion, health protection, prevention of educational risk for a better individual and collective well-being. Such orientation is, on the other hand, highly coherent with the national strategic objec- TRANSFER OF NON-ACADEMIC SKILLS IN ACADEMIC CONTEXT Engagement of the territory concerns the ability to cooperate with lo- cal institutions in redefining the curricula, with the aim of enhancing the experiences coming from below and contaminating the territo- rial fabric, affecting also the citizenship and social belonging areas to somehow influence the economical and productive fabric in the strict 402 MARIA LUISA IAVARONE, FAUSTA SABATANO sense. Starting from this perspective, local authorities could play a significant role in the outside world to support young people’s start- ups, also in order to strengthen the local entrepreneurship. Endorsement of politics and enforcement of regulations refers to the op- portunity to promote educational paths that are created and re-ab- sorbed by the labour market, designing the dialogue between the lifelong educational system and the work and professions system, al- so serving as a service to the society for better social, economic and cultural inclusion. Execution, however, refers to the skill distribution process in the so-called 4.0 society, where the educational system must be aligned with the programming of social and economic-productive poli- cies. Employability is also increased by means of innovative actions based, for example, on practice communities and on accompanying and vocational guidance systems, as well as by means of using active teaching methods – also in academic contexts – that facilitate the de- velopment of self-efficacy skills, such as resilience and the ability to work in a team. 1 A. Canevaro, L’inclusione competente, <https://www.unipi.it/index.php/offerta/ item/download/4032_fd34783ba39ce6b5bdf058a579d6dc3d> (01/2018). 2 The Integra Project, born in 2005 in the Phlegre area of the Campania region to support the difficult lives of children living in areas of high social risk in Campania, in its twelve years of activity involved about 700 children and adolescents. The Project is carried out as an extra-educational training offer at the Regina Pacis Educational Centre chosen by the Bishop of Pozzuoli to host the educational emergency and is en- tirely funded by the Diocese of Pozzuoli and the 8x1000 funds of Caritas Italiana. The local authorities have never had any contribution. 2. The educational plan In order to create pathways centred on the well-being and inclu- sion of subjects in conditions of social marginality, good will, pas- sion, good feelings are not enough, even though they are essential elements that nurture intentionality and educational practice. It is absolutely necessary to structure an indispensable profile of skills in order to respond to the complexity and variety of educational needs. Therefore, implementing an inclusive perspective requires being able to meet competencies and needs. This is a fundamental issue that can be addressed only by reaching greater clarity over the professional profiles that accompany the life of a subject with ordinary or special needs (Canevaro)1. This essay, as mentioned above, springs from the dialogue be- tween educational experiences, gained within the territorial educa- tional contexts, and the university. In particular, starting from the 10-year long experience acquired in three training centres operating in border areas of the city of Naples, a series of ‘key competences’ have been highlighted that are indispensable to the containment of TRANSFER OF NON-ACADEMIC SKILLS IN ACADEMIC CONTEXT 403 social risk disadvantage in an inclusion and well-being project, from an educational point of view. The experiences referred to come from a project created about fifteen years ago2, currently implemented in three centres located in areas of high social risk of Naples (Rione Traiano, Licola mare and Quarto). These structures have an ongoing activity for children be- tween 6 and 13 years old and their families. These are families with many problems, from the moment socio-cultural deprivation prob- lems combine with life paths – in the best cases – at the limit of le- gality, but generally characterised by organised and non-organised local crimes. The project involves 15 educators and three supervisors (psychologists and psychotherapists) who have the task of supervis- ing the educational relationship between the operator and the child. g p p A method has emerged from this research and training experi- ence – the Integra Method – (Sabatano 2011, 2015a, 2015b) to counter social exclusion and stop the poverty, deviance and school drop-out phenomena currently studied by students from different universities and school teachers on the national territory. 3. The skills of the social educator The educator is the person responsible for the delicate and difficult task of implementing the educational experience in the daily reality according to the guidelines and the theoretical perspective chosen, in an attempt to change the practice into improvisation and, thus, to non-scientific action (Bertolini 1988: 299-300). Therefore, educa- tional professionalism is characterised by the «intention and ability to act educationally according to principles and guidelines belonging to the science of education». Not everyone can be an educator. To generally state that this cat- egory includes any person who deals with education at a certain de- gree (parents, animators, teachers) is a widespread and dangerous misunderstanding. The educator is, in fact, the one who must have the knowledge and the useful general and specific know-how, on one hand, in order to be able to make conscious choices and to ori- MARIA LUISA IAVARONE, FAUSTA SABATANO 404 ent himself/herself towards the complex dynamics that influence the educational practice and, on the other hand, to work with methods, techniques and tools consistent with a sense that give the latter mean- ing. This means that even the most specialised skills that he/she may have and, in certain circumstances, must possess, have to pass the fil- ter of a general pedagogical perspective in order to avoid the risk of a crush on action, of a reductionism in the technical sense. Only in this way, practice becomes a fundamental moment of conscious pro- cessuality. In this sense: «theoretical elaborations are supplemented with procedurality and visibility that, in order to be illuminated by thought, become conscious work. General and specific knowledge and skills, therefore, in the theory-practice circle, make the listening modu- lation possible not only to tune in with greater accuracy on known wavelengths, but also to give intuition and perception possible signals» (Gatto 2008). This being said, the idea arises that specialisation is an inner dimension, not an outer one, of educational professionalism. This means it is not a response to specific external conditions (age, disabil- ity, discomfort, deviance), but it rather refers to the research dimension of those tools that are not ready for use, but which have a critical and reflective nature that put the educator in a position to face reality as it comes to attention. 3. The skills of the social educator It is possible to trace four types of tools (Erdas 1991: 156-157) that are essential in the work field in order to man- age the complexity of the educational setting. First of all, the tools of paradigmatic nature, i.e. the assumptions, par- adigms, and beliefs that can be used as perspectives to start from in order to set general problems in relation to which an action program is to be developed. Second, the tools of explicative nature (or epistemological), to be traced in all those conceptual schemes or hypothesis by means of which dif- ferent sciences interpret and explain reality and which have an indis- putable value in operational terms, translated as the need of programs capable of giving answers. p g g Third, the informative tools, i.e. those useful in identifying the con- text in which an educational intervention takes place, making it more effective as it is more responsive to the specific situation. Lastly, the technological tools, understood as the set of models, meth- odological itineraries and, thus, techniques that can be followed in real situations and that guide the practices (e.g. Rogers’ non-mana- gerial model, Ausubel’s meaningful learning, etc.). Therefore, the task of the educator is to master these tools in or- der to know how to use them when the situation requires it, with- out rigidity, listening instead to the expressed and emerging needs.i However, it should be noted that the specificity of certain skills should never be considered closed, in exclusivity. As Canevaro (2013) TRANSFER OF NON-ACADEMIC SKILLS IN ACADEMIC CONTEXT 405 emphasized, the risk of a ‘closed’ skill that eliminates every other re- ality, privileging those seen through its own competence, neglect- ing interactions, relationships with other contexts and experiences. Clearly, it is instead necessary that the educational system – in the various contexts in which it is expressed – is based on the interac- tion between different educational figures. In fact, the complexity of the problems posed requires an indispensable integrated approach, in which the various skills can interact with each other to co-build the educational path. In the perspective of inclusion and well-being, being competent means, therefore, knowing how to communicate through dialogue and exchange, the skills to colleagues and colleagues who do not have the same profile of competence. This means that the abil- ity to make the context competent is included in the competence profile. 3 See, in particular, the document edited by the European Section of the International Association of Social Educators, Common Platform for Social Educators in Europe, January, 2005. 3. The skills of the social educator y p p pi At this point, it seems useful to dwell on the proprium of the so- cial educator: What are the pedagogical skills that characterise this figure? What are the tools and techniques that the educator should master? Sector studies have offered over the years different and many classifications in terms of the skills of the educator in order to define his/her professional profile and to share at European level the criteria for the recognition of titles and skill levels required for the practice of the social educator profession3. As previously stated, this consideration has the purpose to high- light the transferability of skills acquired in the field of educational curricula for the professions of well-being. In particular, the work of observation of the skills of educators in action has allowed to identify those qualities that should character- ise and inform the competent work of care professionals (Bertolini 1988: 308-311). ) In the first place, globality, which derives from reading the edu- cational event in the order and complexity of dimensions and vari- ables that characterise it, pointing to the need for a competence that, as stressed above, does not deplete the understanding of reality in its own perspective, but is characterised by openness and, therefore, is able to interact with other professionals to interpret phenomena and identify possible intervention strategies. Second, the operativeness, understood as the assumption of a con- scious orientation towards the future and, therefore, towards the possible. This takes place in the actions on the field, in helping, stim- ulating, supporting, and encouraging the subjects in training on the MARIA LUISA IAVARONE, FAUSTA SABATANO 406 path that will lead them to be more4 (Freire 2004) and to imagine an otherwise and an elsewhere (Rutter 1985). ( ) Third, the relationality which descends from reading the educational event as something to do with, a live together that feeds on a commu- nication skill, playing in person as the protagonist of the educational relationship. The stories of the subjects (educator and educated) in- tertwine and, thus, each relationship has its own story, which is the story of those people, a story marked by different stages that affect the quality of communication. f q y We are all in relationships and connected to others in different ways. 4 Freire sustains that a context that is satisfied with the existing one generates a shrinkage, a being less. Only a critical approach to reality causes the righteous anger and, therefore, a rebellion against the reality that immobilises and which, thus, betrays and denies the specific vocation of the human being: that of being more. Paradoxically, it is exactly this incompleteness – which man must become aware of – to give him greatness and protagonism in the dialectic of adaptation and integration, to oblige him to seek, improve, become an entrepreneur of culture, history, sociality and politics. 5 Enteropathy is characterised by the ability of the listener – in this case, the edu- cator – to decentralise and put himself/herself in the shoes of the subject educated. According to Bertolini, it is a relational style linked to inclusive knowledge, i.e. to the authentic understanding of the experiences and interpretations that support the actions of the subject educated. This type of relationality allows giving ‘citizenship right’ to subjective meanings that cause behaviours. 4 Freire sustains that a context that is satisfied with the existing one generates a shrinkage, a being less. Only a critical approach to reality causes the righteous anger and, therefore, a rebellion against the reality that immobilises and which, thus, betrays and denies the specific vocation of the human being: that of being more. Paradoxically, it is exactly this incompleteness – which man must become aware of – to give him greatness and protagonism in the dialectic of adaptation and integration, to oblige him to seek, improve, become an entrepreneur of culture, history, sociality and politics. 5 Enteropathy is characterised by the ability of the listener – in this case, the edu- cator – to decentralise and put himself/herself in the shoes of the subject educated. According to Bertolini, it is a relational style linked to inclusive knowledge, i.e. to the authentic understanding of the experiences and interpretations that support the actions of the subject educated. This type of relationality allows giving ‘citizenship right’ to subjective meanings that cause behaviours. 3. The skills of the social educator In th perspective, it is fundamental for educators to recognise the dignit and legitimacy of the explanation models of the subjects educate (parents and children), who are confronted with knowledge, value and ideas proposed, varying the meaning and interpretation base on their experience. Therefore, the problem is not to equip the chil or teenager with a new toolbox, but rather to understand what th toolbox used contains. It is about keeping in mind the principle of pe spective, which concerns the subjective methods of giving a meaning the meaning of each fact, proposition or meeting is related to the per- spective or reference framework based on which it is interpreted […]. Logically, an official educational initiative will cultivate beliefs, skills and feelings in order to transmit and express the ways of interpretation of the social and natural world of the culture that delivers them. Therefore, it follows that an effective education is always in precarious balance, both in culture as a whole and in the groups representing it (Bruner 1997: 26). Thus, the ability of the educator is expressed in educating th children to use the tools they possess, adding new ones based on th experience. To these dimensions it is necessary to add the emotional skill, i. the ability to consciously manage emotions and feelings produce by the educational relationship. The socio-educational work is per meated by very intense emotional experiences, which are not alway easy to decode and manage. Emotions are, at the same time, a bon and a possibility. In fact, on one hand, they often implicitly influenc actions, by governing and guiding them, and on the other hand, aware of them, they are a fundamental resource for dealing with si uations, difficulties and relationships. Emotional intelligence (Gole man 1997), understood as the ability/possibility to know, identif must necessarily look at his/her actions in the social direction, i.e. orienting the educational practices in order to increase the awareness of the youth about being a member of a community that shares rules and culture. This leads to a constant invitation to commitment and personal autonomy, so that children can progressively become self- conscious as members of a community. 3. The skills of the social educator To be with is a proprium of the human being, «man is a social animal», affirmed Aristotle, while Seneca argued that «people are not made to live alone», in order to emphasize how human life is marked in a social sense. Getting into a relationship and improving one’s own abilities is, therefore, a responsibility/opportunity for every individual, but es- pecially for those who play an educational role. Relational skills are embedded in three dimensions: knowing, knowing how to do, knowing how to be. The first dimension refer to knowing, as the interweaving of three levels: scientific, related to the consolidated knowledge and scientific theories held; implicit, connected to beliefs, naive theories, prejudices shared within a culture and which implicitly shape our knowledge; personal, referring to the idea of the world produced by one’s own experience, history, and perception of the self. The second dimension is related to knowing how to do, as the set of skills that out- line the specialist skill of the educator. The third dimension is related to knowing how to be and, therefore, to the ability to listen, empathise and enteropathy (Bertolini, Caronia 2015: 92)5, to the awareness of knowing and feeling, of experimenting emotions (Gaspari 2002: 96).i g g p g ( p ) Finally, the definition of the sense of competence of the social ed- ucator is the integration between the individual and society. The educator TRANSFER OF NON-ACADEMIC SKILLS IN ACADEMIC CONTEXT 407 must necessarily look at his/her actions in the social direction, i. orienting the educational practices in order to increase the awarene of the youth about being a member of a community that shares rule and culture. This leads to a constant invitation to commitment an personal autonomy, so that children can progressively become sel conscious as members of a community. This process is particularl delicate in contexts of discomfort and social deviance, since belong ing is not and inexistent feeling, as one might think, but rather we developed in terms of values, rituals, and behaviours proposed by th devious and illegal culture that individuals, families, the communit the children and adolescents absorb and make their own. As Brune (1997) emphasizes, cultural belonging offers a toolbox, i.e. a set of be liefs, rules, values and visions of the world that outlines those cogn tive patterns upon which knowledge and experience are based. 3. The skills of the social educator This process is particularly delicate in contexts of discomfort and social deviance, since belong- ing is not and inexistent feeling, as one might think, but rather well developed in terms of values, rituals, and behaviours proposed by the devious and illegal culture that individuals, families, the community, the children and adolescents absorb and make their own. As Bruner (1997) emphasizes, cultural belonging offers a toolbox, i.e. a set of be- liefs, rules, values and visions of the world that outlines those cogni- tive patterns upon which knowledge and experience are based. In this perspective, it is fundamental for educators to recognise the dignity and legitimacy of the explanation models of the subjects educated (parents and children), who are confronted with knowledge, values and ideas proposed, varying the meaning and interpretation based on their experience. Therefore, the problem is not to equip the child or teenager with a new toolbox, but rather to understand what the toolbox used contains. It is about keeping in mind the principle of per- spective, which concerns the subjective methods of giving a meaning: the meaning of each fact, proposition or meeting is related to the per- spective or reference framework based on which it is interpreted […]. Logically, an official educational initiative will cultivate beliefs, skills and feelings in order to transmit and express the ways of interpretation of the social and natural world of the culture that delivers them. Therefore, it follows that an effective education is always in precarious balance, both in culture as a whole and in the groups representing it (Bruner 1997: 26). Thus, the ability of the educator is expressed in educating the children to use the tools they possess, adding new ones based on the experience. To these dimensions it is necessary to add the emotional skill, i.e. the ability to consciously manage emotions and feelings produced by the educational relationship. The socio-educational work is per- meated by very intense emotional experiences, which are not always easy to decode and manage. Emotions are, at the same time, a bond and a possibility. In fact, on one hand, they often implicitly influence actions, by governing and guiding them, and on the other hand, if aware of them, they are a fundamental resource for dealing with sit- uations, difficulties and relationships. 3. The skills of the social educator Emotional intelligence (Gole- man 1997), understood as the ability/possibility to know, identify, MARIA LUISA IAVARONE, FAUSTA SABATANO 408 evaluate, and manage one’s emotions, then becomes central to the educational work, representing the basis for rational action, because emotions are driving the thought, and, thus, there is no thinking with- out feeling (Damasio 1995: 9). The development of emotional skills is, thus, a central element of educational professionalism, as the basic condition for being able to put in place an affective relationship be- tween the educator and the educated person, on which the overall effectiveness of the action depends (Rossi 2014). References Bertolini P. 1988, L’esistere pedagogico: ragioni e limiti di una pedagogia come scienza fenomeno logicamente fondata, La Nuova Italia, Firenze.fi Bertolini P. and Caronia L. 2015, Ragazzi difficili. Pedagogia interpretativa e linee di intervento, FrancoAngeli, Milano. Bruner J. 1997, La cultura dell’educazione, Feltrinelli, Milano. Canevaro A. 2013, Scuola inclusiva e mondo più giusto, Erickson, Trento. Damasio A.R. 1995, L’errore di Cartesio. Emozione, ragione e cervello umano, Adelphi, Milano.i De Luca V. and Salvatore D. 2015, La sfida europea. Riforme, crescita e occupazione. FrancoAngeli, Milano. Dunia P. 2017, Competenze e innovazione. Cinque parole per le transizioni dei giovani al lavoro, «Formazione & Cambiamento», <http://www. formazione-cambiamento.it/numeri/2017/5-technology-enhanced- learning-futuro-prossimo/91-gli-articoli/591-competenze-e-innovazione- cinque-parole-chiave-per-le-transizioni-dei-giovani-al-lavoro> (03/18). q p p g ( ) Erdas F.E. 1991, Didattica e formazione. La professionalità docente come progetto, Armando, Roma. Armando, Roma. Freire P. 1968, La pedagogia degli oppressi (tr. it. 2011), EGA, Torino. Freire P. 1968, La pedagogia degli oppressi (tr. it. 2011), EGA, Torino. Freire P. 2004, Pedagogia dell’autonomia. Saperi necessari per la pratica educative, EGA, Torino.i Gaspari P. 2002, Aver cura. Pedagogia speciale e territori di confine, Guerini e Associati, Milano. Gatto F. 2008, Bisogni educativi speciali: criteri per una scuola inclusiva, in M. Baldacci, M. Corsi, Una pedagogia per la scuola, Tecnodid, Napoli, 177-195. Giovannini E. (ed.) 2016, Rapporto ASviS 2016, Gli Obiettivi di Sviluppo Sostenibile. Goleman D. 1997, Intelligenza emotive, Rizzoli, Milano. Iavarone M.L. 2009, Educare al benessere, Bruno Mondadori, Milano. Iavarone M.L. and Iavarone T. 2007, Pedagogia del benessere. Il lavoro educativo in ambito sociosanitario, FrancoAngeli, Milano. Iavarone M.L. and Iavarone T. 2007, Pedagogia del b in ambito sociosanitario, FrancoAngeli, Milano. g Montanari F. and Mizzau L. (eds.) 2016, Modelli di sviluppo territoriale e inclusione sociale, Quaderni della Fondazione Brodolini, 55. g Montanari F. and Mizzau L. (eds.) 2016, Modelli di svilupp sociale, Quaderni della Fondazione Brodolini, 55. Monti L. 2014, Ladri di future, Luiss, Roma. TRANSFER OF NON-ACADEMIC SKILLS IN ACADEMIC CONTEXT * Gaia Gioli, PhD, Post‑doc Researcher, Department of Education and Psychology, University of Florence, Italy. Email: gaia.gioli@unifi.it. 1 See data from the focus groups organized by the Board of Professors of the Master’s Degree in Adult Education, Lifelong Learning, and Pedagogical Sciences of the University of Florence with the Address Committee of the Master’s Degree whose members are the main organizations of the Tuscan social economy in the year 2017-2018. TRANSFER OF NON-ACADEMIC SKILLS IN ACADEMIC CONTEXT 409 Murray R., Grice J.C. and Mulgan G. 2009, Libro Bianco sull’innovazione sociale, <http://www.felicitapubblica.it> (01/2018). p p Pepe D. 2017, Competenze e innovazione. Cinque parole chiave per le transizioni dei giovani al lavoro, «Formazione & cambiamento. Rassegna Trimestrale su teorie e pratiche dell’apprendimento», 5. p pp Pepe D. and Casentini P. 2015, Transizioni verso il lavoro e occupabilità giovanile. Innovazioni, territori e sviluppo, «Osservatorio Isfol», 4, 117-128. pp Rossi B. 2014, Il lavoro educativo. Dieci virtù professionali, Vita e pensiero, Milano. Rossi B. 2014, Il lavoro educativo. Dieci virtù professionali, Vita e pensiero, Milano. Rutter M. 1985, Resilience in the face of adversity: Protective factors and resistance to psychiatric disorder, «The British Journal of Psychiatry», 147, 598-661. Rutter M. 1985, Resilience in the face of adversity: Protective factors and resistance to psychiatric disorder, «The British Journal of Psychiatry», 147, 598-661. Sabatano F. 2011, Crescere ai margini. Educare al cambiamento nell’emergenza sociale, Carocci, Roma. Sabatano F. 2011, Crescere ai margini. Educare al cambiamento nell’emergenza sociale, Carocci, Roma. Sabatano F 2015a Educare per includere in contesti di rischio e marginalità Sabatano F. 2015a, Educare per includere in contesti di rischio e marginalità, «Encyclopaideia», XLIII (19), 21-53. y p ( ) Sabatano F. 2015b, La scelta dell’inclusione. Progettare l’educazione in contesti di disagio sociale, Guerini e Associtati, Milano. Sabatano F. 2015b, La scelta dell’inclusione. Proget disagio sociale, Guerini e Associtati, Milano. Tucci F. 2016, Soft revolution, L’erudita, Roma. Vardisio R. 2017, Verso l’apprendimento ubiquo, reticolare e sinecologico, «Formazione & cambiamento. Rassegna Trimestrale su teorie e pratiche dell’apprendimento», 5. Gaia Gioli (University of Florence)* Abstract: This paper focuses on the impact of employability-oriented modules on the design, planning, and implementation of work transitions. It takes its lead from the PRIN EMP&Co project developed by the University of Florence in 2014-2017, and how its research protocol allows a mapping of the construction of employability during the Master’s Degree Course. Keywords: employability, work transitions, employability-oriented curriculum, PRIN EMP&Co project. 1. Introduction Employers frequently complain that graduates’ standards are not con- sistent with their expectations (Fondazione CRUI 2016: 22), that they lack basic business knowledge and sense of measure in financial issues1. Does this mean that higher education study paths are failing in their mission? At the same time, when talking to higher education teachers, they complain about a lack of knowledge of basic principles and of criti- cal sense and reasoning, abilities that should have already been acquired during college years if not before. g g y One reason is linked to the concept of ‘transitions’ which are always difficult and disconcerting for anyone. In other words, most young grad- uates experiencing their first transition to work… Will have a period of non-competence […] because they will lack ex- plicit and – especially – tacit knowledge of ‘what we do around here’: they will be culturally naïve, reliant on any explicit and formal decla- rations they can find, whereas the reality of communities of practise is one of tacit knowledge, ‘work-arounds’ and local practices. Second […] the knowledge typically rewarded by higher education is quite different from […] expert-like behaviour, that is likely to be more significant in the workplace (Knight, Yorke 2004: 14). 412 GAIA GIOLI This does not mean that higher education alumni lack the intellectual and cultural capital needed for work, but that higher education has been traditionally influenced by a different culture from that predominating in the job market, and it cannot always guarantee the kind of experi- ence, or the social or intellectual capital needed, to acquire the tools to sustain employment and employability. Employability is deeply intertwined with the kind of assistance that higher education can provide students with to prepare their transition into the workplace. For instance, Boffo, Fedeli, Lo Presti, Melacarne and Vianello (2017) have argued that a staunch new relationship between higher education and work can help graduates in their first job since placement is considered the result of an educational process that starts a long time before. It is through traineeships, work-based and work-relat- ed didactics, developed in synergy with higher education, that students can strengthen their employability given that all these practices help the construction of a business-like, professional culture and a knowledge of the job market within the relevant curricula and programmes. 2. 1. Introduction It is in these terms that Lave and Wenger saw learning, as «a relational matter, generated in social living, historically, in social formations whose participants engage with each other as a condition and precondition for existence» (Lave, Wenger 1991: 95) and as a participation process that «can be neither fully internalized as knowledge structures (within individual minds) nor fully externalized as instrumental artefacts or overarching activity structures» (Lave, Wenger 1991: 51). At the basis of learning is always a relationship that influence the learning process and this «is al- ways based on situated negotiation and re-negotiation of meanings in the world. This implies that understanding and experience are in constant interaction – indeed, are mutually constitutive» (Lave, Wenger 1991). y ( g ) Indeed, as clearly explained by Mezirow’s transformative theory, the role of the adult educator is that of a facilitator of metacognition pro- cesses, or of a facilitator who can enhance learners’ ability to reflect on themselves, on cognitive processes, and on social, cultural, relational processes and assumptions. The relationship that the educator is able to create and maintain with the adult learner is as a mentor/friend where it is «the mentor trying to help the friend decide how to deal with a sig- nificant life problem that the friend may not have yet clearly identified as the source of his or her dilemma» (Mezirow 1991: 223). In this way, educators help adult learners identify and critically reflect on the episte- mological, social, and psychological assumptions on which their beliefs, attitudes, opinions, and emotional reactions have been built. These con- stitute the ‘meaning schemes’. Moreover, educators can help learners to transform their meaning perspectives (sets of related meaning schemes), to test them through participation in reflective dialogue (Mezirow 1991). «Learning may be defined as the process of making a new or revised in- terpretation of the meaning of an experience, which guides subsequent understanding, appreciation and action» (Mezirow 1990: 1). In other words, the transformation of the adult is realized, as Mezirow claimed, in correspondence with deep reflection linked to moral devel- opment. And at the right moment, when the learners’ capabilities allow them to function as self-directed learners, then the adult educators’ job has been properly done, since self-directed learning is among the most relevant skills that can be learnt (Mezirow 1985). 1. Introduction Higher Education, the Zone of Proximal Development, and the engagement of students with the world of work The contexts of higher education and of work, as mentioned before, can involve different ways of learning and teaching and result in vari- ous competencies, such as the transfer of knowledge and skills from one context to another: the so-called ‘skill of transfer’ (Scribner, Cole 1973; Bridges 1993). g ) Several scientists have noticed the importance of educational pro- gramme design in the development of the Zone of Proximal Develop- ment (ZPD) (Vygotsky 1978) and of cultural practices (Leontiev 1978). Indeed, Vygotsky defined the Zone of Proximal Development as: «the distance between the actual development level as determined by inde- pendent problem-solving and the level of potential development as de- termined through problem-solving under adult guidance as collaboration with more able peers» (Vygotsky 1978: 85). In other words, the concept highlights how a pedagogical structure and pedagogical guidance help learners move beyond their ZPD and im- prove their competencies. This outlook suggests two different insights. h h d l f l h h h d In other words, the concept highlights how a pedagogical structure and pedagogical guidance help learners move beyond their ZPD and im- prove their competencies. This outlook suggests two different insights. On the one hand, learning in a formal context, such as higher educa- tion, can be improved if the skills and knowledge are embedded within a social context that is essential to learning. For example, if students solve real-world problems, they will be accomplished in a way of thinking that p p ggf g On the one hand, learning in a formal context, such as higher educa- tion, can be improved if the skills and knowledge are embedded within a social context that is essential to learning. For example, if students solve real-world problems, they will be accomplished in a way of thinking that will enable them to understand the cultural practices that can occur in a specific context, i.e., the world of work. 413 THE PRIN EMP&CO. PROJECT On the other, this outlook marks the importance of the involvement of adult educators in instructing young people, in the development of their ZPD. Those who guide and support learners can participate in and influ- ence their process of learning through a dynamic educational relationship. 3. The engagement of Higher Education students with the world of work 3. The engagement of Higher Education students with the world of work In broad terms, there are a lot of ways in which higher education can support students’ learning. For example, if we think about Knight & Yorke’s approach (2004: 199), we can plan to work on: 1) The students’ approach to learning in general, 2) The students’ approach to studying a specific task, 3) The learning environment, i.e., if it is filled with possi- bilities for learning, 4) The internal consistency of the curriculum, i.e., its design.i g Indeed, learning is not only developed following specific instructions to acquire knowledge, but can be fostered in traditional higher-education and non-formal settings, although, as Goodyear remarked: we should recognize that we cannot influence directly the learner’s cog- nitive activity […] the best we can do is help set up some organizational forms or structures that are likely to be conductive to the formation and wellbeing of convivial learning relationships […] Thirdly, we must rec- ognize that the learner has freedom to reconfigure or customize their learnplace (Goodyear 2002: 66). we should recognize that we cannot influence directly the learner’s cog- nitive activity […] the best we can do is help set up some organizational forms or structures that are likely to be conductive to the formation and wellbeing of convivial learning relationships […] Thirdly, we must rec- ognize that the learner has freedom to reconfigure or customize their learnplace (Goodyear 2002: 66). The success of learning can be even greater if the subject of study is taken as a site for a more generic kind of learning, a learning for life, that takes place within a study curriculum. This opens up the possibility of reflecting on employability (Yorke 2006: 3) which can be enhanced through a wide variety of facilitating practices, such as: g y g p • Employability through the whole curriculum; • Employability in the core curriculum; • Work-based or work-related learning incorporated in the curriculum; • Employability-related module(s) within the curriculum; and • Work-based or work-related learning incorporated in the curriculum; • Employability-related module(s) within the curriculum; and • Work-based or work-related learning in parallel with the curriculum (Yorke Knight 2004: 14 20) p y y ( ) • Work-based or work-related learning in parallel with the curriculum (Yorke, Knight 2004: 14-20). • Work-based or work-related learning in parallel with the curriculum (Yorke, Knight 2004: 14-20). 2 Translation from the original Italian version made by the author of the paper. 1. Introduction Indeed, we can affirm that the role of adult educators, or facilitators, runs out when they are able to support adults, to stimulate them in their self-education, in their will to develop their capacity to reveal the prejudices and preconceptions of the social, moral, and economic orders that affect individual percep- tion with respect to need. 414 GAIA GIOLI 3. The engagement of Higher Education students with the world of work These practices can be adopted in the study plan as embedded place- ment models that can lead to learning, enrich students’ CVs, and de- velop their employability. Indeed, these practices can foster students’ learning for, at and from work and thus make sure that employability is caught, rather than taught. Furthermore, they all allow internal processes of self-education that can happen intentionally when encouraged by fa- cilitators or adult educators, as experiences that «combine the acquisi- tion of knowledge, the construction of sense, and the transformation of the Self, and that are developed within social practices throughout life»2 (Biasin 2009: 70). 415 THE PRIN EMP&CO. PROJECT According to Harvey (1999: 2) embedded placement activities can: • take the form of one optional or compulsory activity; • be organized as single block placements (thick sandwich) or multiple block placements (thin sandwich); p • last one year, or one semester, or shorter periods. These forms of contact with the job market should be the common response that higher education and academic programmes offer in the face of claims that there are skills shortages and gaps, that students are not prepared for the world of work and are not equipped with the ‘right’ skills. Indeed, these could bring the subjects taught at a higher education level closer to what employers want now and will need in one or two years’ time, when Master’s Degree students show up on the job market as new graduates. 3. The engagement of Higher Education students with the world of work g Recent research has suggested that the scenario students will have to face as new graduates in the very near future will be characterized by certain major phenomena: j p • mismatching of competences still widespread g p p • transition to employment lasting at least one year (Boffo, Fedeli, Lo P ti M l Vi ll 2017) • transition to employment lasting at least one year (Boffo, Fedeli, Lo Presti, Melacarne, Vianello 2017)l Presti, Melacarne, Vianello 2017) • individuals expected to be more responsive and flexible regarding technological change (Institute for the Future 2011) • higher education institutions considered more accountable than in the past in terms of contributions and efforts to the training of individu- als and the spread of knowledge among future generations of work- ers, since research into the processes of teaching and learning have demonstrated their impact on employment and employability (Boffo, Fedeli, Lo Presti, Melacarne, Vianello 2017) even though criteria to evaluate good quality at a global level are missing. Difficulties in training graduates able to find consistent jobs in the short term have stimulated Italian universities to reflect, research, and work on employability and work-related activities, including projects, case studies, unconventional dissertations, and programmes that can foster a closer link between employers, higher education, and students. These are not only projects in the field of social sciences, but in the educational field. The latter are those that interest us. 4. The PRIN EMP&Co project and the EMAE curriculum The PRIN project was conducted between 2014 and 2017 at the Uni- versities of Padua, Florence, Naples Parthenope, and Siena, with the aim of fostering new strategies, methods, practices, and theoretical constructs 416 GAIA GIOLI of higher education that could support students and young adults during an employment emergency, as a response to the socio-economic crisis. The project worked on designing higher education modernization strate- gies to support students’ employability and learning processes required by the job market (Boffo, Fedeli, Lo Presti, Melacarne, Vianello 2017: XI). of higher education that could support students and young adults during an employment emergency, as a response to the socio-economic crisis. The project worked on designing higher education modernization strate- gies to support students’ employability and learning processes required by the job market (Boffo, Fedeli, Lo Presti, Melacarne, Vianello 2017: XI). jf The part of the research conducted at Florence University focused on the main research questions: How do young people look for a job? How do they approach the transition to work? How do they build their em- ployability during their university studies? The research group limited the field of investigation to a smaller group of students in comparison with the general project: students enrolled in the Master’s Degree study course in Adult Education, Lifelong Learning, and Pedagogical Scienc- es (LM57-85) of the University of Florence, aged between 23 and 29. y g The research yielded two main considerations.i y g The research yielded two main considerations.i The first refers to the idea that the construction of a professional ca- reer should be thought about, reflected on, and prepared during the uni- versity course, and thus that employability itself relates to the years that precede leaving higher education. The idea therefore is that to intervene on employability means working on learning, on the curriculum and the competences that students will have acquired by the end of their study path, which will support them in their professional path. The second relates to the crucial importance of care for subjects and their educational processes. Care should become not only the founda- tion to construct formative pathways or career guidance services, but also the key to reading transitions as care pathways that start within univer- sity courses. Studies have demonstrated that courses at a higher educa- tion level are still too theoretical and have little to do with the world of work (Yorke 2006; Wright 2013). 3 The University of Florence research group was coordinated by Prof. Vanna Boffo (Head of the research team) and included as members Prof. Paolo Federighi, Prof. Giovanna Del Gobbo, Prof. Francesca Torlone, Dr Gaia Gioli. 4 The European SALM research project was conducted to study the issue of the em- ployability of young students aged between 16 and 24 in the social and tourism sectors. The University of Florence team members were Prof. Paolo Federighi (Head of the research team), Prof. Vanna Boffo, Prof. Francesca Torlone (Boffo, Federighi, Torlone 2015). 3 The University of Florence research group was coordinated by Prof. Vanna Boffo (Head of the research team) and included as members Prof. Paolo Federighi, Prof. Giovanna Del Gobbo, Prof. Francesca Torlone, Dr Gaia Gioli. 4 The European SALM research project was conducted to study the issue of the em- ployability of young students aged between 16 and 24 in the social and tourism sectors. The University of Florence team members were Prof. Paolo Federighi (Head of the research team), Prof. Vanna Boffo, Prof. Francesca Torlone (Boffo, Federighi, Torlone 2015). 4. The PRIN EMP&Co project and the EMAE curriculum Academia is not heading in the direc- tion suggested by Dewey in his The School and Society (1899) and Experi- ence and Education (1938), i.e. the connection between experience, work, and education, even though this should be the basis of every educational study course. y The distance between what Academia should do and what it is ac- tually doing is so great that it led the Florence research team3 to work in this direction, following the valuable results of the SALM4 research performed in 2013-2014 within the study course (Boffo 2015: 147-168). 417 THE PRIN EMP&CO. PROJECT The PRIN project has developed a pedagogical instrument that puts employability in a close connection with preparation for the transition to work, with the development of skills and tools required to look for work on the job market. The qualitative research performed adopted the case-study strate- gy (Mortari 2007), while focus groups and semi-structured interviews were the survey techniques chosen and realized following a longitudi- nal approach. pp The data collected involved 10 groups, 110 interviews, and 52 gradu- ates interviewed between June 2014 and November 2016: 39 from Flor- ence University, 2 from Padua University, 9 from the Julius Maximilian University of Wuerzburg, since all these Universities share the same EMAE Master’s Degree curriculum in Adult Education and an interna- tional comparison was therefore possible. A rigid protocol was followed by every researcher, regardless of the role played: interviewer or observer, and the venue, Florence, Padua, or Wuerzburg. Indeed, every interviewee had the same interviewer for the entire longitudinal process, and each interviewer followed a specific re- cursive grid for every interview and focus group. The grid was organized in four main areas aiming at investigating: 1) Volitions, 2) Competences, 3) Channels, 4) Expectations (Boffo, Gioli, Del Gobbo, Torlone 2017: 165-166) in order to map the process of constructing employability in university curricula. Indeed, this was adopted during the focus groups to help students raise their self-awareness and facilitate reflection before the interview, while the interview was conceived as a moment of edu- cational evaluation of the employability process, based on reflection, in- terpretation, and self-education.il p The four fields of reflection set out to identify the youth trends to- wards work, the working directions desired by the graduates, and thus the families and professional figures considered consistent with their volitions. 4. The PRIN EMP&Co project and the EMAE curriculum The life perspectives and aspirations area investigated the ac- tions performed by graduates to plan their life and professional projects, while the reflection area focused on channels that helped the mapping of implicit strategies adopted to sustain transitions, namely: channels, net- works, services, tools, educational and training activities in which the students participated to ease the transition. To observe all these elements means to understand the choices made by young people and the processes that lie behind them: to head in one work direction rather than another because of deep knowledge of one specific economic sector rather than another. Between April 2015 and April 2016, the student-researchers who took part in the SALM project in the academic year 2013-14 discussed their Master’s thesis and joined the PRIN project as graduates. They were asked to answer the grid questions included in the PRIN Project research pro- 418 GAIA GIOLI tocol and the results were interesting, especially since some of the gradu- ates interviewed remarked on the importance of their participation in the SALM project for their ensuing job search and transition to work (Tab. 1). Table 1 – Characteristics of the subjects of the PRIN EMP&Co. research project. [Source: author’s own] Sample Sample size Dissertation year Students’ participation in employability-oriented didactic activities within “Foundations of Adult and Continuing Education” and “Research Methodol- ogy: Basic and Applied to Education and Training” Sample no. 1 6 June 2014 No Sample no. 2 3 November 2014 No Sample no. 3 3 February 2015 No Sample no. 4 10 April 2015 Yes - 1 Sample no. 5 5 June 2015 Yes - 2 Sample no. 6 3 November 2015 Yes - 2 Sample no. 7 1 February 2015 Yes - 1 Sample no. 8 8 April 2015 Yes - 6 Indeed, within the SALM project, they were asked to conduct in- terviews and case studies with the most significant and relevant figures of the social sector, such as Chairs/employers/HR managers/Managing Directors, within the Foundations of Adult and Continuing Education course held by Prof. Paolo Federighi and Research Methodology: Basic and Applied to Education and Training held by Prof. Vanna Boffo in the first year of the Master’s Degree in Adult Education, Pedagogical Sciences and Lifelong Learning in the academic year 2013-14. 4. The PRIN EMP&Co project and the EMAE curriculum The teaching and learning approach followed in the classes was de- signed to promote the development of employability, consistent with the adult education profession and practice in the Tuscan social economy. Students were guided in their understanding of the main professional profiles and related titles, skills, and competences, along with the main organizational models and employment contracts adopted within social economy organizations, to help them identify the main targets to be ad- dressed at the end of their study path. The main results of the interviews are shown in Educational Jobs: Youth and Employability in the Social Economy (Boffo, Federighi, Torlone, 2015: 147-168) but what is interesting to highlight here is the possibility given to students to become the main actors in directly and actively acquir- ing knowledge of the job market and becoming wholly responsible for the investigation. Indeed, they were asked to: 1) identify the actors to interview, 2) contact them by email and telephone, 3) interview them 419 THE PRIN EMP&CO. PROJECT to obtain information regarding the roles and functions they would find in their organizations, 4) transcribe the interview, 5) analyse the inter- view results, 6) interpret and 7) evaluate the transcript of the case studies. p p Their actions took place in a context that enhanced their authentic learning, i.e. learning from real-life situations, based on the assumption that they would benefit more from authentic and problem-based learn- ing, rather than from traditional classes, and could therefore become more employable in the long term. Moreover, the student-centred ap- proach encouraged their active involvement through an organization of work based on small groups, a little intervention by the teacher-facilita- tor, and a major focus on students’ experiences, analyses, interventions and resolutions of problems and critical circumstances. p From the interview analysis, the PRIN EMP&Co researchers were able to identify the positive impact of the methodological choice made: on the one hand, the active involvement of the students within the SALM research project allowed them to acquire new tools, knowledge and skills; on the other, the research protocol helped PRIN graduates to perform a critical self-analysis on the channels used for the active job search and the professional career and personal life, and understand how the work done in previous years had helped them to plan and design the transition. 4. The PRIN EMP&Co project and the EMAE curriculum p y p p g With regard to the part of the interview dedicated to evaluating the channels, the students’ answers showed that the two employability-ori- ented modules, Foundations of Adult and Continuing Education, and Research Methodology: Basic and Applied to Education and Training, allowed them to learn a great deal, although not everybody remarked this (Tab. 2). Table 2 – Percentage distribution of students’ answers regarding the channels and usefulness of employability-oriented modules within the study curriculum. [Source: author’s own] Sample Sample size Active participation in employability-ori- ented didactic activi- ties % Students’ awareness on learning outcomes from employability-oriented didactic activities in Foun- dations of Adult and Continuing Education and Research Methodology: Basic and Applied to Edu- cation and Training Sample no. 4 10 1 100% Sample no. 5 5 2 100% Sample no. 6 3 2 0% Sample no. 7 1 1 0% Sample no. 8 8 6 33% In every case, the interviews allowed students to look back at their study path and the learning activities undertaken: there was a clear sense of usefulness and wellbeing deriving from participation in the educa- 420 GAIA GIOLI tional experience, and this suggests that the graduates were highly ap- preciative, perhaps because they had had time to reflect on the past. Or perhaps because, when facing the real work world, they were obliged to fathom and find all the channels, networks, and tools that would be use- ful to accelerate the transition process from university to their first job. Reflection can be considered an essential element of work-based learn- ing and employability-oriented learning activities.if tional experience, and this suggests that the graduates were highly ap- preciative, perhaps because they had had time to reflect on the past. Or perhaps because, when facing the real work world, they were obliged to fathom and find all the channels, networks, and tools that would be use- ful to accelerate the transition process from university to their first job. Reflection can be considered an essential element of work-based learn- ing and employability-oriented learning activities.if g p y y g The PRIN researchers identified several differentiated paths based on the commitment to memory of the student-researchers involved in em- ployability-oriented didactic activities within study courses. 4. The PRIN EMP&Co project and the EMAE curriculum p y y y From the comparison, what emerged clearly is that the presence of a clear professional identity developed during the study years thanks to personal reflection on the subjects and didactic approach, work expe- rience, and awareness in relation to the channels possessed, can deeply impact the result of transitions (Tab. 3). Table 3 – Impact of employability-oriented modules for the strengthening of professional identity. [Source: author’s own] Graduate – The initial idea, before I enrolled in the Bachelor’s Degree in Philosophy, was to take my BSc and then enroll in the Master’s; then in view of the changed situation … I was never going to enrol in the Master’s Degree Course in Philosophy because of the quality of the path. I looked around and I looked at what was happening inside there [De- partment of Education and Psychology]. I met Prof. Boffo, if I’m not mistaken, in Oc- tober… no, in May 2012, and she pointed me towards the supplementary examinations that I needed to do to pass from Philosophy to the Adult Education Degree Course, then I attended Professor Federighi’s class in 2012 and then… I just took my Bachelor’s De- gree, and enrolled in the Master’s programme without any obligation. I i Y id h i i I ld lik i i h fi ld f y y p y g g Graduate – Yes, I thought of it as being bent on teaching or, in any case, for a following placement in a company… or for the integration between a Bachelor’s and a post-grad certificate; this was my initial idea, in short.i i y […] Interviewer- Then, if we reconstruct the professional profile you built on this path, your initial idea was to become a teacher, at least at the very beginning… i y y g g Graduate – Yes, but my work experience… first in Rome and then in Brussels, in the l year, had a significant impact on changing my perspective… Graduate – Yes, but my work experience… first in Rome and then in Brussels, in the last year, had a significant impact on changing my perspective… Graduate Yes, but my work experience… first in Rome and year, had a significant impact on changing my perspective… y gi p g g y p p Interviewer – Then your professional identity started modifying itself… Graduate – Of course. Table 3 – Impact of employability-oriented modules for the strengthening of professional identity. [Source: author’s own] Graduate – The initial idea, before I enrolled in the Bachelor’s Degree in Philosophy, was to take my BSc and then enroll in the Master’s; then in view of the changed situation … I was never going to enrol in the Master’s Degree Course in Philosophy because of the quality of the path. I looked around and I looked at what was happening inside there [De- partment of Education and Psychology]. I met Prof. Boffo, if I’m not mistaken, in Oc- tober… no, in May 2012, and she pointed me towards the supplementary examinations that I needed to do to pass from Philosophy to the Adult Education Degree Course, then I attended Professor Federighi’s class in 2012 and then… I just took my Bachelor’s De- gree, and enrolled in the Master’s programme without any obligation. Interviewer – You said that at a certain point… «I would like to continue in the field of education» Why? Did you think of Philosophy as being bent on teaching? Graduate – Yes, I thought of it as being bent on teaching or, in any case, for a following placement in a company… or for the integration between a Bachelor’s and a post-grad certificate; this was my initial idea, in short. […] Interviewer- Then, if we reconstruct the professional profile you built on this path, Graduate – The initial idea, before I enrolled in the Bachelor’s Degree in Philosophy, was to take my BSc and then enroll in the Master’s; then in view of the changed situation … I was never going to enrol in the Master’s Degree Course in Philosophy because of the quality of the path. I looked around and I looked at what was happening inside there [De- partment of Education and Psychology]. I met Prof. Boffo, if I’m not mistaken, in Oc- tober… no, in May 2012, and she pointed me towards the supplementary examinations that I needed to do to pass from Philosophy to the Adult Education Degree Course, then I attended Professor Federighi’s class in 2012 and then… I just took my Bachelor’s De- gree, and enrolled in the Master’s programme without any obligation. I i Y id h i i I ld lik i i h fi ld f 4. The PRIN EMP&Co project and the EMAE curriculum Interviewer – You, then the world of cooperatives… cooperation in the social economy… did you know about this or not? y Graduate – No, no. No, at the level of «I really know companies that operate in Florence and the professional profile that is being sought by these associations» no, absolutely not. It was really useful.f y Graduate – No, no. No, at the level of «I really know companies that operate in Florence and the professional profile that is being sought by these associations» no, absolutely not. It was really useful.f y Interviewer- Because you then gave your attention to this type of job offer, didn’t you? Graduate- Yes, yes. Graduate- Yes, yes. At the same time the reflexivity can be guided by the University, which can assume a key role when offering career services inspired by an empirical research project. 4. The PRIN EMP&Co project and the EMAE curriculum Interviewer – How do you see things now? How do you… d Interviewer – How do you see things now? How do you… G d lik f i l i Graduate – But I like to say, I can see… my professional identity as a whole. I’ve worked in the field of politics, then in the structuring of policies and negotiation, in short, in ev- erything that happens behind politics. In fact, I chose to shift my attention to different studies that focused more on the precise dimension of training policies. I began to re- model my future professional interests when I started attending Professor Federighi’s class, when he spoke of training policies, the relationship between educational and working sys- tems and the whole framework of... policies and strategies and measures, in other words. Equally supportive, the participation in empirical work done within a specific module/project designed and carried out or planned within a 421 THE PRIN EMP&CO. PROJECT research project of larger dimensions, such as a European project. This is especially useful for knowledge of the geographical and economic con- text where graduates live (Tab. 4). Table 4 – Impact of employability-oriented modules for the acquisition of new knowledge, channels to support active job searches. [Source: author’s own] Interviewer – And who assisted you, if there was someone who helped you… and if there was no one, what did you do to search for recipients, businesses, communities, services to apply to? pp y Graduate – In Florence I had done… I had participated in a project during the course held by Prof. Boffo and Prof. Federighi. We’d done a project that allowed us to get to know all the situations at the cooperatives that operate in Florence and then I already had a list… no, I didn’t only have a list, I also had a description… rviewer – Because this was a European project… Graduate – Yes, exactly. Yes, yes. And in fact, that was very useful, despite the fact that it was 18 credits when I had to have 6, but it was really very useful. Actually, it was very useful from the beginning, because this opportunity we were given, we received it only in that module, then… Interviewer – You, then the world of cooperatives… cooperation in the social economy… did you know about this or not? 5. Final remarks 5. Final remarks All over the world, young people contribute to the development of society in many ways: as active workers, entrepreneurs, active citizens, and agents of change. Notwithstanding the fact that their potential is of- ten not fully realized because the jobs they are employed in do not match their qualifications and aspirations. As a result, many young people face elevated levels of personal and economic uncertainty (ILO 2017: V). p y ( ) Ever since high-level skills became the basis for being competitive in a global environment, the role of higher education has taken on a fun- damental role. Higher education is critical in this effort, since it not only helps young people acquire the desired values, technical and soft skills that make workers attractive in the job market, but is considered the en- gine of innovation, entrepreneurship (Almalaurea 2017: 30), and produc- tivity. Indeed, it realizes its full potential when it can change flexibly to job market needs through a set of tools and methods (curricula, didactic 422 GAIA GIOLI approach, etc.) that can be adopted to allow students to become employ- able and responsive to the economy (Almalaurea 2015). It can thus be a key driver for both individual and societal growth. Indeed, when con- sidering the transition from higher education to the world of work, we cannot help considering that a difficult transition has long-lasting con- sequences not only on young graduates but on the whole of society, and the future of work itself. This has led us to initiate a link between the higher education sys- tem, curricula, and graduates’ personal attributes, hence their employ- ability, employment and work, i.e. their transition into the working life, and businesses. What the University of Florence has done within the SALM and PRIN projects, especially in reference to employability-oriented and work-re- lated learning − where ‘work-related’ refers to activities different from pure academic subjects (Knight, Yorke 2004: 104), goes in this direction. f p j ( g ) g The research offers a deep insight into the university world, thanks also to the methodological approach adopted that allows us to interpret the lives of graduates, their desires and thoughts, their perceptions about the study curriculum. Above all, they were able to understand, from a longitudinal perspective, the professed and objective impact of the efforts made to modernize the Master’s Degree in previous years. 5) every programme and every teaching moment being devoted to ex- plaining to students – and teaching staff and professors – what is meant by employability and its importance in the transition perspective. 5) every programme and every teaching moment being devoted to ex- plaining to students – and teaching staff and professors – what is meant by employability and its importance in the transition perspective. 5. Final remarks Despite the many positive elements associated with the innovative methodological and didactic approach adopted, the implementation of an employability-oriented curriculum is still viewed as a challenge by many institutions and researchers. For instance, it entails significant ef- fort on the part of academic professors and staff supporting the students, who are asked to play a role that is quite different from that of a tutor or subject expert, a specific kind of training, plus major involvement by employers, academic staff, and tutors, and so on. p yf The initiatives described were undoubtedly excellent, yet they could be perceived as isolated cases or islands, should the quality of the main- stream curricula be perceived at a different level by the students. More- over, excellent initiatives often reach only those who can grasp them and who choose to be reached by them. It would be important to champion a similar path at every university, although this would mean: 1) at an institutional level, new modules and programmes being pro- posed with a specific effort on developing students’ employability; 1) at an institutional level, new modules and programmes being pro- posed with a specific effort on developing students’ employability; 2) at an institutional level, employability becoming an issue and concern for every work unit, i.e. educational research group, career service, and administrative unit, plus every academic department and role; p y p 3) employability being a part of every programme presentation; 4) at a departmental level, employability being the main constituent el- ement of every handbook, website, and assessment criterion; 423 THE PRIN EMP&CO. PROJECT 5) every programme and every teaching moment being devoted to ex- plaining to students – and teaching staff and professors – what is meant by employability and its importance in the transition perspective. References Almalaurea 2015, XVII Indagine. Condizione occupazionale dei Laureati, <http:// www.almalaurea.it/universita/occupazione/occupazione13> (01/2018). Almalaurea 2017, XIX Indagine. Condizione occupazionale dei Laureati, <http:// www.almalaurea.it/universita/occupazione/occupazione15> (01/2018). Biasin C. 2009, Che cosa è l’autoformazione, Carocci, Roma. Blackwell A., Bowes L., Harvey L., Hesketh A. and Knight P. T. 2001, Transforming work experience in higher education, «British Educational Research Journal», XXVI (3), 269-285.f Boffo V. 2015, Employability for the social economy: the role of higher education, in Boffo V., Federighi P. and Torlone F. 2015, Educational Jobs: Youth and Employability in the Social Economy. 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Lave J. and Wenger E. 1991, Situated Learning, Cambridge University Press, Cambridge. 424 GAIA GIOLI Leontiev A.N. 1978, Activity, Consciuousness and Personality, Englewood Cliff, New York. Mezirow J. 1985, A critical theory of self-directed learning, in Brooksfield S. Cristina Lisimberti (Catholic University of the Sacred Heart)* Abstract: Profound processes of change are affecting doctorates all over the world, above all, to provide broader employment prospects. However, the link between the transformations of a doctorate and employability is complex, and entails re-thinking formative pathways for doctorates by focusing on the profes- sional identity of a PhD. Keywords: doctorate; employability; education, professional identity. * Cristina Lisimberti, Post-doc Researcher, Postdoc, Catholic University of the Sacred Heart, Milan, Italy. Email: cristina.lisimberti@unicatt.it. References (ed.) Self-Directed Learning: From Theory to Practice, New Directions for Further education, 25, Jossey-Bass, San Francisco.l Mezirow J. 1990, Fostering critical reflection in adulthood. A guide to transformative and emancipator learning. Jossey-Bass, San Francisco, <https://goo.gl/ PRFyTW> (01/2018). y Mezirow J. 1991, Transformative dimensions of adult learning, Jossey-Bass, San Francisco. Mortari L. 2007, Cultura della ricerca e pedagogia. Prospettive epistemologiche, Bruno Mondadori, Milano. Scribner S. and Cole M. 1973, Cognitive consequences of formal and informal learning, «Science», 82, 553-559. g Vygotsky L.S. 1978, Mind in Society, Cambridge University Press, Cambridge. Yorke M. 2006, Employability in Higher Education: What it is – What it is not, ESECT and The Higher Education Academy, York, <https://goo. gl/3Vi7gH> (01/2018). Yorke M. and Knight P.T. 2004 (reissued 2006), Embedding Employability into the Curriculum, The Higher Education Academy, York, <https://goo.gl/ GsrZ8U> (01/2018). DOCTORATES AND EMPLOYABILITY: NEW PERSPECTIVES FOR DOCTORAL EDUCATION Cristina Lisimberti (Catholic University of the Sacred Heart)* 1. Introduction Despite the centrality of the university in the development of the knowledge society, the focus of pedagogical research for this field is only recent in Italy (Gemma 2006; Orefice, Cunti 2009) and requires more in-depth study. This contribution raises the issue of employability in re- lation to doctorates in their interconnection with professional identity and educational perspectives. Moreover, it concentrates on the current Italian context, from solicitations coming from supranational organiza- tions and from international trends and experiences. In fact, given the complexity of the various dimensions at stake, it is necessary to consider the issues associated with higher education from a ‘glocal’ perspective, where overall scenarios are inseparably interrelated with the peculiari- ties and specificities of national and local contexts (Altbach 2004). Spe- cial attention will be given to formative pathways that can be classed under the ERC Social Science and Humanities area which, even if includ- ed in a common national and international panorama, have peculiarities that nevertheless warrant closer examination, such as: the high number of subjects already working by the time they enter their doctorate pro- gramme (many as state employees) and the scarcity of additional employ- ment prospects with respect to academic careers for those who intend to become involved in research on a professional level (Argentin, Balla- rino, Colombo 2012; ISTAT 2015). The link between the transforma- tions of a doctorate and employability is complex. If, in fact, failure to employ doctorate holders in the university sector at a worldwide level is one of the main incentives that have led to the modification of forma- tive pathways, on the other hand, the employment prospects are multi- 426 CRISTINA LISIMBERTI faceted and not always linear, at least as far as access to academic careers are concerned in Italy. If, therefore, employment opportunities are an indispensable element for reflection on employability, in our opinion, the approach to the issue needs to be broader and more comprehensive, and therefore include formative pathways and, more specifically, the issue of professionalism and the identity of doctorate holders (hereinafter PhDs). 2. PhDs in Italy In recent years, Italy has begun to implement the indications ar- riving from supranational organizations (Ferrara 2015). In fact, it has rapidly passed from a traditional doctorate model, based on the real- ization of a research project under the virtually individual supervision of an academic tutor, to more complex formative pathways, including specialized training sessions (Orefice, Del Gobbo 2011) aimed at ma- turing «the skills required to carry out high-qualification research ac- tivities in public and private entities, as well as qualifying them for the liberal professions, contributing to the creation of the European Higher Education Area and the European Research Area» (Art. 1, Par. 3, MD 45/2013). The benchmark scenario is, however, constantly evolving, and does not always seem to match one single systematic vision (CUN 2017). The most recent instances derive from the implementation notes of the National Research Plan 2015-2020 on innovative doctorates (Prot. 1059, 31/08/2016) which, on the basis of the solicitations already con- tained in the PIDT, provide for the possibility of establishing, also as reciprocal combinations and integrations: Intersectoral (or Industrial) Doctorates, Interdisciplinary Doctorates, and International Doctor- ates, within a framework in which several of the principles indicated by the European Union have been strengthened in a particular manner. On the one hand, these innovative formative pathways seem to offer more specific training proposals, on the other − at least in some cases − they may prove particularly significant for those who do not under- take an academic career precisely because of the greater openness and links with non-academic situations. This approach is consistent with the current working conditions of PhDs in Italy, which it seems use- ful to briefl discuss − they may prove particularly significant for those who do not under- take an academic career precisely because of the greater openness and links with non-academic situations. This approach is consistent with the current working conditions of PhDs in Italy, which it seems use- ful to briefly discuss.i − they may prove particularly significant for those who do not under- take an academic career precisely because of the greater openness and links with non-academic situations. NEW PERSPECTIVES FOR DOCTORAL EDUCATION 427 subjects involved: Research Excellence, Attractive Institutional Envi- ronment, Interdisciplinary Research Options, Exposure to Industry and other relevant employment sectors, International Networking, Transfer- able Skills Training, Quality Assurance. 2. Doctorates in the international Higher Education scenario 2. Doctorates in the international Higher Education scenario To reflect on the issue, we must first consider the vast transforma- tions that higher education has undergone on a global level over the past decade (Altbach, Reisberg, Rumbley 2009) and, more specifically, the profound processes of change affecting PhDs (Nerad 2006). As already mentioned, failure to provide academic employment prospects to PhDs has given a significant impetus worldwide to the transformation of doc- toral formative pathways. These processes, which have been in place for several decades in many Western countries (Auriol, Misu, Freeman 2013), have led to the issue of employment prospects for doctoral students, both in the field of scientific reflection and on the level of national and supra- national policies and strategies.i It is first of all useful to remember the consolidated experience of English-speaking countries where, besides ‘traditional’ doctorates, there are ‘professional’ ones (Professional doctorates) (Neuman 2005) with dis- tinct, sometimes even opposing, characteristics in their outgoing profiles (Taylor 2007). This trend is confirmed by the current proliferation of unique pathways: Kehm (Bao, Kehm, Ma 2016) has, for example, iden- tified nine distinct types in European countries. i yp p In Europe, these transformations (Kehm 2010, 2015) have provided a significant impetus, also in the wake of the efforts of supranational or- ganizations. The Bologna Process in particular had such a decisive role that it was defined as a ‘collector of interest’ on employability (Sin, Neave 2016: 1448). On an institutional level, the European Union has urged universities to «ensure doctoral programmes […] promote interdisciplin- ary education and the development of transferable skills, thus responding to the needs of a vaster job market» (Bergen Conference 2005). The im- portance of professional integration has also been reiterated in numerous subsequent conferences, acting as a stimulus for reforms undertaken in various countries that have joined the European Higher Education Ar- ea (EHEA). Within the framework of the reflection on the moderniza- tion of Higher Education, the so-called Principles for Innovative Doctoral Training – PIDT (European Commission 2011) were also defined, i.e., the strategic elements on which to redefine doctoral training in order to ensure professionalism and a profitable professional integration of the NEW PERSPECTIVES FOR DOCTORAL EDUCATION 2. PhDs in Italy This approach is consistent with the current working conditions of PhDs in Italy, which it seems use- ful to briefly discuss.i In Italy, the employment prospects of PhDs are first-rate (91.5% of doctorate holders find work four years after graduation), confirming the permanence of a competitive advantage associated with the qualification, while improving job positions (23.2%) or income (17.9%) affect only a modest part of those already employed before beginning their doctorate (ISTAT 2015). As far as an academic career is concerned, even though almost all (around 98%) of those who follow such formative pathways declare that they aspire to this (CNVSU 2010), only a handful go on to obtain a permanent position at a university (Tiraboschi 2015). Consid- 428 CRISTINA LISIMBERTI ering that only 6.5% of the research grant holders will be permanently hired by a university, after a lengthy training, it is easy to understand why the rate of PhDs employed in the university sector is extremely limited (ADI 2016). The situation of students enrolled in doctoral programmes in Italy is, therefore, characterized by a significant share (30-40%) of al- ready employed individuals who will return to their habitual job posi- tion (ISTAT 2015). At the same time, only a residual share of subjects will ever enter the academic ranks. Reflecting on the levels of employment achieved (and presumably achievable) by PhDs, therefore, constitutes an extremely important and significant starting point, especially considering the origin (new gradu- ates vs. professionals) and the heterogeneous working prospects (academic vs. extra academic) of doctoral students. The complexity of challenges on both an individual level (full personal fulfilment through work, the possibility of making a contribution to society…), and on a social level (developing talents, bringing individual excellence to the social, cultur- al, and economic development of a country, the return of the econom- ic investment made towards education and training…), in our opinion, force us to consider the triangulation between formative pathways, oc- cupational prospects, and the professional identity of PhDs in integrated terms without excessive simplifications and automatisms. A reflection on education and its status cannot be reduced to a merely technical issue, but forces us to consider how, in overall terms, the identity of a profes- sional is built (Golde, Walker 2006; Lisimberti 2006; Milani 2014). 2. PhDs in Italy To do this, it may be useful to shift the focus of attention from employment to employability, and make use of the vast integrated definitions of em- ployability that include social value and the ramifications of the activi- ties carried out by individual professionals. NEW PERSPECTIVES FOR DOCTORAL EDUCATION 429 leads to employment as a result. From this perspective, employability must, therefore, be investigated by placing the individual at the centre. Focusing on the subjects implies considering them as the active pro- tagonist of their own personal and professional development without disregarding the prevalence of the socio-economic context of refer- ence, the characteristics of the job market, and the crucial role of in- stitutions and training models (Støren, Aamodt 2010). This systemic perspective, which seeks to highlight the complexity of the elements involved, combines well with a pedagogical approach to the issue (Li- simberti 2006), attentive to the dimension of professional development and the professional’s identity. p y With regards to the development of identity, there is a constitutive link with doctoral training that must be considered. The fruition of a doctoral programme induces profound transformations in subjects who must constantly re-evaluate themselves, renegotiating other roles, such as those of being a student, a researcher, or a professional (Harrison 2008; Crossouard, Pryor 2008). The diversification of outbound employment contexts (Hancock et al. 2016) further complicates this process, which is played out in the interaction between university space, personal space, professional space, and workspace (Pratt et al. 2013). According to this view, subjects and their professional and identity development must, there- fore, be placed at the centre of this reflection, with reference to both the definition of formative pathways and to employability. Exclusively considering only the employability of the individual is, however, reductive. In this sense, it seems useful to take the approach indicated by Yorke who, in defining employability as «a set of achieve- ments – skills, understandings and personal attributes – that makes grad- uates more likely to gain employment and be successful in their chosen occupations, which benefits themselves, the workforce, the community and the economy» (Yorke 2006), calls attention to the community and social dimensions of work. Such a concept of employability also makes sense for those who are already employed. After completing their doc- toral education, these subjects are unlikely to advance in their careers, but will instead experience significant development in their profession- alism, which will also have an impact on their work context. From a pedagogical point of view, social and community development cannot, in fact, be separated from the integral development of the person (Ales- sandrini 2012), and constitute an end result. 3. Employability between professional identity and doctoral education The debate on employability in higher education developed interna- tionally in the 1990s, in connection with a growing focus on the quality of higher education (Harvey 2001). As far as doctorates are concerned, employment prospects are the subject of constant ever-growing atten- tion, and are often the focus of discussion. The issue of employability, however, seems to be a less-investigated issue, even in the international literature, to the extent that it is not included among the main fields of research (Kehm 2015).l On a conceptual level, in some cases, reflecting on the product (em- ployment) seems to be confused with the process (employability). Let us assume, in this instance, Harvey’s view that employability is a pro- cess in which the subject occupies a key role (Harvey 2001) and which NEW PERSPECTIVES FOR DOCTORAL EDUCATION 4. Training trajectories for doctorates 4. Training trajectories for doctorates Re-thinking formative pathways for doctorates poses problems that are far from insignificant. These are attributable to the inherent complexity of training subjects who are ready for both an academic career and for other professions. In conclusion, we will touch on some aspects that are closely interconnected, and seem useful to consider from this perspective. y p p Firstly, it seems essential to focus on the subject’s professional devel- opment project (Lisimberti 2006). This implies the need to refrain from proposing standardized models, but rather to begin from individual needs and the resources to be mobilized. Even formative pathways which lie within common and orderly frameworks of reference should be person- alized. In this regard, Green’s perspective appears challenging, namely that «doctoral pedagogy is as much about the production of identity […] as it is the production of knowledge» (Green 2005: 162). We believe that such attention should pervade every formative pathway. However, it as- sumes a significant prevalence for those subjects, particularly numerous in the Social Sciences and Humanities area in Italy, who – most likely – after their doctoral studies, will return to performing their previous oc- cupation (suffice to think of teachers). For them, the risk is higher that a doctorate constitutes a hiatus. In contrast, if appropriately thought out and planned, it could bring benefits to both the individual – in terms of skills and professional development – as well as to their current pro- fession and workplace in terms of developing innovation, networking, and connections with the academic world, plus planning capabilities, advanced research, etc. Secondly, indications from supranational organizations, which incor- porate previous scientific and experiential results, might usefully per- vade the various doctorates and not merely act as a catalyst in specific courses In fact, the international literature has highlighted, in addition to the specificities and undeniable strong points, the limits and poten- tials deriving from the presence of different formative pathways, such as their progressive hybridization and the impoverishment of professional doctorates, often considered inferior in quality to traditional ones (Shul- man 2007; Olson, Clark 2009). Equally risky would be the re-proposal of a ‘monolithic’ model coupled with acceptable yet isolated innovative practices, unable to optimally cope with the needs of employability that originate from the presence of extremely diverse professional and exis- tential trajectories. NEW PERSPECTIVES FOR DOCTORAL EDUCATION Training, from this perspec- tive, must aim towards a full realization of the person. In connection to this, it must also contribute to the development and progress of their immediate community and of society in a broader sense. This scenario forces us to rethink formative pathways for doctoral students in com- prehensive terms, to ensure full personal and professional development through employability. 430 CRISTINA LISIMBERTI NEW PERSPECTIVES FOR DOCTORAL EDUCATION NEW PERSPECTIVES FOR DOCTORAL EDUCATION 431 sponse to requests from the working universe (Ashcrof 2004) It also opens up a greater range of job opportunities (Fallows, Steven 2000), and directly contributes to raising the quality of research (OECD 2012). Transferable skill training can therefore contribute to the improvement of all doctorates, regardless of the origin and employment prospects of the subjects involved. Some Italian universities (almost 30%) are already active in this field and, in some cases, have launched innovative forma- tive pathways which need to be studied in greater depth, in terms of critical analysis, also with a view to identifying possible guidelines (Li- simberti 2017). l The aforementioned aspects, together with a solid theoretical reflec- tion, could be the start of further empirical investigations aimed, amongst other things, at identifying and presenting existing good practices and experiences. This would contribute to the creation of advanced and in- novative training solutions, useful for the development of solid profes- sional identities, ready to cope with the challenges of employability and, more generally, the knowledge society. 4. Training trajectories for doctorates The open challenge is, therefore, to find applicative strategies that can reduce those elements considered strategic within all the formative pathways. 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Introduction A critical assessment of the concepts of employability and competence requires their placement in the wider political and economic framework characterizing the EU. Without this contextualization, the extent of their involvement becomes difficult to understand. Pedagogy cannot lack an awareness of the inherent link between the political dimension and ed- ucational models, not only in the Marxist sense of the relationship be- tween base and superstructure, but also in order to disengage from the inevitable influence that the ‘spirit of the age’ exerts on the socio-cultural organization of society, working «upon weaker minds, through the un- conscious, with an overwhelming force of suggestion that carries them along with it» (Jung 1972, § 653: 340). The interpretative proposal contained in this paper asserts that the concepts of competence and employability are fully intelligible only if placed within a political-economic scenario that has been dominated by the neoliberal economic model since the 1970s. After World War II, politicians decided that the right balance be- tween the various countries would be the result of free competition in the global market. However, until the 1970s, the redistribution of re- sources and the balance between capital and work were regulated by the Keynesian economic-political model. This was backed up by the vari- ous political forces since it conjugated full employment and increasing welfare with robust economic growth. In the 1970s, with the complic- ity of the oil crisis, Keynesian economics were replaced by neoliberal economics that put competition and the market at the centre of social and economic dynamics. These are not, however, an expression of the 436 STEFANO POLENTA nineteenth-century ‘free market hand’ capable of self-regulation, but seemingly impersonal and ‘objective’ rules, behind which lies the game of an increasingly powerful and globalized capitalistic oligarchy. Equi- libriums achieved previously changed in direction of a Growing Unequal, as the OECD recognized in its 2008 report. The replacement of a solidal project with a competitive one is also reflected in the evolution of the European project: the ‘Europe of the Peoples’ promoted by Aldo Moro (which was supposed to lead to the ‘United States of Europe’ and to the centrality of its Parliament) has been replaced by the Europe of austerity and parameters, established in a technocratic way by the Commission. 1. Introduction p y y The concept of competence, and that of employability, which rep- resents the other side of the coin, are understandable if placed within the previously outlined framework. Just as the ‘myth of competition’ within the free global market requires workers’ mobility and flexibility, employability/competence facilitates the homogenization of workers’ profiles, allowing them to be competitive in the job market. However, since globalized competition produces austerity and increases in struc- tural unemployment – as we can see today in Europe, and particularly in Italy – increasing what Marx called the ‘reserve industrial army’ – work- ers will have to compete ‘downwards’ and accept less favourable work- ing conditions. Just as the Euro has achieved not a people’s union but a competition between economies producing austerity, so the concept of employability/competence works as an ‘educational-Euro’, whose goal is to increase workers’ competition, but in a growing condition of un- employment, with the result of reducing labour rights. In the following parts, we will go more deeply, in order, into: ) h h f b f d id h In the following parts, we will go more deeply, in order, into: a) how the concept of competence becomes confused outside the new- ly-delineated interpretation framework; a) how the concept of competence becomes confused outside the new- ly-delineated interpretation framework; b) how the neoliberal model that characterizes the Eurozone, using aus- terity as a government method, allows a distribution of wealth in fa- vour of major capitals and to the detriment of work; c) how the concepts of competence and employability must be critical- ly outlined within the framework considered above, considered as a potential accused of facilitating this process of devaluating work. EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? 437 it – but the fact that it is used to reduce workers’ rights in a context of increasing austerity and capital reallocation in the global economy, on the way to a decline in labour and welfare rights. All the implications of the concept of competence, which we will now limit ourselves to, become difficult to understand outside this sce- nario. Easily intelligible if placed within the business field from which it comes, it becomes vague, especially when trying to ‘translate’ it into the educational field. Norris (1991) argued that «as tacit understanding of the word [competence] has been overtaken by the need to define precisely and [to] operationalize concepts, the practical has become shrouded in theoretical confusion and the apparently simple has become profound- ly complicated» (p. 332). In fact, the concept of competence alludes to something specific, matured within a precise context. In this sense, we can easily understand that a craftsman is competent, as is a lawyer or a manager. But the concept of competence becomes suddenly elusive when it is used to define the processes and products of education. These are profoundly experiential, they are concerned with self-transformation in relation to culture, to the ability to connect a multiplicity of experi- ences to the unity of a person. Their matrices are traceable to the Greek Paideia and the concept of Bildung. They concern the whole dynamic of a personality and not merely some factors of efficacy extrapolable from it and related to certain contexts (which we call skills or competences). So why use the concept of competence also to redefine the processes- products of education? Why force an intrinsically ‘molar’ concept to de- fi h f d i l h hi h ll ‘ l l ’? p So why use the concept of competence also to redefine the processes- products of education? Why force an intrinsically ‘molar’ concept to de- fine the outcomes of educational paths, which are structurally ‘molecular’? Delamare Le Deist and Winterton (2005) pointed out how it is paradoxical that, while management strategists were emphasizing competences that were unique and firm-specific, the HRD [Human Resource Development] literature was more concerned with develop- ing highly transferable generic competences required for the majority of jobs or specific occupations or job roles (Levy-Leboyer 1996; Stasz 1997). There is an inherent tension between the strategy and the HRD approaches. 2. Competence as a fuzzy concept In the introduction we proposed to understand the concepts of em- ployability and competence as the expression of the neoliberal soul that has contaminated the European project. Specifically, their goal is ‘to flu- idify’ the job market, making it more ‘competitive’. The problem is not the competitiveness as such – although cooperation and solidarity be- tween peoples, which are less and less mentioned, should be alongside EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? […] There is such confusion and debate concerning the con- cept of ‘competence’ that it is impossible to identify or impute a coherent theory or to arrive at a definition capable of accommodating and recon- ciling all the diverse ways that the term is used (28-29). it is paradoxical that, while management strategists were emphasizing competences that were unique and firm-specific, the HRD [Human Resource Development] literature was more concerned with develop- ing highly transferable generic competences required for the majority of jobs or specific occupations or job roles (Levy-Leboyer 1996; Stasz 1997). There is an inherent tension between the strategy and the HRD approaches. […] There is such confusion and debate concerning the con- cept of ‘competence’ that it is impossible to identify or impute a coherent theory or to arrive at a definition capable of accommodating and recon- ciling all the diverse ways that the term is used (28-29). The vagueness of the concept of competence is attested by many au- thors. Van der Klink and Boon (2003: 134)) define competence as a ‘fuzzy concept’. It is «an ill-defined concept with no clear content, thus allow- ing ample interpretations. […] Its vagueness is probably at the same time the explanation for its prominent status today, but […] makes it difficult to use the concept as a sound cornerstone for designing HRD and edu- cational practices». 438 STEFANO POLENTA Due to the lack of clarity of the concept of competence, assessment of competencies can be very difficult. Some authors warn against associat- ing competence exclusively with skills, others distinguish between the terms competence and competency, whilst others treat these terms as being synonymous. The essential problem appears to be that these terms are liberally used as general terms to refer to various aspects of job perfor- mance without any attempt being made to give precise definitions of them. While various efforts have been made to arrive at a single defini- tion of the term competence, no agreement has been reached and there is still a wide variation of meaning between various cultures and between different professions. […] It is recommended that if the term competence is being used, the definition of competence being used in the particular context should be stated […] (Kennedy, Hyland, Ryan 2009:1). EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? This brief bibliographic review was not intended to exhaustively inves- tigate the concept of competence – an in-depth analysis of which requires a lot of effort – but to insist on the importance of its contextualization in the current economic-political scenario: only from this can we un- derstand why, despite the ambiguities of the term, educational processes are being reorganized around the concept of competence. The thesis we intend to emphasize, as already mentioned, is that the motivation which has led to the centrality of ‘this kind’ of competence concept is extrinsic to the educational dimension; it meets, precisely, the desire of industry to create a sort of ‘educational Euro’, homogenizing the students’ and workers’ profiles and making them interchangeable in order to create a job market at a global level in which, since the demand of work is high- er than the offer, a reduction in rights and wages can be implemented. EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? 439 the start of the Euro, our per capita income was 20% higher than its aver- age; today it is 20% below average. There are two roads: Germany leaving the Euro, or fixed but adjustable exchange rates. (La Malfa, Savona 2016). For Festa and Sapelli (2014 : 92-93; 149-151) these results are the conse- quences of the assumption behind the EU political-economic model, i.e., the neo-liberal or neo-conservative one. A model through which ‘free market forces’ were supposed to lead the Eurozone towards continuing improvement, by harmonizing the imbalances between wealth and com- petitiveness. Sapelli traces the roots of this liberalism to the German ordo- liberalismus, born between the two wars and opposed to the intervention of the State in the economy, convinced that a free market would lead to a better distribution of wealth and, at the same time, to maximum social in- clusion (hence the definition of ordoliberalismus as a ‘social market economy’). (i y ) However, unlike the nineteenth-century myth of the self-regulated market, neoliberalism imposes an ‘impersonal system of rules’ on the mar- ket (Paggi 2016: 39): austerity policies; obsessive control of inflation (as Sapelli again reminds us, a legacy of the strong devaluation of the curren- cy after WWI, that became marked as a stigma in the Germans’ minds); the fiscal compact and the prohibition to exceed a 3% debt/GDP ratio (which prevents each country from going into debt but also from invest- ing in public works and services, so that Europe has been steadily disad- vantaged in relation to those countries, like the USA and Japan, which are free from these constraints); and, finally, the impossibility of cash trans- fers to the poorest States. These lines of action emphasize a Europe with little interest in social and labour issues, and strongly oriented to capital. This system of rules appears increasingly regulated by the European Commission which «decides in a self-referential way on the basis input […] of a technical structure of tens of thousands of officials and ambassa- dors […], which defines, in turn, macro-objectives and macro-rigidity» (Festa, Sapelli 2014 : 93). The European institutional operation resulting from this is more technocratic than democratic, and the power of the European Parliament appears to be strongly reduced in favour of that of the Commission (Festa, Sapelli 2014). 3. The Eurozone in crisis The Eurozone is in crisis. Recently Savona wrote: «I’m just back from a conference at Oxford University with the participation of eminent economists, political scientists, and sociologists. They all think the Euro is kaput and that the common market itself is at risk» (Spadafora 2016).i Italy is one of the more compromised countries after twenty-five years of EU membership. Looking at the data of the last decade, accord- ing to «Il Sole 24 Ore», our peninsula has lost 31% of industrial produc- tion; unemployment has doubled (from 6.1% to 12.1%), especially youth unemployment (15-24 years, from 21.2% to 40.3%) (Source: ISTAT). The fixed exchange rate destroyed our competitiveness; on the contra- ry, between 1970 and 1995 it had allowed the growth of those successful companies which still allow us to survive. Italy has paid a huge price to the Euro. It has sacrificed 10% of per capita product since 2008; in 1999, at EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? p What should have been a Community project based on a strong and fully legitimate European Parliament has become an economic austerity policy, in the hands of the Commission and obedient to «market disci- pline» (Somma 2016: 40). For these reasons, a European political union seems hard to achieve today. Political unification was supposed to be a parallel path to economic unification. But only the latter took place. The loss of monetary sovereignty which was imposed on the States for their accession to the EU deprived them of the opportunity to decide their own monetary policy, but without having in exchange those public interven- tions for debt financing that only an EU of a political nature could assure, 440 STEFANO POLENTA as the US ‘Federal Reserve’ does, for example. Without their own mon- etary policy, States cannot devalue the currency to make their produce more competitive; so, in order to maintain their monetary equilibrium, they are forced to raise taxes and devalue working, by introducing those policies of flexibility, insecurity, and contraction of wages that have char- acterized the last two decades; but this triggers a vicious spiral that makes public finances worse, and depresses both production capacity and domes- tic demand in a country. This is what we mean by the word ‘austerity’. y y y Austerity does not appear to be an accident, however, but a desired outcome, a method of government aimed at reallocating resources in a different way. Indeed, the scenario that Europe was encouraging after the 1970s was that of the Keynesian expansive economic model in cri- sis. Previously, after the end of WWII, this model had guaranteed a sig- nificant increase in wellbeing, welfare and equilibrium between capital and work, aiming at maximum employment and considering a strong presence of the State in directing the economy and ensuring welfare. In the 1970s, however, strong economic stagnations brought the Keynes- ian expansive model into crisis. For the neoliberalists, the collapse of the Keynesian system was inevitable. For the critics of neoliberal interpre- tation, the crisis was the occasion to replace a capital-oriented equilib- rium model with one in which the rights of workers and citizens have gradually been eroded. EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? 441 global, and strategic. The single currency […] was a way to move do- mestic competition to another higher level and not a way to achieve eco- nomic cooperation and synergy between the EU countries (Valori 2014). For Sapelli, denying States the possibility of minting money repre- sented «a coup d’état carried out by commissions […], transforming a European growth project into a draft European deficit control, i.e. an exportation of the Teutonic model throughout the European area» (Fes- ta, Sapelli 2014 94). From this point of view, the single currency was the last piece through which the possibility of the State to carry out autonomous economic policies was taken away. «This project, “One Money, One Market”, in- stead of making us fly high, has built the foundations for a Europe in a cage, in the Euro trap. […] It has produced growing divergence; it has furthered the strong countries and relegated the weak ones to the mar- gins» (Pini, Somma 2015). g This ‘forced union’ has nothing to do with the creation of ‘a closer union’; on the contrary, it produces aversion when the result is that ‘free mobility’ serves to produce cheap manpower obtained through social dumping and ‘welfare tourism’, destabilizing both the trade union and bargaining systems of individual countries and the functioning of insti- tutions and welfare. (Amoroso, Jespersen 2014: 36). ( J p ) Labour reforms – from the ‘Treu package’ to Renzi’s Jobs Act – are tending to devalue and commodify work (Barra, Caracciolo 2013 178 e ss). The flexibilization and precarization labour policies are inclined to reduce workers’ rights. There is no relation between unemployment re- duction and work reforms, as the former head of the International Mon- etary Fund Blanchard recognized (2006) (see also Brancaccio, Garbellini and Giammetti 2016). For Sapelli too, «it is not acting on the job market that creates employment […] it is investment that creates employment». That besides, for Amoroso, the underlying problem is choosing a non- predatory kind of economic growth, but one that is respectful of all the planet’s populations. EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? In his view, insistence on an ‘economic growth’ is a ‘mystification’ unless a different kind of economy is planned: «economic growth as a synonym of employment is the mantra through which one seeks to obtain the consent of workers and trade unions to create forms of devel- opment aimed at making existing work precarious and deterring or destroy- ing jobs that could be created with a different economy» (Amoroso 2013). EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? For Savona (2012), until the fall of the Berlin Wall, Italian society had been driven by a scheme that aimed at rebal- ancing the forces between capital and work; such a balance then failed, in favour of a return to an older capitalism. p In the 1980s, the denunciation of Italian public spending began; it was considered excessive and a source of corruption (which it was). However, the unilateral insistence on the corruption of the political class and public spending waste has become a way to legitimize the destruction of State intervention with the aim of undermining the country’s sovereignty. All public industries were dismantled to prevent the corrupt political class from making further public investments. The ‘noose’ put around Italy through adherence to the Maastricht Treaty in order to encourage it to discard its defects – as Savona (2014) incisively explained, remembering the words of his teacher Guido Carli – would make sense if the public hand had checked that the process was happening for the good of the country. Instead, only a ‘Eurocratic’ automatic pilot is establishing the direction of national policy. Austerity was a way to reduce the rights that democracies had guaranteed. […] Behind the Euro was a geopolitical, strategic, and financial project; the ruling Italian class of the past, and worse, that of today, did not un- derstand this in all its ramifications which were indeed, geopolitical, 5. The role of the concepts of employability and competence In his numerous publications on this subject, Hirtt (for example: 2000; 2014) has drawn our attention to the pressure exerted by the ‘Work- 442 STEFANO POLENTA group on Education’ of the ‘European Round Table on Industrial Policy’ (ERT) on education policies in Brussels. The ERT is a lobby of busi- ness executives founded in the early 1980s with the aim of helping to strengthen and develop Europe’s competitive capabilities by encouraging the creation of a single European market. In 1989, the ERT published Education and Competence in Europe. The opinion of industrialists is that «responsibility for education must, ultimately, be taken over by industry […] Education must be regarded as a service rendered to the economic world» (ERT 1995). group on Education’ of the ‘European Round Table on Industrial Policy’ (ERT) on education policies in Brussels. The ERT is a lobby of busi- ness executives founded in the early 1980s with the aim of helping to strengthen and develop Europe’s competitive capabilities by encouraging the creation of a single European market. In 1989, the ERT published Education and Competence in Europe. The opinion of industrialists is that «responsibility for education must, ultimately, be taken over by industry […] Education must be regarded as a service rendered to the economic world» (ERT 1995). ( ) Another fundamental game played in the ’70s and ’80s, as we have said, was the redistribution of wealth. Industrialists feared an increase in wages and the welfare level. One of the founders of the ERT, Dekker, stated that in those years «the combined results of the largest 100 com- panies in Europe, excluding the oil companies, showed a profit level of 0%. […] The Community’s relative competitive position deterio- rated because growth in productivity lagged behind wage increases» (Stone 1989: 93). ( ) The theme of the education-industry approach was further developed in Edith Cresson’s White Paper on Education and Training (1995). The ERT participated directly in the White Paper work, and, meanwhile found an easy back-up in Cresson, one of those Socialists who, in the wake of Jacques Delors, after the fall of the Berlin Wall, had quickly abandoned nationalization and measures to safeguard wages to embrace neo-liberal logics. It should be no surprise to read in the White Paper on education about the close link between education and enterprise (pp. 4-38) and that the priority of the economy (pp. EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? 443 of efficacy [skills, competences])? Instead, Dewey emphasized the ex- pansion of meaning that happens in the vital transaction between the individual and the environment, where individuals are never really dis- tinguishable from the environment and the society of which they are a part. This enables thinking of humanism and technological innovation, school, and society together. It is important to emphasize that there is no antithesis between these dimensions, indeed, culture precedes and also sustains economic action. It is true that, today, «school and work are two strange worlds» (Ravaglioli 2010: 14); but we cannot relinquish humanism in the face of the «primacy of technology or instrumental rationality» (p. 32). Neither should the ‘other’ dimension of education be sacrificed to the altar of modernization, because this represents a de- fence against the technicalization that governs our culture, as Magatti (2010: 319-320) stated, re-introducing McLuhan’s concept of ‘counter- environment’. The technical rationality that feeds the culture of skills believes that cultures are worthwhile only if they can increase effica- cy to reality – reducing efficacy to measurable skills and competences. The primacy of practical reason, the centrality of the economy and the neo-liberal credo are aspects of a single vision of the world, whose re- ductive anthropology «perceives life as a means to serve the economy», as Gallino argued (2003: XI). The White Paper also contained the elements of that technocratic framework today characterizing our higher education: we refer to Euro- pean-recognizable credits (ECTS); to skills, and to «personal skills cards» for one’s recognition (p. 35); to employability; to «on-and-off-the-job training» (p. 25); to the granting of educational institutions autonomy but in the context of increasing evaluation policies (pp. 26-7). g p (pp ) Incidentally, the way in which the White Paper’s parameters have been implemented in the Italian university is of particular concern. 5. The role of the concepts of employability and competence 27-28) was assumed as a stringent criterion to impose «the end of the debate on educational principles» (p. 23). The White Paper enjoyed complaining about the reasons for in- novation, competitiveness, priority to technical-scientific disciplines (pp. 6-9) against the «pre-industrial ethos guiltily prevailing in schools» (Ravaglioli 2010: 14). While apparently continuing to give importance to those cultural aspects at the heart of traditional education, the White Paper placed them in a context which, in reality, made them obsolete in favour of technical rationality. y Once the end of the debate on educational principles had been de- clared, the White Paper stated that the cybernetic-cognitivist culture, in which the concept of competence (as a meta-regulator of interaction between organism and environment) was legitimized, and must pre- vail. How much difference was there, incidentally, between this peda- gogical anthropology (fundamentally anti-humanist, being centred on a mechanistic idea of humankind and its relation to the environment) and Dewey’s humanistic pragmatism, which also stressed interaction between individuals and the environment (but without radicalizing the concept of competition, nor the acquisition of individual schemes EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? Beyond the strictly didactic assessment of the benefit of ‘3+2’ and CFU study course reformulation, we must stress that, contrary to the objectives of increasing the funding of universities foreseen by Europe 2020 and in contrast to the European landscape, in Italy, the university reform pro- cess has been combined with a sharp reduction in resources (more than 20% since the 2008 crisis) and with an increase in competitive funds, tied to the delivery of a certain performance, with the obvious risks of destabilizing the system, in sharp divergence from the indications of the ‘European University Association’ (EUA) (Viesti 2016: 47), in particu- lar as regards Southern Universities. In fact, the EUA allows competitive funding only if there are public funding increases. Moreover, as Viesti observes, the indicators that should be the criteria for distributing the competitive fund have been constantly changing, preventing any finan- cial programming (p. 48). In the light of these data we must ask: what political project is following the Italian University? 444 STEFANO POLENTA […] [it] is not the result […] of a transparent project; that was carefully evaluated at least by the leading classes of the Country […] It derives instead from a patchwork of regulations and implementing measures; of regulations […] A largely obscure process; so structured as to escape […] to the understanding not only of citizens or of ‘ruling classes’, but also to the most directly interested ones. A process which is quite inde- pendent from politics; but that, at the same time, has had a substantial continuity also with quite different executives. […] A kind of implicit single thought (Viesti 2016: 9). Returning to the theme of employability/competence, we should note that we need to focus on the overall scenario within which these concepts acquire sense. Along with the cultural aspects, we have highlighted how the concept of competence/employability facilitates downward compe- tition among workers. It is no coincidence that such a reduced level of competitiveness has resulted in greater inequality, as the OECD has ac- knowledged in some of its reports (Divided We Stand: Why Inequality Keeps Rising [2011]; Growing Unequal [2008]). Competitiveness works top-down and produces wage and welfare shrinkage. EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? For Amoroso, the theme of inequality is global, and we are heading towards a Global Apartheid (1999), where only a part of humanity will be the winner in a global compe- tition with the hope of accessing higher incomes and quality services. 6. Final remarks The Eurozone is in crisis. The unilateral insistence on the skills and employability of graduates – in the face of increasing job competitive- ness and flexibility as measures to regenerate the job market – is in sharp contrast to the growing contraction in employment and to the scien- tific evidence that reforms of the job market alone do not produce an increase in employment. To ‘unload’ the burden of economic growth onto individuals’ abilities is not only economic-political mystification but also ethical-cultural, since it contains the idea that the lone atom- ized individual, competing with others, can be an actor of change for society. This inevitably produces a reductionist and guilty anthropol- ogy; it supports the (false) idea that humanistic and technical cultures are at the antipodes, splitting the unity of the person; it eradicates the awareness that a better economic development is impossible by putting graduates in unruly competition with their peers, and that a more just and habitable society is impossible without inclusion, solidarity and co- hesion. Therefore, the concepts of competence and employability must be rethought within a framework different from the neoliberal one, one that is capable of combining culture and development, humanism and EMPLOYABILITY AND COMPETENCE. BUT FOR WHICH EUROPE? 445 technique, solidarity and competitiveness; otherwise, competence and employability will act in a pedagogically reductive manner, simplifying the complexity of human education in the direction of an efficacy that can be spent on the purely economic side. 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He explores the factors that create a concentration of innovative industries in specific hubs. Through a study of the increase in economic differences among 300 areas in the USA over the last 30 years, he points out the main reasons for the birth of those clusters (such as labour market effectiveness, availability of intermediate services and human capital spillovers). From this, he states that the innovative economic sectors (i.e. high-tech or pharmaceutical) generate a multiplier effect that can create in the long- term, for each new job in these sectors, five new opportunities in non-innovative sectors (i.e. services and non-traded activities). References 2010, La grande contrazione: I fallimenti della libertà e le vie del suo riscatto, Feltrinelli, Milano. Norris N. 1991, The trouble with competence, «Cambridge Journal of Education», XXI (3), 1-11. 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Savona, «Formiche.net», <http://formiche.net/2016/04/23/euro-bce-brexit-parla-il-prof-savona- di-carmine-spadafora/> (01/2018). p Stasz C. 1997, Do employers need the skills they want? Evidence from technical work, «Journal of Education and Work», X (3), 205-233. J ( ) Stone N. 1989, The Globalization of Europe: An Interview with Wisse Dekker, «Harvard Business Review», May-June, 90-95. Valori G.E. 2014, Ecco perché il vero problema dell’Italia è la deindustrializzazione, «Formiche.net», <http://formiche.net/blog/2014/06/09/italia- deindustrializzazione/> (01/2018). ( ) Viesti G. 2016, Il declino del sistema universitario italiano, in Fondazione Res, Università in declino. Un Rapporto sugli atenei italiani da Nord a Sud, a cura di G. Viesti, Donzelli, Roma, 3-56. Carlo Terzaroli (University of Florence)* Abstract: The transformations of work are increasingly introducing new sce- narios at a global level. In fact, the disruptive importance of innovation in all workplace contexts anticipates the challenge of skills and capabilities for the work of the future. In this sense, education has a crucial role in supporting the devel- opment of students and future workers. This paper analyses in depth the link be- tween education, work and the pedagogical instrument of work pedagogy that is derived directly from John Dewey’s thought. This theoretical standpoint rep- resents the base for the development of employability in higher education and its future challenges of innovation, development, and social inclusion. Keywords: work pedagogy, employability, innovation, higher education. THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD: WHY EMPLOYABILITY MATTERS Carlo Terzaroli (University of Florence)* 1. The transformation of the world of work In the coming decades, global competition will be about attracting in- novative human capital and innovative companies. The importance of geography and the forces of agglomeration in determining the location of human capital will keep growing. The number and strength of a country’s brain hubs will determine whether it will prosper or decline. Physical factories will keep losing importance, but cities with a large percent- age of interconnected, highly educated workers will become the new factories where ideas and knowledge are forged (Moretti 2012: 2900). This is one of the main conclusions of the economist Enrico Moret- ti’s famous book The Geography of Jobs1 (Moretti 2012) that illustrates the changes in the United States job market. «The New Human Capital Cen- tury» (Moretti 2012: 2900), as he calls it, represents the result of a global 448 CARLO TERZAROLI shift from a traditional manufacturing economy towards a knowledge- intensive one. This trend has potent effects on job markets and people’s professional pathways. As Moretti analyses in his volume, the transfor- mation of work is shaping not just the size and location of industries but the structure of cities as well. In the past, good jobs and high salaries were directly linked to large-scale manufacturing. Industries were the places where economic value was created. Today, the globalization of markets and the relevance of innovation in the creation of products and services has led to a revolution (Ito, Howe 2017): the quality of employ- ment and wages are increasingly related to the realization of innovative ideas, knowledge, and technologies (Moretti 2012). These trends are producing a «Great Divergence» (Moretti 2012: 76) between cities with a high percentage of innovative firms (and skilled workers) and ones that are unable to attract knowledge-intensive ones. However, this phenomenon impacts not just the development of inven- tive companies, but also the environment: «the presence of many college- educated residents changes the local economy in profound ways, affecting both the kinds of jobs available and the productivity of every worker who lives there, including the less skilled. This results in high wages not just for skilled workers but for most workers» (Moretti 2012: 80). In this framework, cities become the fulcrum of opportunities for the development of social interactions that can generate innovative ideas. 1. The transformation of the world of work The agglomeration of smart jobs in the same environment can enhance inno- vation as well as life quality at all levels: moreover, it can attract further innovators and high-skilled workers (Moretti 2012: 232). g The Great Divergence of territories, which could be highlighted both in the USA and Europe, is reflected in employment too. As Paolo Federighi stated, «the changing structure of employment, together with the growing number of jobs that are more knowledge- and skill-inten- sive, increases demand for professional growth among the employed» (Federighi 2013: 13). The study by Ilaria Maselli effectively explains the Job Polarization trend within European job market: indeed, Figure 1 shows that «there is a trend towards polarization on the labour demand side with respect to occupations in most European countries, whereas on the supply side, the trend is towards a linear upskilling of the popu- lation. Depending on the speed of these changes and on the skill con- tent of current demand and supply, there is a risk that in some countries a skill mismatch problem will arise» (Maselli 2012: 26). p ( ) Furthermore, the complexity of this new scenario has been increased by the development of Industry 4.0 (Smit, Kreutzer, Moeller, Carlberg 2016) and will further expand with Industry 5.0 (Rai, Rai 2015). These latest trends impose a crucial role for adult education, conceived as a dis- cipline that «goes beyond the boundaries of the school system and pro- fessional training» (Federighi 1999: 5). THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD 449 Figure 1 – Demand and Supply of Work with Respect to Skills/Tasks in the EU27, 2010- 2020. [Maselli 2012: 26] Figure 1 – Demand and Supply of Work with Respect to Skills/Tasks in the EU27, 2010- 2020. [Maselli 2012: 26] Facing the evolution of work requires the capability to critically anal- yse the context and its evolution, interpreting future tendencies and un- revealed challenges. In this framework, staying in the job market does not mean creating a skills supply that can match the skills demand (or, at least, no longer). The speed at which production settings change drives organizations and individuals to continuously renew their aims, projects, and competences. From this perspective, it could prove inadequate, if not ineff ective, to create formative pathways that simply answer the present skills demand. 1. The transformation of the world of work In fact, the new creation of value is characterised by on- going transformation, mobility of space and contents and a capacity to innovate products, internal relationships, and organizational structures (Ito, Howe 2017). ( ) The brilliant research by Enrico Moretti, which directly reveals the relationship between the presence of innovative fi rms and the develop- ment of cities and employment, suggests a crucial point for adult educa- tion. Indeed, given that innovation has immense importance for high-, medium- and low-skilled workers, the centrality of skills growth, both in fi rms and at an educational level, become a vital challenge for the whole of society. Contributing to increasing the educational rates, skills, and capabilities of people is not just a matter of one part of the work- ers. On the contrary, the challenge of up-skilling is a task of the pro- duction and educational systems, especially in this knowledge-intensive context which is, today more than ever, centred on human added value (Samans, Davis 2017). Care of these processes is one of the aims of adult education, not just in the workplace, but also within schools and higher education facili- ties (Boff o 2015, 2017) where it seems more complex. University is not simply the place where people acquire competences for entering the job 450 CARLO TERZAROLI market. It is a space for human education that allows students to grow up globally as subjects, citizens, and workers (Boffo 2012, 2015). How- ever, what matters most is the new relevance of higher education in the advance of innovation. Given the capability of creating better work op- portunities and higher wages (Moretti 2012) increasingly related to the value of humans’ creativity, the future of our communities is increasingly linked to skills development and entrepreneurial attitudes. Which is why, over the last decades, educational institutions have been charged with the new mission of creating new relationships with multiple stakeholders such as public bodies, companies, associations, and non-profit organiza- tions. The Third Mission of Universities (ANVUR 2015) is intended to enhance and disseminate knowledge and technologies to local, national, and international socio-economic contexts to improve the quality of the economy, work, and human life. 1. The transformation of the world of work These efforts could generate great ben- efits to all the actors involved: graduates could be equipped with skills to enter the job market and contribute with innovative work; industries could integrate new knowledge into their production processes; com- munities could take advantage of the presence of innovative firms that can also create opportunities for the low- and medium-skilled. market. It is a space for human education that allows students to grow up globally as subjects, citizens, and workers (Boffo 2012, 2015). How- ever, what matters most is the new relevance of higher education in the advance of innovation. Given the capability of creating better work op- portunities and higher wages (Moretti 2012) increasingly related to the value of humans’ creativity, the future of our communities is increasingly linked to skills development and entrepreneurial attitudes. Which is why, over the last decades, educational institutions have been charged with the new mission of creating new relationships with multiple stakeholders such as public bodies, companies, associations, and non-profit organiza- tions. The Third Mission of Universities (ANVUR 2015) is intended to enhance and disseminate knowledge and technologies to local, national, and international socio-economic contexts to improve the quality of the economy, work, and human life. These efforts could generate great ben- efits to all the actors involved: graduates could be equipped with skills to enter the job market and contribute with innovative work; industries could integrate new knowledge into their production processes; com- munities could take advantage of the presence of innovative firms that can also create opportunities for the low- and medium-skilled. THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD 451 This is why nowadays it is extremely crucial to understand the new trends in the economy and job market: through these analyses educators could better plan pathways to offer students the opportunity to acquire capabilities for their future lives. This is crucial since, as Dewey had al- ready illustrated in 1899, the first transformation that impacts the struc- ture of education is the economic and industrial one: the change that comes first to mind, the one that overshadows and even controls all others, is the industrial one – the application of science re- sulting in the great inventions that have utilized the forces of nature on a vast and inexpensive scale the growth of a world-wide market as the object of production, of vast manufacturing centres to supply this mar- ket, of cheap and rapid means of communication and distribution be- tween all its parts (Dewey 1932: 5). It is almost indispensable to point out that these factors have become vastly more disruptive in the current situation, considering the innova- tions in technologies and industries. Following Dewey’s standpoint, these trends have progressively generated a radical metamorphosis of society. We could spend a long time discussing how the past structure was able to better create social and community links or a more settled relationship between people and the world, «but it is useless to bemoan the departure of the good old days […]. It is radical conditions which have changed, and only an equally radical change in education suffices» (Dewey 1932: 9). We must take note of those in progress to give people the right tools to avoid suffering from their effect. Work completely permeates human life, in its deepest meaning and its organization of time (Boffo 2012). In line with this, Dewey suggested including manual activities within the school curriculum, to integrate life into the educational process: in fact, «we must conceive of work in wood and metal, of weaving, sewing, and cooking, as methods of liv- ing and learning, not as distinct studies» (Dewey 1932: 11). These kinds of activity must be considered «as instrumentalities through which the school itself shall be made a genuine form of active community life, in- stead of a place set apart in which to learn lessons» (Dewey 1932: 11). What are the implications for pedagogy? 2. The relationship between education and work: why employability matters In this framework, the debate on the development of students’ work capabilities arises as a key issue for the growth of our countries. The spe- cific contribution of adult education could inform the processes through which young adults acquire capabilities for their personal and profession- al life. In this sense, it seems interesting to highlight the specific added value of a pedagogical and educational perspective on this relationship alongside the traditional approach elaborated by economic and sociologi- cal studies (Gazier 1998). ( ) The connection between education and work originally referred to John Dewey’s studies. The American philosopher laid great stress on the relevance of this relationship. In his volume The School and Society (1899) he suggested going beyond the conception of school through the mere standpoint of teachers and children. As he clearly showed, school has been transformed by the new patterns of economy and society in broad- er terms: «the modification going on in the method and curriculum of education is as much a product of the changed social situation, and as much an effort to meet the needs of the new society that is forming, as are changes in modes of industry and commerce» (Dewey 1932: 4). In this sense, the innovation in education can be considered «as part and parcel of the whole social evolution, and, in its more general features at least, as inevitable» (Dewey 1932: 5). THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD How could we plan formative pathways, at all levels, that can «consider the relationship of the school to the life and development of the children in the school» (Dewey 1932: 31)? This is a tough task for the whole discipline, especially for adult educa- tion, which is fundamentally based on subjects’ growth in their working and living contexts. This challenge reflects the research into the most effective ways through which to generate learning linked to human life needs (in any individual, personal, or social scope considered). As Dewey lucidly stated in Experience and Education (1938), 452 CARLO TERZAROLI it is part of the educator’s responsibility to see equally to two things: first, that the problem grows out of the conditions of the experience be- ing had in the present, and that it is within the range of the capacity of students; and, secondly, that it is such that it arouses in the learner an active quest for information and for production of new ideas. The new facts and new ideas thus obtained become the ground for further expe- riences in which new problems are presented. The process is a continu- ous spiral (Dewey 1976: 79). The relevance that the American philosopher assigned to the genera- tion of innovative ideas is extremely relevant in our contemporary con- text. The new patterns of global economies and job markets (Moretti 2012) outline the vital role of creativity in work. The ability to imagine innovative projects, to face new situations, and to renovate the plans of individual careers or organizational business plans are progressively stra- tegic characteristics (Ito, Howe 2017). Education has the task of support- ing the development of those skills, both in school and in other learning contexts. How can we do this? How can we integrate the free and dis- ruptive dimensions of play and creativity with the structure of adults’ learning pathways? We are in front of a changing paradigm. g p y g g p g Traditionally, the ancient philosophers divided the meaning for these actions through different words: public affairs and work (negotium) were separated from free time (otium), where play could happen. It seems that they were referring to different fields of life, quite clearly separate. THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD 453 work feeds on creativity and resourcefulness, this division wanes imme- diately. How can higher education support this process within its specific pathways? This is one of the key points for present and future research. p y y p p What we should be focusing on is the deeper meaning of work, con- ceived as profession and career, and its relationship with the formation of the self. Dewey provided a precious reflection on this in Democracy and Education (1916): A vocation means nothing, but such a direction of life activities as ren- ders them perceptibly significant to a person, because of the conse- quences they accomplish, and also useful to his associates. The opposite of a career is neither leisure nor culture, but aimlessness, capriciousness, the absence of cumulative achievement in experience, on the personal side, and idle display, parasitic dependence upon the others, on the so- cial side. Occupation is a concrete term for continuity. It includes the development of artistic capacity of any kind, of special scientific ability, of effective citizenship, as well as professional and business occupations, to say nothing of mechanical labour or engagement in gainful pursuits (Dewey 1930: 358-359). The concept of ‘employability’, which is going to be analysed in the next chapter, slots neatly into the pedagogical framework traced by John Dewey throughout the nineteenth century. What is the relation between school and work? How can we help students develop skills and capabili- ties to better create their own personal and professional pathway? What are the global changes that people need to tackle in their career? How could higher education support both economies and citizens, while in- creasing the quality of life? These questions, and many others, represent the foundations of the educational reflection on the concept of employ- ability based on Dewey’s pedagogical instrument. THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD On the contrary, what Dewey outlined reveals a unique perspective on the relationship between these two aspects: «in their intrinsic meaning, play and industry are by no means so antithetical to one another as is often assumed, any sharp contrast being due to undesirable social conditions. Both involve ends consciously entertained and the selection and adap- tations of materials and processes designed to effect the desired ends» (Dewey 1930: 237). y In this sense, the integration of these dimensions in education could generate new patterns for the development of skills and capabilities. The active occupations represent interesting forms of generating learning, in- cluding work-related learning. In fact, if work permeates people’s lives and their meaning, and this work is increasingly characterised by cre- ativity, mobility, and other forms of innovation (both for business and career), it is meaningless to maintain this separation. Dewey’s contribu- tion to pedagogical reflection clashed with the antithesis «between edu- cation in preparation for useful labour and education for a life of leisure» (Dewey 1930: 293). Furthermore, it added that «the bare terms useful labour and leisure confirm the statement already made that the segre- gation and conflict of values are not self-enclosed, but reflect a division within social life» (Dewey 1930: 293). In the present situation, in which THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD 2 The Higher Education Academy started working on employability in 2006 through a series of papers. These works, coordinated by Prof. Mantz Yorke, were car- ried out by Professor Peter Knight (Open University), Professor Lee Harvey (Sheffield Hallam University), Professor Stephen McNair (Surrey University), Dr Brenda Little (CHERI), Professor Kate Purcell (University of the West of England), Mike Hill (Graduate Prospects) and Val Butcher from the Higher Education Academy. 3. Higher Education facing the challenge of work: the concept of employability The category of employability arose along with the evolution of job market trends at a global level. It has been considered a new construct for the development of students’ and graduates’ work capabilities, especially in countries with high levels of unemployment. In this sense, it represents a reaction to the mismatch between education and employment to in- crease the support to transitions towards the job market (Boffo, Fedeli, Lo Presti, Melacarne, Vianello 2017). Economists and sociologists were the first who already discussed employability as a way to reduce mismatches in the twentieth century (Gazier 1998). However, these forms ignored the development of the subject and the deep perspective of the construc- tion of skills and capabilities within the course of life. In 1998, Hillage 454 CARLO TERZAROLI and Pollard recovered the individual perspective, focusing on subjects’ capability to stay in the job market thanks to their knowledge, skills, and aptitude: «In simple terms, employability is about being capable of get- ting and keeping fulfilling work. More comprehensively employability is the capability to move self-sufficiently within the job market to realise potential through sustainable employment» (Hillage, Pollard 1998: 3). Anyhow, the pedagogical elaboration of the concept mainly refers to important scholars who operated within the Enhancing Student Employabil- ity Co-ordination Team (ESECT) research group at the Higher Education Academy in York (UK)2. Lee Harvey, Mantz Yorke and Peter Knight guided the team that intensively analysed many aspects of employability. In fact, the ESECT traced an interesting didactic and pedagogical ap- proach aimed at transforming higher education to tackle innovation and the changes in the current social and economic situation. The problem is not merely the power distribution within the uni- versity-economy relationship, or an instrumental view of higher educa- tion. On the contrary, the added value of the pedagogical standpoint on employability concerns the university as a place where young adults can develop critical and reflective thought: this may help them deal with the challenges of their lifespan as global citizens, workers, and active partici- pants in the learning process as well (Harvey 1999). Lee Harvey clari- fied this point thus: Employability raises fundamental questions about the purpose and struc- ture of higher education. Employability is not about training or provid- ing add-on skills to gain employment. On the contrary, employability is about how higher education develops critical, reflective, empowered learners. THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD From their perspective, employability meant: «[…] a set of achievements – skills, understandings and personal attributes – that make graduates more likely to gain employment and be successful in their chosen occu- pations, which benefits themselves, the workforce, the community and the economy» (Yorke 2006: 8).i This definition outlines a crucial connection between learning and employability and can determine innovative methodologies. This point considers not just the students’ perspective, but calls into question the construction of curricula, didactic methods, interdisciplinary contents, but also career services. In fact, it is a learning process, curricular and extra-curricular, that connects, within educational action, concrete ex- periences, and key aspects for personal and professional growth. In con- clusion, the category of employability is extremely disruptive in the pedagogical field since it highlights the formative process of subjects in relation with the social value of traditional university missions such as research and didactics in conjunction with the Third Mission. 3. Higher Education facing the challenge of work: the concept of employability Despite appearances to the contrary, the real challenge is not how to accommodate employability but how to shift the traditional bal- ance of power from the education provider to those participating in the learning experience (Harvey 1999: 13). This is why Harvey emphasized how the conception of employabil- ity, as a simple indicator, is reductive in measuring academic perfor- mance. Yorke & Knight found themselves within the same framework (Yorke, Knight 2006). They described a competence-centred approach (European Commission/EACEA/Eurydice 2014) that has been wide- ly adopted as a reference point for the creation of specific programmes. THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD 455 4. Final remarks The job market is changing extremely rapidly. If we acknowledge the trend of the last ten years (especially since the 2008 economic crisis), we can only imagine the extent of the changes we are going to meet in the next decades (Moretti 2012). Today’s challenge for the university is not simply the ability to answer the mismatch by strengthening the skills supply. In fact, the timeframe of education does not coincide with the current work demand, which is constantly changing: the risk is to continually (and ineffectively) chase after the economy and the market. Besides, this is not the role of the university, even today when it is be- ing asked to bring more and more added value in terms of innovation (Ito, Howe 2017). As Ulrich Teichler stated, «Higher education has to take care primarily to avoid the danger of «ivory tower knowledge de- livery». It does not express warnings against the opposite possible disas- ter of higher education, i.e. to subordinate itself simply to the presumed demands of the employment system» (Teichler 2004: 7-8). The Academy should look to the future, trying to understand the current trends also in terms of skill (Davies, Fidler, Gorbis 2011), and above all training people able to always deal with different challenges at personal, social, and political levels. The change, for the future of countries, starts from higher education’s ability to prepare people who can contribute to innovation, develop- ment, and social inclusion (Boffo, Gioli, Terzaroli 2017). If work really does represent the tool by which people build sense and their place in the world (Boffo 2012), then people should progressively develop the ability 456 CARLO TERZAROLI to create new and better opportunities, for as many people as possible, to increase the quality of their life. This is primarily the task of Univer- sities, for today and for the future as well. References ANVUR 2015, La valutazione della terza missione nelle università italiane. Manuale per la valutazione. 13 febbraio 2015, <http://www.anvur.org/attachments/ article/26/M~.pdf> (01/2018). f p ( ) Boffo V. 2012, A Glance at Work, Firenze University Press, Firenze.f Boffo V. 2015, Employability for the social economy. The role of higher education, in Boffo V., Federighi P. and Torlone F., Educational Jobs. Youth and employability in the social economy, Firenze University Press, Firenze, 147-168.f Boffo V., Fedeli M., Melacarne C., Lo Presti F. and Vianello M. 2017, Teaching and Learning for Employability. 4. Final remarks New Strategies in Higher Education, Pearson, Milano-Torino. Boffo V., Gioli G. and Terzaroli C. 2017, The Development of Student Employability in Higher Education: a comparative perspective on university approaches at European level, «Educational Reflective Practices», 1, 7-29. Davies A., Fidler D. and Gorbis M. 2011, Future Work Skills 2020, Institute for the Future for the University of Phoenix Research Institute, Palo Alto. y Dewey J. 1930, Democracy and Education, The Macmillan Company, New York. Dewey J. 1932, The School and Society, The University of Chicago Press, Chicago. Dewey J. 1976, Experience and Education, Macmillan Publishing Company, New York. Federighi P. 2013, Adult and further education in Europe. Using public policy to secure a growth in skills, Publications Office of the European Union, Luxembourg. Federighi P. 1999, Glossary of Adult Learning in Europe, UNESCO Institute of Education, Hamburg. Gazier B. (ed.) 1998, Employability: Concepts and policies, European Employment Observatory, Berlin. Harvey L. 1999, New realities: the relationship between higher education and employment, Keynote presentation at European Association of Institutional Research, Lund, <https://goo.gl/5JnchK> (01/2018). Hillage J. and Pollard E. 1998, Employability: Developing a Framework for Policy Analysis, Institute for Employment Studies, London. Ito J. and Howe J. 2017, Al passo col futuro. Come sopravvivere all’imprevedibile accelerazione del mondo, Egea, Milano. Maselli I. 2012, The Evolving Supply and Demand of Skills in the Job market, «Intereconomics», 1, 22-30. fl Moretti E. 2012, The Geography of Jobs, Houghton Mifflin Harcourt, Kindle version, Boston-New York. i Rai S. and Rai A. 2015, Review: Nanotechnology – The secret of fifth industrial revolution and the future of next generation, «Nusantara Bioscience», VII (2), 62-66. THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD THE ROLE OF HIGHER EDUCATION IN A CHANGING WORLD 457 Samans R. and Davis N. 2017, Advancing Human-Centred Economic Progress in the Fourth Industrial Revolution, World Economic Forum, Cologny-Geneva. Smit J., Kreutzer S., Moeller C. and Carlberg M. 2016, Industry 4.0, European Parliament – Policy Department A: Economic and Scientific Policy, Brussels. Teichler U. 2004, Changes in the Relationships Between Higher Education and the World of Work on the Way Towards the European Higher Education Area, Keynote speech at the EUA Conference “University and Society: Engaging Stakeholders”, Marsiglia, 1-3 April 2004. g p Yorke M. 2006, Employability in higher education: what it is – what it is not, The Higher Education Academy, York. Yorke M. and Knight P.T. 2006, Embedding employability into the curriculum, The Higher Education Academy, York. Francesca Torlone (University of Florence)* Abstract: Certain young people are being propelled towards a more promis- ing future than in the past, due to – amongst other things – continuous learn- ing, which guarantees their productive capabilities. Employers are called upon to arrange a cogent value proposition to support young people in their constant growth that would also help them create an autonomous professional identity. Keywords: employee learning value proposition, youth transitions, profession- al identity. * Francesca Torlone, PhD, Researcher in Adult Education, Department of Education and Psychology, University of Florence, Italy. Email: francesca.torlone@unifi.it. THE VALUE PROPOSITION OF ORGANIZATIONS FOR YOUNG GRADUATES AND THEIR EMPLOYABILITY Francesca Torlone (University of Florence)* 1. Introduction In this contribution, we consider the pedagogical dimension of or- ganizations linked to the definition of policies, levers, and instruments whose combination can be a value for young people passing through them, from work experience and apprenticeship to normal working relationships. p Consequently, we are going to look at the results of the PRIN Emp&Co research (Employability and Competencies, 2012-2016) using the Employee Value Proposition (EVP) paradigm, i.e. everything that people experience and receive within a working relationship (Pavar, Charak 2015; Minchington 2010; Michaels et al. 2001), to understand how young people build their own set of values, from those offered by the organizations where they pass through the management of train- ing processes and to which they are exposed (in transitions prior and subsequent to graduation). This is because EVP includes not only pay and economic benefits, but also – amongst other things – the ELVP – Employee Learning Value Proposition, i.e. the provision of learning ac- tions – above all informal – contextualized and aimed at the acquisi- tion and development of skills and knowledge of all kinds, as well as certain behaviour and attitudes. Such opportunities can be directly created by the factors of value proposition of the organizations or simply stimulated by them. It is the young person’s task to seize them to build their own growth path. 460 FRANCESCA TORLONE The research set out to investigate the educational or disadvantaged valency of learning processes as a result of the value proposition of work- ing contexts, especially in view of the number of organizations through which young graduates pass just after graduating. In the research we did not investigate personal and social factors, which also affect the profes- sional future of individuals, to focus on the educational dimension of factors that promote or hinder transitions. 1. Introduction p Our hypothesis is that a value proposition structured in instruments that create affiliation and quality in the required work promote recognition and benefit – as well as learning – career development; that they pro- vide transparent pay policies (Browne 2012), as well as attracting talented young people and expressing high potential for keeping their job; that they stimulate individuals, inducing them to make the best of themselves, to build (also autonomously) sense in their work actions, starting from reactive learning opportunities (Eraut 2000), dependent on the growth and development paths of the professional self to act in the context of work. Conversely, a poor value proposition, lacking in learning valency risks re-routing such paths and adversely affecting the management of internal and external transitions to the job market. j The contribution is subdivided into an introductory part that illus- trates the adopted paradigm (paragraph 1), followed by paragraph 2, which provides brief notes of a methodological character, further elaborated in Chapter 4 of this volume. In paragraph 4, we describe the types of transitions that emerged dur- ing the research as typifying the period immediately following gradua- tion. Paragraph 4 provides the empirical evidence that we have read and interpreted through the learning value proposition approach, on which we finally founded our conclusions (paragraph 5). 2. The first transitions of young graduates towards work found in the research 2.1 Methodological nods During the research the data was collected using records of semi-struc- tured interviews, according to a rigid research protocol (see Gaia Gioli, Em- ployability-Oriented Curriculum: Strategies and Tools to Train Young Graduates. The PRIN EMP&Co. Project). The analysis unit was the learning (adverse and favourable) action directed at young people during their work experience. 2.2 Descriptions of the first transitions Starting from the analysis of the interviews, we show the main data, re- ferring to what we might define as the first three transitions towards work: THE VALUE PROPOSITION OF ORGANIZATIONS FOR YOUNG GRADUATES 461 1. internships and work experience during the university coursei 1. internships and work experience during the university coursei 2. the first work experiences after graduation, not necessarily consistent with preparation or professional aspirations i 3. the first work activity consistent with the higher education received and with the professional family corresponding to the aspirations of the young graduate. The succession of the three transitions is indicative of a path that we might consider virtuous, but not generalized. In fact, the third transi- tion – which usually occurs at least 12 months after the end of studies – was only found in a part of the young respondents. There are, in fact, two other groups that were distinguished by not being a part – after one year – of any work environment or for not having reached any form of stabilization of their work. Below we provide some basic information on the basic features of the value proposition which characterised the work experiences encoun- tered by the respective professional paths. The aim is to understand whether there could be a correlation between the professional results achieved in the third transition and the value proposition of previous work experience. Cluster 1 – Young people not in any business environment by the end of the Third Transition. Second transition Cluster 2 – Young people in a precarious/casual working environment at the end of the Third Transition. First transition Internship experience con- ducted in an activity that is consistent with person- al interests, in conjunction with non-homogeneous work experiences, neither with personal interests nor with career paths and career expectations (waiter/ress, host/ess). The value proposi- tion activated by the organi- zation sometimes serves to develop some skills, espe- cially transverse (relational, managerial). On the other hand, it is not very decisive in terms of professionalism and attractiveness. Higher education experi- ences compensate for the skills not acquired in previ- ous experiences. At this stage young people have job op- portunities and are engaged in activities whose content is highly professional. There are still no compensation, ca- reer development, and reward instruments. Young people still have cas- ual employment relation- ships. In many cases they are simultaneously engaged in multiple activities and with various organizations. Pro- fessional growth and new perspectives often result from the networks of relationships that young people build. It is through “small jobs” and the networks Higher education experi- ences compensate for the skills not acquired in previ- ous experiences. At this stage young people have job op- portunities and are engaged in activities whose content is highly professional. There are still no compensation, ca- reer development, and reward instruments. Young people still have cas- ual employment relation- ships. In many cases they are simultaneously engaged in multiple activities and with various organizations. Pro- fessional growth and new perspectives often result from the networks of relationships that young people build. It is through “small jobs” and the networks that young people deepen their knowledge of the spe- cific job market (e.g. third sector, international co- operation) and their po- tential future employment opportunities. Cluster 3 – Young people in a work context in a structured way at the end of the Third Transition. First transition Second transition First transition Second transition Second transition The value proposition of in- ternship experience is poor: young people are looking by themselves for opportuni- ties to acquire further areas of specialization (e.g. Mas- ter in Clinical Pedagogy, research PhD). Transition to internship and closure of the university course are accompanied by work experiences non-coherent with young people’s pro- jects for work placements, for a variety of reasons (de- sire to know the contexts of work, activate compensation measures independently and construction of professional networks). Job or opportunity research is focused on small locali- ties and local markets and is not guided by an analysis of demand in the job market. Failures also in non-relevant job search with their own aspirations and professional paths follow each other. This does not always result in dis- couragement. In some cases, there is a strong motivation to complete specialization inter- ventions because of a more fa- vourable professional future. Component management of value proposition is predomi- nantly in the hands of the young person due to the ab- sence of an involvement in an organization. For extended periods of time there are sea- sonal work experiences that are repeated without stabil- ity over the years (e.g. being educator in summer centres). They contribute to the de- velopment of a professional identity and skills that organ- izations are not interested in stabilizing internally. Component management of value proposition is predomi- nantly in the hands of the young person due to the ab- sence of an involvement in an organization. For extended periods of time there are sea- sonal work experiences that are repeated without stabil- ity over the years (e.g. being educator in summer centres). They contribute to the de- velopment of a professional identity and skills that organ- izations are not interested in stabilizing internally. Job or opportunity research is focused on small locali- ties and local markets and is not guided by an analysis of demand in the job market. Failures also in non-relevant job search with their own aspirations and professional paths follow each other. This does not always result in dis- couragement. In some cases, there is a strong motivation to complete specialization inter- ventions because of a more fa- vourable professional future. 462 FRANCESCA TORLONE Cluster 2 – Young people in a precarious/casual working environment at the end of the Third Transition. First transition Internship experience con- ducted in an activity that is consistent with profession- al aspirations (e.g. training officers and managers for private organizations, nurs- ery educators, family peda- gogues, ministerial advisers for innovation policy in ed- ucation), along with casual work experiences, consist- ent and not (leaflet delivery, classroom tutor, babysitter, back office in bank). Consistent work experience in the same field of training; in some cases, the indicator of compensation is built on the initiative of the young per- son who continues in paral- lel with multiple, inconstant work experiences. Organizational behaviour supports the professional growth processes of young people, for whom organi- zations provide challenging tasks (e.g. the creation of an HR office for early child- hood services), relationships networks, career prospects and, at times, rapid develop- ment paths and consequent rewarding instruments. Organizational behaviour supports the professional growth processes of young people, for whom organi- zations provide challenging tasks (e.g. the creation of an HR office for early child- hood services), relationships networks, career prospects and, at times, rapid develop- ment paths and consequent rewarding instruments. p The indicator of the value proposition on the content of work and on professional challenges is often developed autonomously by the young person who builds on own initiative opportunities for growth of skills (Master’s de- gree, elaboration of research projects for PhD candidates). g In other cases, pathways aimed at building self-em- ployment projects are being developed. THE VALUE PROPOSITION OF ORGANIZATIONS FOR YOUNG GRADUATES 463 3. The ‘employee value proposition’ for managing the transitions of young graduates We now analyse how the EVP’s educational dimension affects orga- nizations in the progression of transitions. 3.1 The EVP in general terms 3.1 The EVP in general terms In recent years, we have witnessed a change of approaches, terms, and contents that help to expand an organization’s offerings to the people in- volved. The concept of EVP, initially used in managerial literature and human resources, and today of interdisciplinary interest (marketing, com- munication, etc.), generally includes (Minchington 2010): Compensation (remuneration policies); Benefits (incentive schemes including monetary and non-monetary elements, fixed and variable remuneration, incentives to increase the baggage of skills, etc.); Work content (recruiting, train- ing, assessment, growth policies that affect the more or less challenging and attractive content of the work assigned to each worker); Career as- sistance (career development policies); Affiliation (loyalty policies to the organization). 3.2 The pedagogical dimension of the EVP 3.2 The pedagogical dimension of the EVP Pedagogical reflection relates to the way in which a work experience (an apprenticeship, internship – of any form – and more or less regular work experience) are learning opportunities through which young peo- ple capitalize on their ‘achievements’ (Yorke 2006) for the autonomous construction and implementation of their professional project through interaction with the contexts they are a part of. In fact, the growth and construction of one’s own realization depend not least on the confidence that young people feel to be what they want, and the actions they plan and set in order to become what they want to be. y It is essentially to conceive the value produced by organizations as an opportunity for young people to reflect on their identity – profes- sional and personal – and to bolster this based on the experiences that they are offered by life, or slowly build freely through a conscious in- tegration of every aspect of the professional and training experience (networks, Master’s courses, professional tasks, etc.). Young people on a first work experience, before being competent in productive action, learn to be competent in reflective and learning action and can bring about transformations that influence the definition of even short paths to explore organizations. Through the various experiences that the job market offers (not always fulfilling), young people acquire knowledge, grow, transform their behaviour, nurture proactivity or resistance (in- fluenced by the contexts and the way they live). Especially through these, young people learn to read the here of now and define the here of tomorrow. In other writings, we have dealt with the subject in the penitentiary context (Torlone 2016). Here we analyse the role that or- ganizations – not just penitentiary – have, through EVP and ELVP, to promote processes of awareness, self-development, self-design, re- search, and development of the professional self into a renewed social context in which family membership/ professional profiles and identi- ties are no longer immutable as in the past or guaranteed by enduring lifelong work, but constructed in a more modest and negotiable way (Bauman 2005).l It is indisputable that being part of an organizational context, influ- ences an individual’s private and personal sphere in various ways, their ability to give a sense and unique meaning to experiences, even soon- er than the ability linked to working productively. First transition It has a direct impact on the proposed learning value, as we shall see in the paragraphs below. i EVP therefore incorporates, but goes beyond, the benefits of monetary compensation (from compensation to total reward) that an organization provides to its employees in exchange for their time, commitment, tal- ent, and performance. The focus is on attraction and retention towards those who help organizations achieve their missions and business goals. This is from the perspective of the organizations. p p g The theme, analysed from the perspective of young people look- ing for work, raises questions of pedagogical importance since they are bound to identify actions that allow young people to build and manage themselves, to employ their talents, to act on knowledge, skills, and behaviour – hidden or lacking – with the support of the organizations to which aspirations, knowledge, attitudes, and time are available. In other words, pedagogical problematics lie in the way young people are able to build a proposal that is valuable in terms of positioning within companies, organizations, and the free market. This means analysing the meaning of EVP for a young trainee, apprentice, worker, i.e. the educational dimension of the EVP (i.e., the ELVP), depending on the transitions into and out of the job market, the way the learning actions which take place during a work experience support young people or not in managing transitions and building their own professional life project (aware that the process of ‘employability’ begins much earlier, Boffo et al. 2017). 464 FRANCESCA TORLONE 3.2 The pedagogical dimension of the EVP In this context, an ELVP is successful whenever it prepares young people – at least the most ambitious and those likely to initiate change – for the autonomous and targeted management of their transition into the job market (Tab. 1) through involvement in learning actions be they ‘favourable’ or ‘adverse’ (Federighi 2016), re-worked autonomously by the young person, in tune with their new career aspirations. THE VALUE PROPOSITION OF ORGANIZATIONS FOR YOUNG GRADUATES 3.2.1 The educational dimension of incentives for growth Talented young people, with a high degree of agency ability (Ban- dura 1977) and the desire to make things happen, reveal a high capacity to recognise and exploit the (reactive, deliberative, intentional) learning generated by (all) living contexts, from meeting others and using them to build one’s own professional life project. Young people can cultivate experience and relate to people in their Zone of Proximal Development, which is expanding as new acquisitions stimulate new learning; they are able to invent their own professional identity, solicit and create it by acti- vating decision-making processes constructed autonomously or through the available networks. By means of these processes, identity can also change in advancing experiences; professional itineraries are hypothesized and defined and experienced in the light of the learning experiences that the young person creates and lives critically, if not made available by the working contexts they are travelling through. All of which requires a young person to have a high capacity to «imagine and desire something that is not yet given; to identify goals for achieving it, starting with what is available; to give rise to something new; to reconstruct strategies and aims discursively» (Costa 2013: 111). Otherwise, the value creation pro- cess loses its advantages and benefits. Young people, independently – if not accompanied by organizations – have their own learning resources, and differentiate their training and learning experiences (also with personal and economic sacrifices and re- nunciations), enhancing their ability to translate negative or little chal- lenging ELVPs into opportunities to grow elsewhere. THE VALUE PROPOSITION OF ORGANIZATIONS FOR YOUNG GRADUATES It is in this way that the young professional worker who acts, communicates, interprets, seeks, studies, 466 FRANCESCA TORLONE takes interviews and asks questions, leveraging his transformative reflex- ivity (Mezirow 2003).i takes interviews and asks questions, leveraging his transformative reflex- ivity (Mezirow 2003).i y Of the five components of EVP, we shall now consider the most im- portant ones from a pedagogical point of view. 3.2.1 The educational dimension of incentives for growth THE VALUE PROPOSITION OF ORGANIZATIONS FOR YOUNG GRADUATES 465 Table 1 – The effects of EVP on the progression of the transitions of young people in the construction of a professional identity. Effects of EVP / ELVP on young people Types of EVP / ELVP Young proactive, ambitious in looking for employment. Young little proactive, little ambitious in looking for employment. Young not proactive nor ambitious in looking for employment. Rich, positive EVP (referring to all com- ponents of the EVP) Attraction. Retention. Vertical mobility (internal/external). Attraction. Resistance to transitions. Attraction. EVP poorly rich, negative (in reference only some of the 5 com- ponents, they satisfy the young person) Attraction. External mobility. Attraction. Retention. Resistance to transitions. Possible external mobility. Attraction. Retention. Resistance to transitions. EVP poor, negative (referring to all com- ponents of the EVP) Attraction. External mobility. Attraction. Retention. Resistance to transitions. Possible external mo- bility (immediate/ postponed). Attraction. Retention. Resistance to transitions. Possible ex- ternal mobil- ity (immediate/ postponed). Table 1 – The effects of EVP on the progression of the transitions of young people in the construction of a professional identity. The quality of the organization’s value offering affects the professional future of young people in terms of mobility for further professionaliza- tion and employment, sought elsewhere. In this regard, the first transi- tion (to ‘small jobs’ not relevant to the study path or career aspirations, see section 2) is important because it gets young people used to seeking what satisfies them elsewhere, to seizing learning values in every context they travel through, to build paths and abilities in analysis, reading, and selecting organizational contexts. g g Conversely, a negative or only partially positive EVP/ELVP may acti- vate external mobility paths or hold on to those who have poorly defined professional projects and little attention to the discovery of alternative occupational contexts. f The young person, in management of the EVP/LVP offering (wheth- er positive or negative), faces the need, not always properly accepted, of thinking personally while something changes, meanwhile transform- ing within the context of the job, to become ‘another professional’. The need, specifically, is to steer the change going on or about to be activated and of which they must be protagonists right from the start, triggering the set of abilities and skills possessed. 3.2.2 Challenging work contents Opportunities for growth and wellbeing for young people in the workplace can be produced directly by the value proposition of orga- nizations. Nowadays, the jobs available are many, varied, increasingly rich in intellectual content, and fewer physical and varied efforts in the world. Young people have the opportunity (unknown to young peo- ple in the past) to travel, to specialize, frequently change work and or- ganizations, to deepen their knowledge and enrich their relationships (Federighi, Torlone 2013). They are, however, called for a commitment to become autonomous, to grow, become involved, to define their own professional aspirations (not necessarily identical to those built on their THE VALUE PROPOSITION OF ORGANIZATIONS FOR YOUNG GRADUATES 467 university course), to improve in their work and the jobs they are doing, or to seize the growth potential that each organization offers (Learn- ing Value Proposition). The more solid and rich the learning potential of the workplace that the young person experiences (Federighi 2013), the greater the potential for building professional advancement because of stimulation in discovering the path and a search for the work (rath- er than a job) or activities suited to their professional expectations and consistent with who the young person is and wishes to be. This is the time when, both in work and through it, young people can exalt their creative and propositional abilities, experience restricted ambitions of autonomy, challenge their imagination, turn to success and profession- al conferment, encourage and be encouraged, exalt their sense of en- trepreneurship in terms of a personal interpretation of the role being played, steer organizational behaviour towards value creation. Young people who have unclear professional development, poor motivation, and little ambition, are limited to living the working environment for what it is and can give. g The success of young workers at work is not without its obstacles, suffering and difficulties, but it can be accompanied by a process of sense-making that relieves them and helps them rediscover the work en- vironment as a place for self-improvement, for ‘self-increase’ (Rossi 2009: 75), and self-realization, according to the professional goals in evolution. Job growth opportunities – wherever we look – can also be stimulated, driven by the ELVP of the organizations where the work experience is taking place. In this case, it is the young people who are responsible for evolving their professional self, seeking other experiences with greater challenges and learning content. 3.2.3 Recompense In some cases, a poor ELVP allows young people to fi nd employ- ment opportunities that are economically more fulfi lling than those in the past. Usually it is not until the third transition that a level of com- pensation in line with the salary expectations the young person has ma- tured is achieved. 3.2.2 Challenging work contents The difficulty, in these cases, is to rec- ognise a lack of learning gains, learning disadvantages, damage and losses to internalize and implement change, the transition to a new occupa- tional status. When the acquisition takes place, the young person looks for further work with more attractive ELVPs, considering the first too restrictive, unattractive, and promising experiences compared to a job insertion plan that is increasingly defined or requires additional stimuli (internalized by the young person). The preparation of vocational curri- cula and job interviews are experienced differently each time and waiting becomes educational, all critical processing opportunities for a prepared path. To avoid involution, cognitive decline, loss of acquisition, imper- sonal slightly improper awareness, young people can choose between staying in saturated organizational contexts of knowledge development and new learning, less likely to build a professional identity to experi- ence and enrich in full autonomy, and the opportunity to open up to the future and venture into the (re)construction of their own working history – often with investments to be made. 468 FRANCESCA TORLONE 3.2.3 Recompense 4. Final remarks Based on the data presented, the following becomes apparent: 1. The employment prospects of young graduates are related to the type of work experience of young people. This depends on the learning value of the professional and the relational content present in such experiences, which help to develop the individual’s ‘calling’. Aspira- tions and interests change and alter due to work experiences. 2. Young people cannot expect to immediately begin a professional career consistent with their aspirations and studies. In all cases, completing their basic skills and knowledge of job demand require a commitment from young people to improve, both through positive work experi- ences and through a further post-graduate commitment. Young peo- ple are required to be «active, inventive and resourceful, not just on one occasion, but constantly, day after day» (Beck, Beck-Gernsheim 2002: 23-26), during the paths that are built and experienced. The less active, proactive, and ambitious are those most penalized.f p p 3. The differences between young people in terms of professional out- come, which can be traced back to one year after graduation, are pa- tently obvious. The professional future of young people affects personal and social factors that we have not investigated in depth in this research. In all cases of failure or partial fulfilment of aspirations, there is a per- sistence of working experiences with poor educational content. THE VALUE PROPOSITION OF ORGANIZATIONS FOR YOUNG GRADUATES 469 themselves (e.g. a post-graduate or Master’s degree), or receives from the contexts they are passing through (e.g. on-the-job training, or courses). y p g g ( g j g ) The relevant work experiences accumulated by young people take on more importance than unobtrusive or unsuccessful university pathways with vacancies (Humburg et al. 2013). Those more careful understand this in time and set to work to make their career path attractive. 3.2.4 Career development In the absence of advancement and security prospects, it is the young person who must build these to meet the skills demand that they begin to discover in the fi eld, to know how to look at and select more than in the past. Commitment to career development and continuous improvement (Fig. 1), with diff erent and non-generalizable results and timeframes, is the result of young people’s individual choices – sometimes infl uenced by personal situations, and not least by suff ering – which matures with creativity, intelligent awareness, adaptability, knowledge management skills within networks that are created and available; innovations com- pared to initial experiences (internship). In some cases, this is the time when the young person enters a long- term occupation (third transition), with satisfactory remuneration. Figure 1 – The career progression of the young graduate. 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Alessandra Vischi (Catholic University of the Sacred Heart)* Abstract: The acceleration of changes underlines the need to enhance our efforts to adapt education to the dynamics of the current economic situation and the issue of employment. In the framework of the circular economy, pedagogy, which is based on the educability of individuals, takes into consideration forms of educational planning to identify a long-lasting balance between economic prosperity, social wellness, and environmental development. The challenge of the future is the pos- sibility of increasing youth employment; this calls for pedagogical expertise and organizational planning to ensure that everyone’s development is authentic and holistic. To this end, the MSc Degree programme in Educational Planning and Human Resource Development offered by the Catholic University trains gradu- ates to become professional figures with expertise in coordinating and managing the development of human resources (guidance, selection, personal services); the professional training and retraining of project managers in social and educational contexts for both academic and corporate spheres. The guiding vision behind the MSc in Educational Planning and Human Resource Development is fully in line with the Catholic University of the Sacred Heart’s educational project, to support a culture of responsibility and creativity, entrepreneurism and collabo- ration, multi-disciplinary knowledge and skills, and scientific research for the purpose of holistic human development. Educational planning, in a period of socio-economic and social change, involving the whole planet in many respects, can relaunch an ‘integral model of development’, based on long-term wellbe- ing, technological innovation, ‘human development’, and the dignity of work. Keywords: employability, educational planning, work. EMPLOYABILITY AND TRANSITIONS TOWARDS WORK: MSC DEGREE PROGRAMME IN EDUCATIONAL PLANNING AND HUMAN RESOURCE DEVELOPMENT, CATHOLIC UNIVERSITY OF THE SACRED HEART OF BRESCIA Alessandra Vischi (Catholic University of the Sacred Heart)* 1. The circular economy, work, educational planning In response to the current socio-economic situation in Italy, marked by a high rate of unemployment (ISTAT 2016), the education sector must reflect on the employability models and interventions adopted by universities. The acceleration of changes highlights the need to enhance our efforts to adapt education to the dynamics of the current econom- ic situation and the issue of employment. The debate on education, which is based on the educability of individuals, takes into consid- eration forms of educational planning that can characterize a long- 472 ALESSANDRA VISCHI lasting balance between economic prosperity, principles, and human achievement. The value of education is undeniable, since «develop- ment is now complicated to an unprecedented degree, and requires multiple approaches and interpretations, which must come together in a humanist and pedagogical cultural synthesis, essential for authen- tic implementation» (Maritain 1947: 30). An ethical and educational interpretation of the transitions to work and employability, seek the identification of a positive relation- ship between socio-cultural processes and individual development. According to the available data, which, accompanied by an ongoing increase in registration figures, reveal high placement rates, the edu- cational ‘construct’ of the MSc Degree programme in Educational Planning and Human Resource Development is a relevant case in point for this issue. p We must make people capable of expressing their own talents and making the most of their knowledge to create innovation; we must ensure that policies are designed to pursue sustainability, both local- ly and globally, and are connected to and based on principles of col- laboration and responsibility, so that the financial resources invested generate human value and not short-term profit. g pi The challenge facing Italy and the international community is to reduce unemployment and facilitate economic growth, while en- couraging wellbeing and care for the environment. The importance of education arises from this perspective, especially the education of young people, as a means of teaching them how to enjoy responsi- ble freedom in pursuit of justice and the common good. The Europe 2020 Strategy (<http://ec.europa.eu/europe2020>) aims to create the conditions for a more competitive economy along with smart growth, by investing in teaching, research and innovation; sustainable growth, by protecting the environment and encouraging responsible choices; and supportive growth, to increase employment and reduce poverty. EDUCATIONAL PLANNING AND HUMAN RESOURCE DEVELOPMENT 473 possible, we must be aware that a «gradual investment in humanity as a strategic resource goes beyond simply cognitivization of work»; it means appealing to the human resource within a culture of work, through which people «operate, learn and collaborate in redefining the entrepreneurial systems of the global market, with an ever-closer connection to the deepest meanings of the ego» (Bocca 1998: 101). This postulates an ethical convergence towards the ‘good’ of the or- ganization, which requires long-sighted, strategic planning that is in- formed and imaginative and involves economic consensus, with the enhancement of personal competences and training-related elements as an opportunity to spread and share workplace culture. Within the framework of the circular economy, an economic model targeted at «using resources in the most efficient way to guarantee the continuation of such efficiency, so as to bring important econom- ic benefits», Europe issued the communication: «Towards a circular economy: A zero waste programme for Europe» in order to raise awareness among citizens and promote interventions for the gradual elimination of waste, in line with the Europe 2020 Strategy. Please see: <http://eur-lex.europa.eu/legal-content/IT/TXT/?uri=CELE X%3A52014DC0398R(01)>. ( ) In this context, of note are the so-called green jobs, which either involve new professional profiles, purely technical in nature, or a re- training in existing competences within existing professional fields, as long as these relate to sustainability in the broad sense. According to the Green Jobs: Towards Decent Work in a Sustainable, Low Carbon World 2008 report promoted by the United Nations Environment Pro- gramme, green jobs can be defined as jobs in various fields, from agri- culture to transport, energy efficiency and construction, that address environmental impact, avoid all forms of waste and protect biodiversity and ecosystems (<http://ec.europa.eu/environment/enveco/jobs/>). In Europe by 2012, 3.4 million people were employed in green jobs and the figure is continually increasing; in Italy in 2015, there were 2,964,000 with competences relating to the green sector, correspond- ing to 13.2% of the employed population in the country as a whole. Please see: <http://www.symbola.net/assets/files/GreenItaly%20.Pre- sentazione%20(con%20testi).DEFINITIVA_1476974725.pdf>. Despite the positive impact of green jobs within the professional landscape, we are obliged to focus our attention on the critical ques- tion of youth employment: in Italy, in July 2016, the level of unem- ployment fell overall by 11.4%, a decrease of 0.1 percentage points since June, however, the number of employed people also fell at the same time. 1. The circular economy, work, educational planning Educational expertise must be applied to the question of the heuristic scope of transformations underway, the governance of which require economic, cultural, and ethical changes, in which universities, pro- fessions, and businesses must play their part. The professional sphere contributes to achieving long-term inte- grated development objectives, reinforcing social dialogue and public/ private partnerships, while encouraging the sharing of responsibility with regard to sustainable production and consumption methods. Or- ganizations as educational contexts, work as a means of ‘taking care’ of one another, and the talent and experience of individuals, can all serve as the matrix upon which to establish new forms of governance and professional responsibility, which recognise the ontological value of human beings. To encourage individual development as much as EDUCATIONAL PLANNING AND HUMAN RESOURCE DEVELOPMENT EDUCATIONAL PLANNING AND HUMAN RESOURCE DEVELOPMENT While in June there was an increase (+1.3%), in July, the monthly estimation of unemployment levels fell by 1.3% (–39 thou- sand). This fall concerned both men (–1.4%) and women (–0.2%), 474 ALESSANDRA VISCHI and all age groups except the 15-24 (+23 thousand) and 25-34 (+38 thousand) (See <http://www.istat.it/it/>). The reduction seems to be related to an increase in inactive people, i.e. people not engaged in seeking work, categorized as NEET (Rosina 2015), i.e. young peo- ple between the ages of 15 and 29 who are not working and are no longer enrolled on a study course (Not in Education, Employment or Training). There are more NEETs in Italy than in any other Eu- ropean country. The OECD, which analyses social wellbeing and its trends in the 34 industrialized countries each year, found the percent- age of NEETs in Italy prior to 2007 to be around 20%, or 4 points above the average, as published in their Society at a Glance 2016 – A Spotlight on Youth report on Italy (see <https://www.oecd.org/italy/ sag2016-italy.pdf>). Between 2007 and 2014, this rate increased to 27%. Since 2015, the rate has fallen slightly, to roughly 2.5 million NEETs, however it remains above pre-crisis levels and almost double the OECD average (15%). Italy has the highest proportion of young people with low literacy skills (20%) and the second highest rate of young people with low numeracy skills (26%); another factor behind the increase in NEETs is a lack of jobs: 11.4% of NEETs in Italy are unemployed. The number of young people who have left the educa- tion system and are without work constitutes a ‘significant economic cost’ – emphasises the OECD – of between 360 and 605 billion dol- lars, or 0.9%-1.5% of the global wealth. g The challenge of the future is to address the NEET phenome- non and the possibility of increasing youth employment; this calls for pedagogical expertise and organizational planning to ensure that everyone’s development is authentic and holistic, smart and special- ized, green and supportive. Educating the younger generations today involves recognizing the importance of creating educational mod- els and processes to develop not only the competences currently re- quired, but also critical awareness within a culture of sustainability and responsibility. EDUCATIONAL PLANNING AND HUMAN RESOURCE DEVELOPMENT p y With a view to increasing employment, also in Italy, in 2014 the European Union Council launched the Youth Guarantee plan, through which the state and regions undertook to make young peo- ple under 25, who neither study nor work (NEETs), a high-quality offer of work, further study, apprenticeship, internship, or another form of training within four months of the beginning of their un- employment or of their leaving formal education. There are two targets within the NEET group: young people registering as unem- ployed or first-time jobseekers, who are not following a course of education or training, totalling 537,685 for young people between 15 and 24, and 906,694 for young people between 15 and 29; and young unemployed people who leave a course of education or train- EDUCATIONAL PLANNING AND HUMAN RESOURCE DEVELOPMENT EDUCATIONAL PLANNING AND HUMAN RESOURCE DEVELOPMENT 475 ing early and do not register as unemployed or first-time jobseek- ers, but are seeking work (even if not actively) or are unavailable for work, amounting to 390,282 for young people aged 15-24, and 659,247 for young people aged 15-29: For details on Italy (Garanzia Giovani), see: <http://www.garanziagiovani.gov.it/Documentazi- one/Documents/Piano-di-attuazione-italiano-della-Garanzia-per- i-Giovani.pdf>. p The plan is also an important opportunity for companies, who, benefiting from the subsidies allocated to the various Italian regions, can invest in motivated young people, offering on-the-job training and self-entrepreneurship opportunities to achieve the programme’s objectives. The Ministry is also involving companies through agree- ments with the main trade associations to promote internships and apprenticeship contracts, as well as publishing job and workplace- training opportunities on Italy’s Youth Guarantee national portal (Garanzia Giovani). Recent Italian legislation on the right to work emphasizes the importance of supporting young people, either school- or univer- sity-educated, throughout their first professional experience; work- based training and encouraging the ‘talents’ of individuals, including those who struggled in the traditional education system, are now key objectives. Schools and universities, together with institutions and companies, have a responsibility to promote the acquisition of competences, with a view to engaging young people in the world of work; this must be- come an educational context in which individuals can express their own creativity and productivity in the form of ideas, actions, and prod- ucts. «Work-related learning can bring about effective improvements in professional contexts, with a particular focus on youth cultures» to combine professionalism and responsibility, «embedding education and training in the job itself» (Alessandrini 2010: 266). g j ( ) Investing in the education of individuals and developing a spe- cific culture of initiative in an integrated way is currently the most eagerly awaited mid- and long-term project to take advantage of the negative experiences behind the current economic crisis and navi- gate towards promising entrepreneurship and an ethically sound ho- rizon: «Old models are disappearing, but promising new ones are taking shape on the horizon» (Benedict XVI 2009: no. 40); we must «place training at the heart of innovation» (Margiotta 2016), along with knowledge, professions and productive processes. Managerial creativity, inter-generational responsibility, and values shared by both companies and stakeholders are all key elements for encouraging so- cio-cultural progress, the benefits of which are supportive and long- lasting for the younger generations of today and tomorrow. 476 ALESSANDRA VISCHI 2. Educational planning, human resource development and employability In recent years, the world of work and the education professions (Federighi 2010) have undergone significant transformations; work- ing environments, characterized by rapid technological innovation and high levels of uncertainty and precariousness, are calling for a brand-new approach to educational planning to promote the devel- opment of organizations along with new competences. p g g p The Catholic University of the Sacred Heart has increasingly fo- cused on the challenges of the job market in a cosmopolitan con- text, by means of academic courses and scientific research, networks throughout the region, and an international outlook. g g To this end, the MSc Degree programme on Educational Plan- ning and Human Resource Development offered by the Catholic University: (<http://brescia.unicatt.it>) trains graduates to become professional figures with expertise in coordinating and managing the development of human resources (guidance, selection, personal services); the professional training and retraining of project manag- ers in social and educational contexts for both the academic and cor- porate spheres. This programme, classed as LM 50 under the Italian degree classification system, is multidisciplinary in nature, combin- ing theory and practice, planning activities, and the development of competences. p In agreement with local organizations, this programme provides graduates with expertise in: the educational coordination of services targeting children, adolescents, young adults, adults and the elderly; human resource development and management in for-profit and non- profit organizations; educational planning in the region (project man- agement) to promote and manage a network of interventions involving various stakeholders (schools, business, entities and associations); com- bining relational skills with knowledge of innovative techniques in educational and vocational guidance interventions, empowerment and professional retraining; valorizing gender-based differences and par- ticipatory leadership in welfare projects in the field, from needs analy- sis to the assessment of changes in an ever-changing context, be they socioeconomic (Circular Economy, Smart City) or legislative (extended work-related learning,) in nature. The programme lasts two years. To pass the programme, students must gain at least 120 university cred- its. For each module students achieve a certain number of credits and a mark out of thirty, which is determined by means of an exam. 2. Educational planning, human resource development and employability The integrated teaching programme includes curriculum-based apprentice- ships and workshops: on the two-year course, the curriculum-based apprenticeship consists of 200 hours in total, of which 140 are carried out in an organization outside the university, and 60 inside the uni- EDUCATIONAL PLANNING AND HUMAN RESOURCE DEVELOPMENT 477 versity, including group sessions and individual meetings. These ac- tivities have been taking place in the Faculty of Education since the academic year 1992-1993 on the Degree programme in Education, at the time the Facoltà di Magistero or Teaching Faculty, and has always been considered an integral part of the university curriculum and «in order to meet requirements, must maintain a sound and ongoing con- nection with the theory and discipline of the Degree programme to which it belongs» (Pati 2012: 7). Activities related to apprenticeships require the university to «update the system governing its own edu- cational offering, learning to skilfully combine theory and practice», and meanwhile local organizations define the practical knowledge, offering students an experience of «the culture of educational work/ training» undertaken (Pati 2012: 11-12). Workshop activities, which consist of one workshop in the first year and one in the second year, are led by experts from the working world, enabling students to learn about specific issues in a practical way. i Graduates of this programme, benefitting from knowledge across several disciplines, find employment in the following fields: social and educational, human resources development, professional guidance and training; consulting and supervision services for local organizations, companies and training providers; and educational planning, both on a local and international scale. They enter and grow professionally within organizations (for-profit, non-profit and public) as experts in educational planning; educational coordinators; training managers; educational consultants for public and private organizations; directors and project managers of educational and cultural interventions (con- ception, planning, implementation and evaluation); human resource managers in corporate settings; inter-institutional coordinators work- ing between social areas covered by public and private organizations; experts in assessing educational and socio-educational interventions; tutors in professional training and on staff-training courses; and di- rectors in the area of public-sector staff management. pf g The placement rate for graduates is high: within a year of graduat- ing 84.3% of graduates have found work, rising to 91% within three years; 80% of the graduates state they are satisfied with the Degree programme and would re-enrol, while 100% confirm that the work- load is manageable. 2. Educational planning, human resource development and employability Beyond ministerial obligations, this programme maintains a con- stant relationship with its social partners, through events held at least three times a year dedicated to the target professional role/s and pro- fessional competences, in order to remain up to date and meet the de- mands of the job market: the inaugural event Sogni e Bisogni (‘Dreams and Needs’) in October, the Career Day in spring and Graduation Day in July. 78 ALESSANDRA 478 ALESSANDRA VISCHI The first event − Sogni e bisogni (Dreams and Needs) is a seminar on sharing routes and experiences, takes place in the month of October during the first week of lectures, and sees the participation of local or- ganizations from the for-profit and non-profit sectors, as well as insti- tutions. The university, represented by the professors and students from the Degree Course, meets and engages in dialogue with stakeholders who, for their part, collaborate with the training course in question. Following a well-established and respected tradition, an orienta- tion and networking event is also held: the Career Day. This event is intended to facilitate the transition to work from higher education and as a contribution to offer new professional contexts by creat- ing networks between the local culture, the city, the region, and the students of the Catholic University of the Sacred Heart. The Career Day is addressed to those who have already enrolled, those who are about to enrol, and those who wish to do so, in order to build their own career paths by playing a central role in a dynamic and innova- tive professional field. pi The course delivers knowledge and skills across several disciplines, and actively engages first-year students at all stages, combining rela- tional competences and techniques for interventions in services across all ages, from children and adolescents, to adults and the elderly; and within for-profit and non-profit contexts, which are collaborating in- creasingly often these days. All within the field of educational plan- ning and human resource development. Graduation Day takes place in July outside the university: this is a day of festivities and celebra- tions, but at the same time an important opportunity for reflection on the themes of education, pedagogical planning, and economic and social development. 2. Educational planning, human resource development and employability p The representatives of the social partners, who contribute to the study course in numerous ways, show appreciation for the profes- sionality demonstrated by the educational planners, whose energy has enabled them to enter multiple professional areas, from for-profit to non-profit or public administration. These representatives, from the main entrepreneurial and non-profit organizations in the region, emphasize how important it is to further enhance the collaboration between the educational and production spheres, in order to continu- ally improve opportunities for networking and planning training ac- tivities and workshops for the benefit of the community. Companies require a professional figure capable of noticing and bringing to light the developmental needs of the organization and the demands of the region with the intention of drafting targeted interventions for change. The guiding vision behind the MSc in Educational Planning and Human Resource Development is fully in line with the Catholic Uni- versity of the Sacred Heart’s educational project, to support a culture EDUCATIONAL PLANNING AND HUMAN RESOURCE DEVELOPMENT 479 of responsibility and creativity, entrepreneurism and collaboration, multi-disciplinary knowledge and skills, and scientific research for the purpose of holistic human development (Giuliodori, Malavasi 2016). fi The results achieved demonstrate the efficiency of its governance choices: models and interventions that the course pursues to improve the employability of its students in numerous ways (Boffo, Federighi, Torlone 2015). It is equally important to further reinforce the dialogue between universities and the world of work, in order to identify new patterns of research and partnerships, thereby ensuring that the course framework (teaching modules, apprenticeships, workshops and public events) is constantly improving in terms of how integrated and strate- gic it is, against a dynamic and uncertain socio-economic backdrop. g g y p «Planning is a key issue for all professions involved in education and training, obliging us to remember the past and commit to the future, taking into consideration the roots of the problems, the de- mands, the desires, and the changes of the interlocutors involved, referring back to imagination and ideals that are the drivers of peda- gogical action» (Birbes 2012: 132). Educational planning, in a period of socio-economic and social change, involving the whole planet in many respects, can relaunch an integral model of development (Pope Francis 2015), based on long-term wellbeing, technological innova- tion, human development (Gennari 2001), and the dignity of work. * Vanna Boffo, PhD, Associate Professor of General Pedagogy, Department of Education and Psychology, University of Florence, Italy. Email: vanna.boffo@unifi.it; Monica Fedeli, PhD, Associate Professor of Teaching and Learning, and Organizational Development, Department of Philosophy, Sociology, Pedagogy and Psychology - FISPPA, University of Padua, Italy. Email: monica.fedeli@unipd.it. References Alessandrini G. 2010, Elementi dello scenario e ruolo dell’approccio di pedagogia del lavoro, «Pedagogia Oggi», 2, 264-266. g g gg Bertagna G. 2011, Lavoro e formazione dei giovani, La Scuola, Brescia. Bocca G. 1998, Pedagogia del lavoro. Itinerari, La Scuola, Brescia. Birbes C. 2012, Progettare competente. Teorie, questioni educative, prospettive, Vita e Pensiero, Milano.f Boffo V., Federighi P. and Torlone F. 2015, Educational Jobs: Youth and Employability in the Social Economy. Investigations in Italy, Malta, Portugal, Romania, Spain, UK, Firenze University Press, Firenze. p y Fabbri L. and Rossi B. (eds.) 2010, Pratiche lavorative. Studi pedagogici per la formazione, Guerini Studio, Milano. F d i hi P 2010 P f i i d ti l P d i O i 1 70 86 f Federighi P. 2010, Professioni educative e lavoro, «Pedagogia Oggi», 1, 70-86.i Gennari M. 2001, Filosofia della formazione dell’uomo, Bompiani, Milano. Gennari M. 2001, Filosofia della formazione dell’uomo, Bompiani, Milano. Giuliodori C and Malavasi P (eds ) 2016 Ecologia integrale Laudato sì Gennari M. 2001, Filosofia della formazione dell uomo, Bompiani, Milano. Giuliodori C. and Malavasi P. (eds.) 2016, Ecologia integrale. Laudato sì. Ricerca, formazione, conversione, Vita e Pensiero, Milano. Giuliodori C. and Malavasi P. (eds.) 2016, Ecologia integrale. Ricerca, formazione, conversione, Vita e Pensiero, Milano. Malavasi P. 2007, Pedagogia e formazione delle risorse umane, Vita e Pensiero, Milano. 480 ALESSANDRA VISCHI Margiotta U. 2016, Formazione e innovazione: un binomio generativo, in Costa M., Capacitare l’innovazione La formatività dell’agire lavorativo, FrancoAngeli, Milano, 217-228. g Maritain J. 1947, La personne et le bien commun, Desclée de Brouwer, Paris. d h d Maritain J. 1947, La personne et le bien commun, Desclée de Brouwer, Paris. Pati L. (ed.) 2012, Tutorship e attività di tirocinio: tra pensieri, vissuti, azioni, EDUCatt, Milano. Pati L. (ed.) 2012, Tutorship e attività di tirocinio: tra pensieri, vissuti, azioni, EDUCatt, Milano. Pope Francis 2015, Lettera enciclica Laudato si’. Sulla cura della casa comune. Pope Francis 2015, Lettera enciclica Laudato si’. Sulla cura della casa comune. Rosina A. 2015, NEET. Giovani che non studiano e non lavorano, Vita e Pensiero, Milano. Rosina A. 2015, NEET. Giovani che non studiano e non lavorano, Vita e Pensiero, Milano. United Nations Environment Programme (UNEP) 2008, Green Jobs: Towards Decent Work in a Sustainable, Low-Carbon World. Vischi A. 2016, Entre Pedagogía Y Trabajo. Circular Economy, Jóvenes Y Desarrollo De Los Recursos Humanos, Pensa Multimedia, Lecce. Vanna Boffo (University of Florence), Monica Fedeli (University of Padua)* Vanna Boffo (University of Florence), Monica Fedeli (University of Padua)* The PRIN2012LATR9N project began more than 5 years ago, when we found ourselves drafting the research map in a cultural and pedagogi- cal context rather far-removed from the themes we wished to address. In fact, to speak of Work-Related Learning, widespread apprentice- ship programmes, callings, the relationship between education and work, and employability, could have seemed to be steering the project’s reflec- tion away from the pedagogical-educational contexts in question. What we can say is that the development of the project began as a challenge: starting to deal with matters seldom investigated and uncommon in the university community of Italian pedagogy meant stubbornly tackling content that belonged to other disciplines ‒ according to the academic and cultural traditions. Therefore, in terms of innovation, this could be considered the first step: dealing with themes partially adhering to pedagogical disciplines with the intention of expressing full membership of the paths on skills and employability in the contexts of educational and training knowledge. The project title already contained the fundamental points of the re- search design: «To design innovative programmes for higher education, to promote personalized learning, to build on job competences, to valo- rize talents to create new work opportunities. Positive strategies in higher education to support young adults during their employment emergency, as a response to the socioeconomic crisis and as a citizenship action». We broke it down into five consecutive lines. We dealt with higher edu- cation, indeed, with learning in higher education, and we tied togeth- er themes and reflections in a much-frequented international context. In recent years, these themes have become familiar, the problem of the transformation of the university and of its role in and for civil society has 482 VANNA BOFFO, MONICA FEDELI become crucial. The debate is ongoing, inspired especially by numerous micro-changes that have occurred through MIUR Directives and AN- VUR Assessments. We could say that the change has become manifest in the details of the Decrees, in the assessment regulations of the study courses, or in the indicators that monitor PhD courses. In this context, year after year, cultural change has taken shape and has become part of a debate which is increasingly acknowledged. In the light of the epochal changes in which we are immersed, Work, Job Placements, Professions, and Professionalism become the fulcra to reflect on higher education’s role today. Vanna Boffo (University of Florence), Monica Fedeli (University of Padua)* i In the first instance, John Dirx, of Michigan State University, on the line of Teaching and Learning has emphasized the centrality of a change in perspective and focal point in the contexts of Education: students ought to be put at the centre of the learning process, but above all, of the teach- ing processes. The role of university teaching is key if we really wish to propose a future for this institution which is supposed to be the engine of a country’s knowledge: Over the last half-century, teachers in higher education have gradual- ly begun to incorporate active and engaged forms of learning in their practices. They have found that shifting from passive learning and a reli- ance on recitation and lecturing to more student- and learning-centred approaches actively involves students in the learning process, enhances their motivation to learn, fosters higher-order thinking skills, and creates more meaningful learning experiences for them. Changing from a long tradition of passive learning, however, to more student- and learning- centred approaches takes time, knowledge, skill, and commitment It will be necessary to highlight research-based principles to actively engage students in the learning process and to explore strategies for promoting change in teaching practice at individual and organizational levels in higher education (Emp&Co. Conference 2017). Instead, if we observe the exponential change arriving from the worlds of scientific research and industry, might we ask ourselves how to promote such a change in the global university? What can be done to give all the universities in Italy the same possibility to better prepare their students. According to Soong Hee Han of the University of Seoul: Higher education, different from others, develops and delivers cutting- edge innovations in setting new societal visions, restructuring, technol- ogies, and professional skills development to implement and maintain them. It includes not only high-tech industries but also fundamental and structural changes in society and economy, as we would expect from the Fourth Industrial Revolution. I argue that the macro changes in indus- try and the labour market will further rely on the active role of higher education in both long-term academic competences, and ‘re-skilling’ continuing education for current employees. Vanna Boffo (University of Florence), Monica Fedeli (University of Padua)* From here, Didactics, Guidance, Calling, Traineeship, and Employability become focal points for the development of education, but above all to define a matrix of innovation and renewal for the uni- versity system. y y Accordingly, we could say that this chain of reflection represents the second passage for process innovation that even basic research can produce. The research that we put together aims to present the results along three main lines: • the study of developments and changes in university students’ subjec- tivity in relation to their personal formation (non-formal and informal contexts) and university education and training (formal); • the study of developments and changes in university students’ subjec- tivity in relation to their personal formation (non-formal and informal contexts) and university education and training (formal); • the impact that the teachings at university ‒ meaning a structure pro- viding knowledge, practice, reflection, and experience ‒ have in ac- companying and developing the necessary skills for people to enter professional and life contexts and self-orient their formative process • the organizational dimensions and the university practices of place- ment, in the conviction that altering the system context is one of the engines for change that can respond to the new requirements that the world of professions and work demands from young graduates. The research project was split into six phases. The research addressed the main moments in the students’ academic life: career guidance up- on entry, personalized teaching, formation of the professional vocation, profession-building activities such as internships and work experiences, and lastly job placement. Within the areas shown hereafter, each unit developed and investigated each of these topics in parallel. The research also dealt with the micro, macro, and mega dimensions, and this takes us to the third point of reflection on the outcome of the innovation achieved. Along with reflection on the subjects, students, grad- uates, and young adults, we elaborated reflection on the educational struc- tures – where normally only traineeship is recalled, and in addition, we explored the potential of educational transformation which the university can and must look to through reflection on competences and employability. As three distinguished colleagues pointed out at the closing confer- ence on our work, the prospects from which to depart are many. We INNOVATION AND KNOWLEDGE TRANSFER OF THE RESEARCH 483 shall summarize these by using these reflections which can also take us into an international scenario. Vanna Boffo (University of Florence), Monica Fedeli (University of Padua)* Also, as recently basic in- come, robot tax, or jobless growth gain keener attention, the issue of employability and transition should be actively combined with new so- cial platforms like the shared economy, and the new way of combining work and life, which will also dramatically change the role of higher education in weaving new social textures of the issues of employabil- ity and social inclusion, as well as personal development. I will deliver three key changes to consider, from the international perspectives, that 484 VANNA BOFFO, MONICA FEDELI (1) the ecosystem of higher education, both in Europe and Asia is be- ing quickly reshaped; (2) Key competences in the workplace, facing the emergence of Super-Intelligence and Super-connection, are increasingly seeking the values of liberal arts and pure sciences, cultivated in higher education, which are being fused and interconnected to meet the com- plex world; (3) The emergence of artificial intelligence will diminish job opportunities, and ‘employability’ and ‘social inclusion’ will not be separate from one another, creating a new social and economic platform where the concept of ‘employment’ will be dramatically re-conceptual- ized (Emp&Co. Conference 2017). Future perspectives on this issue tell us that Eastern Asia, unlike Italy and Europe, is building new higher education models precisely to antici- pate the complex social changes that we shall shortly have to face. The question has to do with the development of individual skills through new curricula to initiate the creation and development of new higher education ecosystems. From a political perspective, Maria Slowey of Dublin City University has asked us to reflect on the role of the university for the development of a country in the light of the last ten years of economic-financial cri- ses and social, civil, and political austerities, which have fuelled the de- bate on the guidelines to be given and on the value of higher education: Higher education (comprising not only universities but also a diverse range of other tertiary institutions such as specialist colleges, polytech- nics, and professional institutions) is increasingly expected to fulfil a wide variety of roles for society and, more narrowly, for the economy ‒ such as widening access, meeting labour market needs, technology transfer, contributing to regional development and civic engagement. It is pos- sible to explore a central paradox ‒ or, as Collini (2012) interprets it, a ‘Faustian pact’. Vanna Boffo (University of Florence), Monica Fedeli (University of Padua)* To the extent that universities succeed in offering a ‘ser- vice provider’ role to important stakeholders (in particular, the state and employers) is this at the cost of effectively abandoning its transformative mission? This mission has traditionally involved generating new knowl- edge and reflecting critical analytic, independent perspectives back to the society of which universities are a part. As Collini put it: One be- gins to wonder whether societies do not make a kind of Faustian pact when they set up universities: they ask them to serve various purposes, but if they are to be given the intellectual freedom necessary to serve these purposes, they will always tend to exceed or subvert those pur- poses. (Collini 2012: 7). There are fundamental questions about the role of higher education in contemporary society: in particular, the balance between private and public good. The global expansion of higher edu- cation coupled with the development of the ‘knowledge economy’ or ‘knowledge society’ has led to higher education being increasingly at the centre of policy interest at both international levels (through bodies such as the EU, OECD, World Bank, and UNESCO) and national lev- INNOVATION AND KNOWLEDGE TRANSFER OF THE RESEARCH 485 els. The paradox is thus that while Higher Education is seen as increas- ingly important for the successful development of society, its core values and purposes are, arguably, facing unprecedented threats (Emp&Co. Conference 2017). In conclusion, we could say that we need to think about the research results precisely to interpret tomorrow, and what will happen in and for university contexts. y The educational and pedagogical sense of our work lies in the fact of having conducted our disciplines in unexplored territories to try to com- bine the speed of change and stay within the innovation and prospects for a future that is already ‘today’. If the research has taught us anything, it is that a large dose of courage is necessary to remain dissatisfied. In fact, in the face of so many changes, we need to be able to transform our way of being. While some styles of thinking, language, and professions are disappearing, others are appearing. We must be able to understand and model the transformations, to avoid producing ‘injustices and creating new marginalization’. This is the task of the research and this is the task of the transfer of knowledge and learning. Clancy P. 2015, Irish higher education: a comparative perspective, Institute of Public Administration, Dublin. Collini S. 2012, What are universities for?, Penguin Books, London. Di k J 2017 T hi d L i i Hi h Ed i h // i Slowey M. 2017, European and International Policies on Higher Education: Conflicting Priorities- a ‘Faustian Pact’?, <http://empeco.prin.it/book-of- abstracts-international-conference/> (01/2018). Dirkx J. 2017, Teaching and Learning in Higher Education, <http://empecoprin. it/book-of-abstracts-international-conference/> (01/2018). Clancy P. 2015, Irish higher education: a comparative perspective, Institute of Public Administration, Dublin. Han S. 2017, Employability and Transitions from an International Perspective, <http://empeco.prin.it/book-of-abstracts-international-conference/> (01/2018). , Collini S. 2012, What are universities for?, Penguin Books, London. References Clancy P. 2015, Irish higher education: a comparative perspective, Institute of Public Administration, Dublin. Collini S. 2012, What are universities for?, Penguin Books, London. Dirkx J. 2017, Teaching and Learning in Higher Education, <http://empecoprin. it/book-of-abstracts-international-conference/> (01/2018). Han S. 2017, Employability and Transitions from an International Perspective, <http://empeco.prin.it/book-of-abstracts-international-conference/> (01/2018). Slowey M. 2017, European and International Policies on Higher Education: Conflicting Priorities- a ‘Faustian Pact’?, <http://empeco.prin.it/book-of- abstracts-international-conference/> (01/2018). LIST OF AUTHORS Nicola Andrian, PhD Student, Department of Philosophy, Sociology, Pedagogy and Applied Psychology (FISPPA) of the University of Pad- ua, Italy, under joint supervision of the PhD Course in Education and Contemporaneity of the University of the State of Bahia, Brazil. Email: nicola.andrian@phd.unipd.it. Barbara Barbieri, PhD, Assistant Professor of Psychology of Human Re- sources, Department of Social Sciences and Institutions, University of Cagliari, Italy. Email: barbara.barbieri@unica.it. Marina Barioglio, PhD, Post-doc Researcher in Education, Department of Human Sciences “Riccardo Massa”, University of Milano-Bicocca, Italy. Email: marina.barioglio@unimib.it. Sergio Bellantonio, PhD, Post-doc Researcher in General & Social Peda- gogy, Department of Physical Education, University of Naples “Parthe- nope” Italy. Email: sergio.bellantonio@uniparthenope.it. Natascia Bobbo, PhD, Researcher in General, Social and Health Pedago- gy, Department of FISPPA, University of Padua, Italy. Email: natascia. bobbo@unipd.it. Vanna Boffo, PhD, Associate Professor of General Pedagogy, Depart- ment of Education and Psychology, University of Florence, Italy. Email: vanna.boffo@unifi.it. Ilaria Bortolotti, PhD Student, Department of Social and Developmental Psychology, Sapienza University of Rome, Italy. Email: ilaria.bortolot- ti@uniroma1.it. Marianna Capo, PhD, SInAPSi Centre, University of Naples Federico II, Italy. Email: marianna.capo@unina.it. 488 EMPLOYABILITY & COMPETENCES Valentina Paola Cesarano, PhD Student, Department of Humanistic Studies, University of Naples Federico II. Italy, Email: valentina- paola.cesarano@unina.it. Donatella Cesareni, PhD, Associate Professor in Educational Stud- ies, Department of Social and Developmental Psychology, Sapienza University of Rome, Italy. Email: donatella.cesareni@uniroma1.it. Massimiliano Costa, PhD, Associate Professor of General Pedagogy, Cà Foscari University, Venice, Italy. Email: maxcosta@unive.it. Antonia Cunti, PhD, Full Professor of General & Social Pedagogy, Department of Physical Education, University of Naples “Parthen- ope”, Italy. Email: antonia.cunti@uniparthenope.it. Anna Dalla Rosa, PhD, Post-doc Researcher in Industrial and Or- ganizational Psychology, University of Padua, Italy. Email: anna.dal- larosa@unipd.it. Gilda Esposito, PhD, Post-doc Researcher in Adult Learning and Ed- ucation, University of Florence, Italy. Email: gilda.esposito@unifi.it. Monica Fedeli, PhD, Associate Professor of Teaching and Learning, and Organizational Development, Department of FISPPA, Univer- sity of Padua, Italy. Email: monica.fedeli@unipd.it. Paolo Federighi, PhD honoris causa at Vest Timisoara University, Full Professor of Adult Education, Department of Education and Psychology, University of Florence, Italy. Email: paolo.federighi@ unifi.it. Daniela Frison, PhD, Post-doc Researcher, Department of FISPPA, University of Padua, Italy. Email: daniela.frison@unipd.it. Glenda Galeotti, PhD, Post-doc Researcher, Department of Educa- tion and Psychology, University of Florence, Italy. Email: glenda. galeotti@unifi.it. Andrea Galimberti, PhD, Post-doc Researcher in Education, Depart- ment of Human Sciences “Riccardo Massa”, University of Milano- Bicocca, Italy. Email: andrea.galimberti1@unimib.it. LIST OF AUTHORS Maria Benedetta Gambacorti-Passerini, PhD, Post-doc Researcher in Ed- ucation, Department of Human Sciences “Riccardo Massa”, Univer- sity of Milano-Bicocca, Italy. Email: maria.gambacorti@unimib.it. 489 LIST OF AUTHORS Gaia Gioli, PhD, Post-doc Researcher, Department of Education and Psychology, University of Florence, Italy. Email: gaia.gioli@unifi.it. Maria Luisa Iavarone, PhD, Full Professor of General and Social Ped- agogy, University of Naples “Parthenope”, Italy. Email: marialuisa. iavarone@uniparthenope.it. Silvia Lazzaro, PhD, Health Professional Educator, Department of FISPPA, University of Padua, Italy. Email: lazzaro.silvia@gmail.com. Cristina Lisimberti, PhD, Post-doc Researcher, Catholic University of the Sacred Heart, Milan, Italy. Email: cristina.lisimberti@unicatt.it. Francesco Lo Presti, PhD, Assistant Professor of General and Social Pedagogy, University of Naples “Parthenope”, Italy, Email: franc- esco.lopresti@uniparthenope.it. Daniela Maccario, PhD, Associate Professor of General Didactics, De- partment of Philosophy and Education Sciences Department, Uni- versity of Turin, Italy. Email: daniela.maccario@unito.it. Pierluigi Malavasi, PhD, Full Professor of General and Social Pedago- gy and Director of the School of Advanced Studies in Environmen- tal Studies (ASA) - Catholic University of the Sacred Heart, Italy. Email: pierluigi.malavasi@unicatt.it. Claudio Melacarne, PhD, Associate Professor of General and Social Pedagogy, Department of Education, Humans Sciences and Inter- cultural Communication, University of Siena, Italy. Email: claudio. melacarne@unisi.it. Tania Morgigno, PhD, Master Degree of Pedagogical Sciences Trainee- ship Tutor, Department of Human Sciences “Riccardo Massa”, Uni- versity of Milano-Bicocca, Italy. Email: tania.morgnigno@unimib.it. Cristina Palmieri, PhD, Researcher in General and Social Pedagogy, Department of Human Sciences “Riccardo Massa”, University of Milano-Bicocca, Italy. Email: cristina.palmieri@unimib.it. Maria Papathanasiou, Volunteer Researcher, Department of Humanistic Studies, University of Naples Federico II, Italy. Email: mpapsal@gmail.com. Stefano Polenta, PhD, Associate Professor of General Pedagogy, Depart- ment of Education, Cultural Heritage and Tourism, University of Mac- erata, Italy. Email: stefano.polenta@unimc.it. 490 EMPLOYABILITY & COMPETENCES Alessandra Priore, PhD, Post-doc Researcher, Department of Motor Sciences and Health, University of Naples, Italy. Email: alessandra_ priore@yahoo.it. Alessandra Priore, PhD, Post-doc Researcher, Department of Motor Sciences and Health, University of Naples, Italy. Email: alessandra_ priore@yahoo.it. Alessandra Romano, PhD, Researcher of General Didactics and Special Education, Department of Scienze della formazione, scienze umane e della comunicazione interculturale, University of Siena, Italy. Email: alessandra.romano2@unisi.it. Bruno Rossi, Honorary Professor of General and Social Pedagogy, University of Siena, Italy. Email: bruno.rossi@unisi.it. Fausta Sabatano, PhD, Post-doc Researcher in General & Social Peda- gogy, Department of Physical Education, University of Naples “Par- thenope”, Italy, Email: fausta.sabatano@uniparthenope.it. Anna Salerni, PhD, Associate Professor, Sapienza University of Rome, Italy. Email: anna.salerni@uniroma.it. LIST OF AUTHORS Nadia Sansone, PhD Student, Department of Social and Develop- mental Psychology, Sapienza University of Rome, Italy. Email: na- dia.sansone@uniroma1.it. Marco Schiavetta, PhD Student, Department of Educational Sciences, University of Genoa, Italy: marco.schiavetta.348@psypec.it. Patrizia Sposetti, Research Fellow, Sapienza University of Rome, Italy. Email: patrizia.sposetti@uniroma.it. Sonia Startari, PhD, Research Fellow at Department of Educational Sciences, University of Genoa, Italy: sonia.startari@unige.it. Andrea Strano, Research Assistant at International Research Centre for Education and Advanced Studies - CISRE, Cà Foscari University, Ven- ice, Italy. Email: andrea.strano@unive.it. Maura Striano, PhD, Professor of General and Social Pedagogy, De- partment of Humanistic Studies, University of Naples Federico II, Italy. Email: maura.striano@unina.it. Giordana Szpunar, PhD, Assistant Professor of General Pedagogy, De- partment of Social and Developmental Psychology, Sapienza Univer- sity of Rome, Italy. Email: giordana.szpunar@uniroma1.it. 491 LIST OF AUTHORS Carlo Terzaroli, PhD Student, Department of Education and Psychology, University of Florence, Italy. Email: carlo.terzaroli@unifi.it. Concetta Tino, PhD, Post-doc Researcher, Department of FISPPA, University of Padua, Italy. Email: concetta.tino@unipd.it. Francesca Torlone, PhD, Researcher in Adult Education, Department of Education and Psychology, University of Florence, Italy. Email: franc- esca.torlone@unifi.it. Michelangelo Vianello, PhD, Associate Professor of Work and Organiza- tional Psychology, University of Padua, Italy. Email: michelangelo.vi- anello@unipd.it. Alessandra Vischi, PhD, Researcher in General and Social Education and Training Coordinator at the School of Advanced Studies in Envi- ronmental Studies (ASA) − Catholic University of the Sacred Heart, Brescia, Italy. Email: alessandra.vischi@unicatt.it. Silvia Zanazzi, PhD, Research Fellow, Sapienza University of Rome, Italy. Email: silvia.zanazzi@uniroma.it. Studies on Adult Learning and Education Federighi P., Boffo V. (a cura di), Primaria oggi. Complessità e professionalità docentef Boffo V., Federighi P., Torlone F., Educational Jobs: Youth and Employability in the Social Economy. Investigations in Italy, Malta, Portugal, Romania, Spain, United Kingdom Federighi P., Torlone F. (a cura di), La formazione al rispetto dei diritti umani nel sistema penale p Torlone F., Vryonides M. (edited by), Innovative Learning Models for Prisoners Torlone F. (a cura di), Il diritto al risarcimento educativo dei detenuti Sava S., Novotný P. (edited by), Researches in Adult Learning and Education: The European Dimension Slowey M. (edited by), Comparative Adult Education and Learning. Authors and Textsf Boffo V., Fedeli M. (edited by), Employability & Competences. Innovative Curricula for New Professions Employability & Competences The book is the final report of the researches, discussions, conversations around and about the Project PRIN Employability & Competences which took place on March 9th-‐11th, 2017 within an International Conference at the University of Florence. It was the final event of the project PRIN2012LATR9N which aims were: «to design innovative programs for higher education, to promote personalized and learner-centered teaching and learning, to build on job competencies, to value talents to create new work-‐opportunities, to support young adults during their employment emergency, as a response to socio-‐economic crisis and as a citizenship action». The research activities concerned the main phases of the students’ academic life: career guidance upon entry, personalized teaching, career calling, professional vocation, profession-‐building activities such as internships and work related experiences, and lastly job placement. VANNA BOFFO, Ph.D., Associate Professor of General Pedagogy at the University of Florence, she is Rector’s Delegate for Job Placement and Program’s Coordinator of the Master Degree Course in Adult and Continuing Education and Pedagogical Sciences. MONICA FEDELI, Ph.D., Associate Professor of Learning and Teaching Adults and Organization Development at University of Padua, she is Advisor for Didactic Innovation and E-learning and Program’s Coordinator of the Master Degree Course in Management of Educational Services and Continuing Education.
W4390667785.txt	https://www.e3s-conferences.org/articles/e3sconf/pdf/2024/05/e3sconf_incasst2024_03002.pdf	en		Numerical investigation on the effect of blunt body deflector on darieus turbine performance	E3S web of conferences	2,024	cc-by	2,721	E3S Web of Conferences 475, 03002 (2024) InCASST 2023 https://doi.org/10.1051/e3sconf/202447503002 Numerical investigation on the effect of blunt body deflector on darieus turbine performance Ahmad Syafiq Rofi’i1, Aditia Aulia1, Muhammad Ferry Fadri1, Muhammad Haidar Syarif1, Elvira Rosmawati Rahman1, and Gunawan Dwi Haryadi2 1 Department of Mechanical Engineering, Republic of Indonesia Defense University, IPSC Area, Sentul, Bogor, Indonesia 2 Department of Mechanical Engineering, Diponegoro University, Semarang, Indonesia Abstract. One of the renewable energy source that is abundant but has not been used optimally is wind energy. Roads are currently the main transportation infrastructure along with the increasing number of motorized vehicles. Wind is one of the best renewable energy sources that can be utilized on the highway where it can be used as a power source to drive the Vertical Axis Wind Turbine (VAWT). Darrieus type turbines can operate at low wind speeds and do not require a specific wind direction. However, the installation of this wind turbine on the highway can be hampered due to the presence of highway light poles. We simulate a light pole as a blunt object placed in front of the turbine which will then be observed for the ratio of torque produced to compare the performance of the turbine with a blunt body and without a blunt body. This study aims to determine the effect of the diameter ratio and distance between the turbine and the blunt object on the torque generated on the rotor. This research uses CFD simulation on NACA 4415 foil. The results showed that placing a blunt object in front of the Darrieus turbine increased the turbine torque. The most optimal increase in torque is obtained at a distance of 700 mm with a blunt body diameter of 0.6 DT or 0.3 DT which can increase the torque output of the turbine. 1 Introduction Road transportation has been the primary transport method in Indonesia. Roads have been a crucial element in connecting rural and urban areas. Road transport is considered the most reliable means of transport in the country, as other means of transport such as railroads have not yet fully developed and are only available in big cities, and the country relies heavily on passenger vehicles. Based on the data from the national statistics center, the total number of motor vehicles increased from 105,303,318 vehicles in 2015 to 141,992,573 vehicles in 2021 [1] and these numbers are expected to increase in the following years. The exact type of vehicles as well as their quantity can be seen in the table 1 below. Corresponding author: aditya.aulia@idu.ac.id © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 475, 03002 (2024) InCASST 2023 https://doi.org/10.1051/e3sconf/202447503002 Table 1. Vehicle type and quantity in Indonesia from 2015-2021. Data taken from BPS (https://www.bps.go.id/indicator/17/57/2/perkembangan-jumlah-kendaraan-bermotor-menurutjenis.html, accesed on 14 August 2023) This growth of motor vehicles means an increased need for roadways, specifically highways. According to the Ministry of Public Works and Public Housing, the government had built 2,832 km of operational highways by the end of 2022. This number is expected to increase to 4,761 km by 2024. These roads needed power to operate their lights and traffic signs. However, using regular fuel power plants to power these roads is less economical in the long run [2]. Thus, these roads need to be able to sustain itself. One possible means of achieving this is by using a wind turbine installed on the road’s divider where it would be spun by passing vehicles. In this research, Vertical Axis Wind Turbine (VAWT) is used. This turbine configuration is chosen due to the relatively low wind velocity of the passing vehicles which produces an average wind speed of 4.4 m/s [3]. VAWT only required a minimum wind speed of 2 m/s compared to its Horizontal Axis Wind Turbine (HAWT) counterpart which needed a minimum wind velocity of 6 m/s [4]. Other advantages of the VAWT configuration are lower noise and less required space, making it suitable for urban applications [4]. The VAWT configuration is also favorable for use on highways as it can pick up wind from both sides (left and right) when installed on the road divider [5]. However, the installation of the turbine may be hampered by highway light poles that can affect the performance of the turbine. This paper aims to investigate the effect of highway light poles on the turbine’s performance. CFD software will be used to simulate the poles’ effect on the turbine. The light pole will be simulated as a blunt body object placed in front of the turbine. The resulting torque of the turbine with the blunt body will be compared to the turbine’s performance with no blunt body. 2 Methods and procedures This study relied on the use of CFD software. Specifically, SolidWorks Flow Simulation was chosen to conduct the necessary calculations. This approach is commonly used as a way to reduce the risk of failure and minimize expenses before carrying out actual experiments. Additionally, in certain situations, simulations can actually serve as a replacement for experiments, especially when dealing with particularly intricate models. Solidworks Flow Simulation Use Navier-Stokes Equation to solve the flow problem [6]. Navier Stokes equation is a formulation of mass, momentum, and energy conservation. 2 E3S Web of Conferences 475, 03002 (2024) InCASST 2023 https://doi.org/10.1051/e3sconf/202447503002 SolidWorks Flow Simulation utilizes the modified k-ε model for turbulence prediction. The aim of this model is to simplify the prediction of turbulence. The modified k-ε model explains the behavior of homogeneous fluids with turbulent, laminar, and transitional flows. It takes into account the conservation laws of turbulence. Where −𝜌𝑢𝑗 ′ 𝑢𝑖 ′ is the Reynolds stress model, ui is the velocity component, P is fluid pressure, ρ is fluid density, τ is deviatoric stress tensor, ε is the dissipation of turbulent kinetic energy, k is turbulent kinetic energy, h is enthalpy, μ is dynamic viscosity of fluid, μt is eddy viscosity, QH is the amount of heat, and S is entropy The aim of this simulation is to investigate the effect caused by highway light poles considered as a blunt body in front of the turbine shown in Figure 1. This simulation involves the use of the NACA 4415 airfoil, with a blunt body positioned in front of the turbine at varying x-axis distance between 700 mm and 1600 mm with 100 mm increment. The diameter of the blunt body ranges from 0.1 times turbine diameter DT to 1 times DT. The inlet acts as a velocity inlet, with an approximate velocity of 4.4 m/s. This velocity is based on the data from Nassir et al. The outlet functions as a wind outlet with a pressure of 101325 Pa. A schematic of this configuration is shown in Figure 2. Fig. 1. Simulation setup with blunt body. 3 E3S Web of Conferences 475, 03002 (2024) InCASST 2023 https://doi.org/10.1051/e3sconf/202447503002 Fig. 2. Boundary condition setup. The SolidWorks Flow Simulation uses an immersed body mesh approach, which consists of rectangular cells adjacent to each other and to the external boundary of the computational domain. The mesh creation process is independent of geometry and the cells can intersect the boundary between solid and fluid as needed. This Cartesian-based mesh approach offers several advantages such as simplicity, speed, and robustness of the mesh generation algorithm, minimal local truncation errors, and robustness of the differential schemes [6]. The mesh domain of the simulation is shown in Figure 3. Fig. 3. Simulation meshing. To achieve precise results with limited computational resources, a specific type of mesh is used. An automatic global meshing method was used to conduct multiple simulations efficiently, with a level 6 mesh and level 4 refinement for local meshing. This approach allowed for collecting highly accurate data while maximizing the available resources. 4 E3S Web of Conferences 475, 03002 (2024) InCASST 2023 https://doi.org/10.1051/e3sconf/202447503002 3 Result and discussion The simulation is performed by varying the x-axis distance with 100 mm increment from 700 mm to 1600 mm, while for every 100 mm the blunt body diameter is varied from 0,1 times DT to 1 times DT in 0,1 times DT increment. The torque produced by the turbine, as depicted in Figure 5, reveals a noteworthy trend. It is evident from the data that the presence of a blunt body placed in front of the turbine leads to a decrease in torque production. This decrease is particularly pronounced as the distance between the blunt body and the turbine increases. In other words, the further the blunt body is positioned in front of the turbine, the more significant the reduction in torque output. This finding highlights the crucial relationship between the placement of the blunt body and the resulting torque, suggesting that the placement of the turbine from such obstructions has a significant impact on the turbine’s performance. Fig. 4. Schematic of force diagram acting on the turbine blades. The rotation of the Daeriuss turbine is primarily achieved through the utilization of lift force, which is a pivotal component in its operation. In addition to the lift force, each blade of the turbine incorporates an airfoil design that inevitably generates a complementary drag force. A visual representation of the forces acting upon the turbine can be found in Figure 4, providing valuable insights into its dynamic performance characteristics. Upon closer examination of Figure 4, it becomes evident that blade number 1 contributes negatively to the overall torque generated by the turbine, effectively resisting its rotation. In stark contrast, turbine blades numbered 2 and 3 exert a positive torque, actively propelling the turbine's rotation. An intriguing facet emerges as we delve deeper into this dynamic interplay between forces and torques: the magnitude of torque generated by these blades exhibits a direct correlation with the prevailing wind velocity. Specifically, higher wind speeds correspond to increased torque production by blades 2 and 3, amplifying the turbine's rotational power. Conversely, for blade number 1, this relationship is inverse, resulting in diminished torque production as wind velocity escalates. Understanding the intricate mechanics of the Daeriuss turbine's operation requires an appreciation of the dual forces at work – lift and drag. The lift force serves as the primary driver behind the turbine's rotation, harnessed through the airfoil-shaped blades. However, it is essential to acknowledge the presence of drag force as well, which acts in opposition to the turbine's motion, presenting a resistance that must be overcome for efficient energy extraction. Figure 4 serves as a visual aid, offering a comprehensive depiction of the forces influencing the turbine's performance. A closer examination of this figure reveals that blade number 1 plays a counteractive role, generating a negative torque that resists the turbine's 5 E3S Web of Conferences 475, 03002 (2024) InCASST 2023 https://doi.org/10.1051/e3sconf/202447503002 rotation. In stark contrast, turbine blades 2 and 3 contribute positively to the overall torque, actively enhancing the turbine's rotational motion. The intriguing facet to note is the dynamic relationship between wind velocity and torque production. As wind speeds increase, blades 2 and 3 respond with heightened torque output, bolstering the turbine's efficiency in harnessing wind energy. On the other hand, blade number 1 exhibits an inversely proportional response to wind velocity, leading to reduced torque generation as the wind accelerates. This intricate interplay between forces, torques, and wind velocity highlights the need for a nuanced understanding of the Daeriuss turbine's performance to optimize its energy conversion under diverse environmental conditions. As we can see from Figure 6, the addition of blunt body decreases the wind velocity on the turbine blades. Due to this, the turbine produces negative torque instead of positive and preventing it from rotating back to its original position and generating electricity. The graph of the torque output is shown on Figure 5. Fig. 5. Torque performance graph. The graph illustrates that when a blunt body is placed in front of a turbine, it effects the turbine’s performance. Most of the time the addition of a blunt body in the front of the turbine will decrease the turbine’s torque. This result is consistent with the experiment by Satrio et al, in which the obstacle placed at the 0º position (right in front of the turbine) will decrease the turbine’s performance [7]. However, this is not always the case. At a distance of 700 mm and diameter of 0.6 DT, the turbine produces the highest output torque at 0.19 Nm, a slight increase from a non-blunt body turbine at 0.14 Nm. This increase also happens at 700 mm distance and 0.3 DT, which produces torque of 0.16 Nm. This phenomenon happens as there is a small increase in wind velocity of blade number 2 as shown in Figure 6. This increase in the wind velocity happens due to the creation of the wake behind the obstacle and therefore increases the positive torque in the co-rotating zone of the turbine as stated by Zidane et al [8]. Based on the chart, it appears that the blunt body has the greatest impact when positioned between 1000 mm and 1200 mm. Nonetheless, as the distance deviates from this range, the impact gradually diminishes, and the torque regains its strength. Eventually, the torque levels off with that of the non-blunt body, suggesting that the effect of the blunt body has dissipated. Another detail from the chart is every graph produced by each distance is similar. The biggest effect of the blunt body occurs when its diameter is 20%, 50%, and 100% of the diameter of the turbine. 6 E3S Web of Conferences 475, 03002 (2024) InCASST 2023 https://doi.org/10.1051/e3sconf/202447503002 Fig. 6. Velocity contour plot of the simulation. As we move past 700 mm distance, the effect that the blunt body has on the turbine will cause the torque value to lower even more. The lowest produced torque value is at 1200 mm distance and 1 DT which produced a torque of -0.39 Nm. Past 1200 mm however, the decrease in torque is not as severe. According to the obtained data, it is recommended to place the turbine 1.6 meter or further to minimize the decreasing torque. However, if the distance between the obstacles is less than 1.6 meter, it is advisable to ensure that the turbine is positioned at a minimum distance of 0.7 meters from any obstacles that are less than 1.6 meters in proximity, and that the turbine diameter is 67% larger than the diameter of the obstacle. 4 Conclusions From the simulation performed, it was found that light poles will effect the torque produced by the turbine. The effect depends on the x-axis distance of the pole from the turbine and the pole diameter. Generally, the placement of the light poles will decrease the torque of the turbine. This is due the decreased wind velocity around the turbine blades caused by the blunt body. The safest placement distance of the turbine is 700 mm in the x-axis from the blunt body with blunt body diameter of 0.6 DT or 0.3 DT as it can increase the turbine’s torque output albeit not much. These results indicate that the placement of the turbine in relation of other objects, such as light poles, needed to be taken into account such that it will not impair the turbine’s performance. References 1. 2. 3. Directorate of Distribution Statistics, Land Transportation Statistics 2021 (BPS RI, Jakarta, 2022) E.H. Bani-Hani, A. Sedaghat, M. Al-Shemmary, A. Hussain, A. Alshaieb, H. Kakoli, Ener. Eng. 115, 61-74 (2018) S.A.H. Nassir, F.M. Zawawi, M.Z. Mazlan, S. Samion, M.A.W.M. Yazid, U. Abidin, J. Adv. Res. Fl. Mech. Th. Sci. 57, 175-184 (2019) 7 E3S Web of Conferences 475, 03002 (2024) InCASST 2023 4. 5. 6. 7. 8. https://doi.org/10.1051/e3sconf/202447503002 T. Hidayat, Wind Power in Indonesia: Potential, Challenges, and Current Technology Overview, in Indonesia post-pandemic outlook, 109-132 (2022) K.M. Saqr, M.N. Musa, J. Ener. Pol. 39, 3873-3877 (2011) A. Sobachkin, G. Dumnov, Numerical Basis of CAD-Embedded CFD, In Proceedings of the NAFEMS World Congress, NWC13, 9-12 June 2013, Salzburg, Austria (2013) D. Satrio, Suntoyo, F. Albatinusa, S. Junianto, S. Musabikha, Madi, F.O. Setiawan, Effect of Positioning a Circular Flow Disturbance in Front of the Darrieus Turbine, In Proceedings of the The 7th International Conference on Marine Technology, SENTA 2022, 20 October 2022, Surabaya, Indonesia (2022) I.F. Zidane, H.M. Ali, G. Swadener, Y.A. Eldrainy, A.I. Shehata, Alexandria Eng. J. 63, 175-189 (2023) 8
https://openalex.org/W1976982913	https://europepmc.org/articles/pmc4282878?pdf=render	English	null	Differentiating allergic and irritant contact dermatitis by high-definition optical coherence tomography: a pilot study	Archives of dermatological research	2,014	cc-by	7,750	Arch Dermatol Res (2015) 307:11–22 DOI 10.1007/s00403-014-1492-4 Arch Dermatol Res (2015) 307:11–22 DOI 10.1007/s00403-014-1492-4 ORIGINAL PAPER Differentiating allergic and irritant contact dermatitis by high-deﬁnition optical coherence tomography: a pilot study Selected HD-OCT features corresponded well with severity of visual scoring. These features might help to quantify the degree of inﬂammation in inﬂammatory skin conditions. HD-OCT might help in optimizing visual patch test scoring in some situations. Keywords High-deﬁnition optical coherence tomography Patch test scoring Transition metals Allergic contact dermatitis Irritant contact dermatitis Differentiating allergic and irritant contact dermatitis by high-deﬁnition optical coherence tomography: a pilot study Marc A. L. M. Boone • Gregor B. E. Jemec • V. Del Marmol Received: 13 April 2014 / Revised: 24 June 2014 / Accepted: 18 August 2014 / Published online: 4 September 2014 The Author(s) 2014. This article is published with open access at Springerlink.com Received: 13 April 2014 / Revised: 24 June 2014 / Accepted: 18 August 2014 / Published online: 4 September 2014 The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Differentiation of allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) is important because of different management requirements. Various non-invasive tests have been used in an attempt to improve diagnosis. In irritant dermatitis, thickening of the epidermis has been a constant ﬁnding. High-Deﬁnition Optical Coherence Tomography (HD-OCT) is a non-invasive real- time three-dimensional imaging technique with cellular resolution for which an adapted algorithmic method for pattern analysis discriminating inﬂammatory skin diseases has been proposed. The aim of this study was threefold. (1) To evaluate the correlation between HD-OCT features and clinical scores of allergic and irritant patch test reactions. (2) To explore the potential of HD-OCT in optimizing the visual patch test scoring. (3) To assess in vivo the cytological and 3-D micro-architectural differences in skin reaction types between doubtful positive ACD and ICD. Twenty-two vol- unteers were patch tested using potassium(VI)dichromate, cobalt(II)chloride, nickel(II) sulfate and palladium(II)chlo- ride. Visual patch test scoring and HD-OCT assisted patch test scoring were performed at 48 and 96 h after patch test application according to ECDRG guidelines. Selected HD- OCT features correlated well with clinical severity scores. HD-OCT assessment improved the visual patch test scoring although not signiﬁcantly. Increased epidermal thickness observed in ICD at ﬁrst reading was a signiﬁcant ﬁnding useful in differentiating doubtful (??) ACD from irritant (IR) ICD reactions. In conclusion, HD-OCT might be a unique tool for in vivo non-invasive real-time three-dimen- sional epidermal thickness measurements helping to differ- entiate IR from doubtful (??) reactions in patch testing. Selected HD-OCT features corresponded well with severity of visual scoring. These features might help to quantify the degree of inﬂammation in inﬂammatory skin conditions. HD-OCT might help in optimizing visual patch test scoring in some situations. (IR) ICD reactions. In conclusion, HD-OCT might be a unique tool for in vivo non-invasive real-time three-dimen- sional epidermal thickness measurements helping to differ- entiate IR from doubtful (??) reactions in patch testing. G. B. E. Jemec Department of Dermatology, Roskilde Hospital, Health Sciences Faculty, University of Copenhagen, Copenhagen, Denmark M. A. L. M. Boone (&) V. Del Marmol Department of Dermatology, Hoˆpital Erasme, Universite´ Libre de Bruxelles, 808 Route de Lennik, 1070 Brussels, Belgium e-mail: dr.boone@scarlet.be Introduction Contact dermatitis (CD) is an inﬂammatory eczematous skin disease mostly caused by chemicals or metal ions that exert toxic effects without inducing a T cell response (contact irritants) or by small reactive chemicals that modify proteins and induce innate and adaptive immune responses (contact allergens) [24]. Irritant and allergic contact dermatitis represent a major health problem. In general, irritant contact dermatitis (ICD) is more frequent than allergic contact dermatitis (ACD) [10]. The preva- lence of CD is high as it is suggested that almost 20 % of the general population suffers from CD to at least one chemical, most commonly nickel (Ni) [19]. Differentiation of ACD and ICD is important because of different thera- peutic and management options. In most cases contact dermatitis is suspected from anamnestic and clinical ﬁnd- ings. Diagnosis is conﬁrmed by patch testing. Recording of patch test reactions follows the guidelines of the Interna- tional Contact Dermatitis Research Group as shown in M. A. L. M. Boone (&) V. Del Marmol Department of Dermatology, Hoˆpital Erasme, Universite´ Libre de Bruxelles, 808 Route de Lennik, 1070 Brussels, Belgium e-mail: dr.boone@scarlet.be 12 3 3 Arch Dermatol Res (2015) 307:11–22 12 selected for this study. Inclusion criteria were (1) suspected contact allergy to transition metals (2) patch testing on the back using the European Standard Series including potas- sium dichromate (VI) Cr2K2O7 (in 0.5 % petrolatum), cobalt (II) chloride hexahydrate CoCl2 . 6H2O (1 % petro- latum) and Nickel(II) sulfate hexahydrate NiO4S.6H2O (5 % petrolatum) AND extended at least with palladium (II)chloride PdCl2 (2 % petrolatum) (Chemotechnique Diagnostics, Vellinge, Sweden). (3) Positive irritant or allergic reaction(s) at ﬁrst and/or second reading to tran- sition metal(s), (4) availability of good quality HD-OCT imaging of reactions at ﬁrst and second reading, and (5) all subjects provided informed consent for HD-OCT imaging. Table 1 Recording of patch test reactions according to the interna- tional Contact Dermatitis Research Group (ICDRG) [4] Table 1 Recording of patch test reactions according to the interna- tional Contact Dermatitis Research Group (ICDRG) [4] tional Contact Dermatitis Research Group (ICDRG) [4] ?? Doubtful reaction; faint erythema only ? Weak positive reaction; erythema, inﬁltration, possibly papules ?? Strong positive reaction; erythema, inﬁltration, papules, vesicles ??? Extreme positive reaction; intense erythema and inﬁltration and coalescing vesicles - Negative reaction IR Irritant reaction of different types NT Not tested Table 1 [33]. Clinical assessment: (Tables 1, 2) The allergic patch test reactions were scored by a board- certiﬁed specialist of dermatology experienced in reading patch test results, using the visual grading scale recom- mended by the International Contact Dermatitis Research Group and the North American Contact Dermatitis Group [33]. Irritant reactions were scored according the irritant reaction types proposed by Wahlberg et al. [33]. The aim of this pilot study is (1) to evaluate the correlation between selected HD-OCT features with clinical severity of patch test reactions (2) to explore the possible optimization of the visual patch test scoring of reactions to transition metals by HD-OCT and (3) to assess in vivo and real time the cytological and 3-D micro-architectural differences in skin reaction types of doubtful positive ACD and ICD. Cross-sectional (CS) and en-face (EF) imaging by high- deﬁnition optical coherence tomography Introduction There is rarely disagreement concerning the reading of obvious irritant and ?? or ??? allergic reactions. By contrast the reading of ?? and ? reactions and some irritant reactions may cause difﬁculties. Skin biopsy is of little help in distinguishing the two because histopathologic changes and inﬂammatory inﬁltrate are often similar at this stage [21]. The selected transition metals are capable of inducing both ICD and ACD reactions and therefore were consid- ered particularly suitable for this study. Tests were applied using Finn Chambers according to guidelines (SmartPractice, Phoenix, USA). The patch test substances were removed after 48 h and evaluated by clinical assessment and HD-OCT imaging 20 min after removal (ﬁrst reading). A second reading was performed 48 h after removal of the patch (Table 2) Both readings are essential because of difference in kinetics of allergic and irritant contact reactions in vivo [4, 19]. A control site of normal skin and a site exposed to petrolatum (vehicle) only were also evaluated. Clinical photographs of skin reactions were taken under standardized conditions with a digital camera. Different non-invasive tests have therefore been used in an attempt to improve diagnostic speciﬁcity [8, 11, 29, 30]. Among these, reﬂectance confocal microscopy (RCM) has been shown to be a promising tool for the differentiation of acute allergic and irritant contact dermatitis in vivo [31]. Relevant RCM features for diagnosis of ACD and ICD have been published recently [3]. Conventional optical coher- ence tomography (OCT) seems to be useful as an objective parameter in grading severity of patch test reactions [11]. Moreover, in irritant contact dermatitis thickening of the epidermis was a constant ﬁnding using different techniques and which could be monitored over time [34]. All clinical and HD-OCT assessments have been per- formed by the same investigator. An obligate irritant agent has not been added because increased epidermal thickness in the case of irritant reactions has already been docu- mented quantitatively by RCM, conventional OCT and high-frequency ultrasound [1, 23, 34] and qualitatively by immunohistochemical ﬁndings [22, 23]. High-deﬁnition optical coherence tomography (HD- OCT) is a more recently introduced non-invasive three- dimensional imaging technique with cellular resolution [4– 7]. An adapted algorithmic method for pattern analysis in inﬂammatory skin diseases has been suggested based on inﬂammation related HD-OCT features [7]. Materials and methods Patients and protocol design High-deﬁnition optical coherence tomography (Skintell Agfa Healthcare Belgium) uses a combination of parallel time-domain interferometry and adaptive optics resulting in a constant homogenous high resolution of 3 lm in all Twenty-two Caucasian subjects with Fitzpatrick skin phototype II or III, aged between 18 and 69 years, were 123 Arch Dermatol Res (2015) 307:11–22 13 Table 2 Visual (V) and HD-OCT assisted (H) patch test scoring # Subject First reading after 48 H Second reading After 96 H Cr Co Ni Pd Cr Co Ni Pd V H V H V H V H V H V H V H V H 1 – – – – ?? ?? ?? ?? – – – – ?/?? ?/?? ? ? 2 ?? ?? IR IR – – – – – – IR IR – – – – 3 IR IR IR IR – – – – IR IR IR IR – – IR 1? 4 ?? ?? ?? ?? ? ? – – ?? ?? ?? ?? ? ? – – 5 – – – – IR IR – – – – – – ? ? IR 1? 6 ?? IR – – – – – – IR IR – – IR IR IR IR 7 IR IR IR IR – – – – – – IR IR – – – – 8 – – – – ? ? – – – – – – ? ? – – 9 – – ? ? ??? ??? ? ? – – ? ? ??? ??? ? ? 10 – – IR IR – – – – – – IR IR – – – – 11 IR IR IR IR ?? ?? ?? IR IR IR IR IR ? ? IR IR 12 IR IR IR IR IR IR – – IR IR IR IR IR IR – – 13 IR IR IR IR IR IR – – IR IR IR IR – – IR IR 14 – – IR IR – – – – – – – – ?? IR – – 15 – – – – ?? ?? – – – – – – ? ? ?? ?? 16 IR IR – – ? ? IR IR – – – – ? ? – – 17 IR IR – – – – – – IR IR – – IR 1? Materials and methods – – 18 IR IR – – – – – – IR IR – – – – IR 1? 19 IR IR – – ?/?? ?/?? ?? ?? – – – – ?? ?? IR 1 20 – – IR IR ? ? ? ? – – ?? IR ?? ?? ? ? 21 – – – – ? ? IR IR – – – – ?/?? ?/?? IR IR 22 IR IR IR IR IR IR – – IR IR IR IR IR IR – – Irr/All ratio 10/3 11/2 10/2 10/2 4/10 4/10 2/5 3/4 8/1 8/1 8/3 9/2 4/12 4/12 8/4 4/8 First reading Second reading Total number of reactions 46 Total number of reactions 48 Irritative reactions visual 26 Irritative reactions visual 28 Irritative reactions HD-OCT 28 Irritative reaction HD-OCT 25 Allergic reactions visual 20 Allergic reactions visual 20 Allergic reactions HD-OCT 18 Allergic reactions HD-OCT 23 Misclassiﬁcation ?? ? IRa 2 Misclassiﬁcation ?? ? IRa 2 Misclassiﬁcation IR ? ??b 0 Misclassiﬁcation IR ? ??b 5 a Italic misclassiﬁed lesions: visual (??) ? HD-OCT (IR) b Bold misclassiﬁed lesions: visual (IR) ? HD-OCT (??) Table 2 Visual (V) and HD-OCT assisted (H) patch test scoring High-Deﬁnition Optical Coherence Tomography was performed on the patch test, control patch test and unaf- fected clinically normal looking paralesional skin. The generated images were analyzed with respect to the fol- lowing criteria: visualization of individual cells in the stratum corneum, stratum granulosum, stratum spinosum and morphology of dermo-epidermal junction, papillary dermis and superﬁcial reticular dermis. directions and dimensions, allowing visualization of cells in the epidermis, papillary dermis and superﬁcial reticular dermis in their micro-architectural context at up to 570 lm in depth. A detailed description of the HD-OCT has been published [4–7]. Relevant RCM features [3] for diagnosis of ACD and ICD were studied on the EF HD-OCT images (Table 3). This set of morphological features described for RCM is very similar to those described for HD-OCT [7]. In contrast to RCM, the in-buildt HD-OCT slice (CS) mode identiﬁes the exact 3-D position of the selected EF image. Epidermal thickness (ET) measurements have been performed using the measurement toolbar of the Skintell program. Normal looking skin assessed by HD-OCT (Fig. 1) Measurements were carried out in a climate-controlled room, at 24 C and 40 % relative humidity. On each of the three individual spots ET mea- surement has been repeated three times. The average of the nine measurements was calculated corresponding to the mean ET. All HD-OCT measurements were performed by the same investigator. Materials and methods Each voxel of the 3-Dimensional HD-OCT image 12 3 Arch Dermatol Res (2015) 307:11–22 14 Table 3 Relevant RCM features for diagnosis of ACD and ICD respectively RCM feature ACD ICD 2d 4d 2d 4d Superﬁcial epidermal changes Stratum corneum disruption/ detached corneocytes (-/?) (-/?) (???) (??) Parakeratosis (?/-) (?/-) (???) (??) Spongiosisa of SG (??) (???) (??) (?) Spongiosis of SS (??) (???) (??) (?) Exocytosis SG (??) (???) (??) (???) Exocytosis SS (??) (???) (??) (???) Vesicle formationb SG (??) (???) (?) (?) Vesicle formation SS (??) (???) (?) (?) Necrosis (?) (?) (???) (???) Increased epidermal thickness (-/?) (?) (??) (???) Basal layer brightness (-/?) (-/?) (??) (?) Blood vessel dilatation (?) (??) (?) (?) Superﬁcial dermal inﬂammatory inﬁltratec (?) (??) (?) (??) Parakeratotic changes are seen as highly reﬂective polygonal cells at the level of SC with a central dark or bright nucleus Intradermal necrosis seen as circumscribed dark spaces with irregular borders with detached KC ddays after patch test placing a Depending on the severity of the clinical reaction: mild, moderate or marked spongiosis b Vesicle formation may be microfocal, diffuse or widespread c Inﬂammatory inﬁltrate either as aggregates or as single cells Table 3 Relevant RCM features for diagnosis of ACD and ICD respectively sample t test or Welch’s t test for unequal sample size and unequal variances. The same parametric procedure was used to evaluate the optimization of the visual patch test scoring by HD-OCT. Pearson’s correlation coefﬁcient between clinical scores and selected HD-OCT features was determined. sample t test or Welch’s t test for unequal sample size and unequal variances. The same parametric procedure was used to evaluate the optimization of the visual patch test scoring by HD-OCT. Pearson’s correlation coefﬁcient between clinical scores and selected HD-OCT features was determined. HD-OCT features of allergic contact dermatitis (Figs. 2, 3, 4, 5) Cross-sectional and EF HD-OCT features of ACD were similar as those described previously for acute spongiotic dermatitis [7]. Darker areas relative to the surrounding epithelium of the stratum spinosum corresponding to spongiosis were seen on EF HD-OCT images. In these areas larger intercellular spaces between round to polygo- nal mildly reﬂective keratinocytes were observed. On CS HD-OCT images an increase in epidermal thickness but Results Visual patch test scoring at ﬁrst and second reading (Table 2) Visual patch test scoring at ﬁrst and second reading (Table 2) The overall irritative/allergic reactions ratio was 26/20 at the ﬁrst visual reading and 28/20 at the second visual reading. The individual irritative/allergic reactions ratio for each transition metal was 10/3(Cr), 10/2(Co), 4/10(Ni) and 2/5(Pd) at ﬁrst reading and 8/1(Cr), 8/3(Co), 4/12(Ni) and 8/4(Pd) at second reading. Normal looking skin assessed by HD-OCT (Fig. 1) Cross-sectional and EF HD-OCT features of normal skin were similar as those described previously [4]. In the CS mode stratum corneum was deﬁned as a hyporeﬂective ‘‘band’’ limited by two thin hyperreﬂective lines. The upper one corresponded with the entrance signal and the lower one corresponded with an interference signal due to the difference in refraction indices between stratum corneum and stratum granulosum. If stratum corneum was very thin both hyperreﬂective lines merged to become one thicker line. The epidermis was seen as a more reﬂective homog- enous band below the stratum corneum. The sub-basal membrane area appeared as a dark zone in between the basal cell layer and the higher reﬂective papillary dermis. On EF mode the corneocytes and the grainy cells were identiﬁed. The typical honeycomb structure of the stratum spinosum cells could be noticed. The basal cells around the dark dermal papillae could be observed. Adnexal structures and blood vessels were differentiated from other dermal microstructures. Parakeratotic changes are seen as highly reﬂective polygonal cells at the level of SC with a central dark or bright nucleus is deﬁned by a unique set of x, y, z coordinates. These coordinates permit to measure with great accuracy in vivo and real time the epidermal thickness three-dimensionally and non-invasively. The ET measurement was assessed at the suprapapillary plate in between adnexal epithelium on three different spots on CS image: one with the visual highest ET, one with the visual lowest ET and one spot with intermediate ET. The measurement procedure is demonstrated in Fig. 1. Measurements were carried out in a climate-controlled room, at 24 C and 40 % relative humidity. On each of the three individual spots ET mea- surement has been repeated three times. The average of the nine measurements was calculated corresponding to the mean ET. All HD-OCT measurements were performed by the same investigator. is deﬁned by a unique set of x, y, z coordinates. These coordinates permit to measure with great accuracy in vivo and real time the epidermal thickness three-dimensionally and non-invasively. The ET measurement was assessed at the suprapapillary plate in between adnexal epithelium on three different spots on CS image: one with the visual highest ET, one with the visual lowest ET and one spot with intermediate ET. The measurement procedure is demonstrated in Fig. 1. Statistical analysis (3) Stratum corneum is deﬁned as a hyporeﬂective ‘‘band’’ limited by two thin hyperreﬂective lines (red arrows). The upper one corresponds with the entrance signal and the lower one corresponds with an interference signal due to the difference in refraction indices between stratum corneum and stratum granulosum. If stratum corneum is thin both hyperreﬂective lines merge to become one thicker line (yellow arrow ‘‘3’’). (4) Epidermal thickness is then measured between this thicker line and the blue line by using the measurement toolbar of the Skintell(R)program (yellow arrow ‘‘4’’ pointing to the yellow accolade) ‘‘band’’ limited by two thin hyperreﬂective lines (red arrows). The upper one corresponds with the entrance signal and the lower one corresponds with an interference signal due to the difference in refraction indices between stratum corneum and stratum granulosum. If stratum corneum is thin both hyperreﬂective lines merge to become one thicker line (yellow arrow ‘‘3’’). (4) Epidermal thickness is then measured between this thicker line and the blue line by using the measurement toolbar of the Skintell(R)program (yellow arrow ‘‘4’’ pointing to the yellow accolade) Thisincreasewassigniﬁcant comparedtodoubtfulpositive (??)ACD and normalskin atthe ﬁrstreading (p \ 0.001) and second reading (p \ 0.001) (Welch’s t test, unequal sample sizes and unequal variances). The ET of normal skin of the back was 47.38 lm [?/-1.07 lm; 95 % conﬁdence interval (CI)]. In ICD at ﬁrst reading, the ET increased with 12.57 lm (?/-2.48 lm; 95 % CI) compared to 2,51 lm increase (?/- 3.71 lm; 95 % CI) in doubtful positive (??) ACD. At second reading, the ET increases in ICD measured 9.87 lm (?/- 2.33 lm; 95 % CI) and in doubtful positive (??) ACD 2.89 lm (?/- 2.56 lm: 95 % CI). decrease in reﬂectivity could be observed. Smaller but higher reﬂective cells, single or in small aggregates were observed at the level of the stratum spinosum in both EF and CS modes. Intercellular edema stretching apart kerat- inocytes sometimes resulted in the formation of intraepi- dermal vesicles. This vesicle formation secondary to focal areas of spongiosis could be noticed on both EF and CS modes. These vesicles presented as dark round to poly- lobulated areas. In case of severe allergic reactions macro- vesicles were observed. Cells could be observed in- and outsides these cavities. Superﬁcial epidermal morphological and cellular chan- ges were also found in ICD compared to ACD. Statistical analysis Numerical variables such as epidermal thickness mea- surements were analyzed using the independent two- 123 123 15 Arch Dermatol Res (2015) 307:11–22 Fig. 1 Epidermal thickness (ET) measurement procedure by High- Deﬁnition Optical Coherence Tomography 3-D imaging: (a) Cross- sectional (CS) mode and (b) En-face (EF) mode. Each voxel of the 3-D HD-OCT imaging is deﬁned by a unique set of x, y, z coordinates. (1) First the basal cell layer at the suprapapillary plate will be searched for in the EF mode at the cross of green and red line (yellow arrow ‘‘1’’). (2) The exact position of the basal cell layer in the CS mode can be determined at the cross of green and blue line (yellow arrow ‘‘2’’). (3) Stratum corneum is deﬁned as a hyporeﬂective Fig. 1 Epidermal thickness (ET) measurement procedure by High- Deﬁnition Optical Coherence Tomography 3-D imaging: (a) Cross- sectional (CS) mode and (b) En-face (EF) mode. Each voxel of the 3-D HD-OCT imaging is deﬁned by a unique set of x, y, z coordinates. (1) First the basal cell layer at the suprapapillary plate will be searched for in the EF mode at the cross of green and red line (yellow arrow ‘‘1’’). (2) The exact position of the basal cell layer in the CS mode can be determined at the cross of green and blue line (yellow arrow ‘‘2’’). (3) Stratum corneum is deﬁned as a hyporeﬂective ‘‘band’’ limited by two thin hyperreﬂective line upper one corresponds with the entrance signa corresponds with an interference signal due t refraction indices between stratum corneum and If stratum corneum is thin both hyperreﬂective lin one thicker line (yellow arrow ‘‘3’’). (4) Epiderm measured between this thicker line and the blu measurement toolbar of the Skintell(R)program pointing to the yellow accolade) Fig. 1 Epidermal thickness (ET) measurement procedure by High- Deﬁnition Optical Coherence Tomography 3-D imaging: (a) Cross- sectional (CS) mode and (b) En-face (EF) mode. Each voxel of the 3-D HD-OCT imaging is deﬁned by a unique set of x, y, z coordinates. (1) First the basal cell layer at the suprapapillary plate will be searched for in the EF mode at the cross of green and red line (yellow arrow ‘‘1’’). (2) The exact position of the basal cell layer in the CS mode can be determined at the cross of green and blue line (yellow arrow ‘‘2’’). HD-OCT features of irritant contact dermatitis (Figs. 2, 3) HD-OCT features of irritant contact dermatitis Statistical analysis These changes could be observed in combined (3-D) CS and EF HD-OCT mode. The changes consisted of (1) signiﬁcant parakeratosis whereby parakeratotic cells appeared as bright highly reﬂective round to oval nucleated structures centrally located within stratum corneum, (2) stratum corneum disruption with disrupted corneocytes presented as single or sheets of multiple discrete mildly bright cel- lular structures larger than normal keratinocytes without identiﬁable nucleus, (3) necrotic keratinocytes presented on HD-OCT as polygonal structures brighter and larger than surrounding keratinocytes. Edema in the dermis appeared to lower dermal reﬂec- tivity. In severe forms no dermal reﬂectivity remained. In moderate forms, dilated signal free cavities in the dermis corresponding to blood vessels were noticed. In these forms the dermal perivascular inﬂammation was marked with small highly reﬂective cells. HD-OCT features of irritant contact dermatitis A signiﬁcant increase in epidermal thickness (ET) was found in ICD by CS HD-OCT measuring the thickness of the suprapapillary ET. 12 3 3 Arch Dermatol Res (2015) 307:11–22 16 n with clinical/visual mation by HD-OCT ribing inﬂammation could be consisted of features linked to i i i t id l i l superﬁcial dermal inﬂammatory inﬁltrate associated with blood vessel dilatation. The degree of epidermal changes related to water accumulation correlated well with clinical severity scores. The degree of dermal edema also corre- lated with clinical severity scores. The more severe the d l d th l th bl d l dil t ti d Fig. 2 Visual patch test scoring graded and corresponding HD- OCT images 48 h after patch application: (a) ?? for Ni(II) and (b) IR for Cr(VI).On the corresponding CS (slice) HD- OCT imaging a difference in epidermal thickness above the suprapapillary plate is observed between both conditions (green versus red vertical line). On the IR CS HD-OCT image a disruption of the stratum corneum is noticed (yellow arrow). On the IR EF image a parakeratosis (green arrow) is noticed as well as several necrotic keratinocyte (magenta arrow). On EF images of both conditions spongiosis is present (yellow circles). Z- values = depth in lm of the en- face image values = depth in lm of the en- face image HD-OCT features: correlation with clinical/visual severity. Grading of inﬂammation by HD-OCT Two groups of features describing inﬂammation could be determined. The ﬁrst group consisted of features linked to water accumulation: spongiosis, intraepidermal vesicle formation and dermal edema. The second group consisted of features related to cellular inﬁltrate: exocytosis and superﬁcial dermal inﬂammatory inﬁltrate associated with blood vessel dilatation. The degree of epidermal changes related to water accumulation correlated well with clinical severity scores. The degree of dermal edema also corre- lated with clinical severity scores. The more severe the dermal edema, the less the blood vessel dilatation and superﬁcial dermal inﬂammatory inﬁltrate could be per- ceived Figs 4 5 HD-OCT features: correlation with clinical/visual severity. Grading of inﬂammation by HD-OCT HD-OCT features: correlation with clinical/visual severity. Grading of inﬂammation by HD-OCT HD-OCT features: correlation with clinical/visual severity. Grading of inﬂammation by HD-OCT superﬁcial dermal inﬂammatory inﬁltrate associated with blood vessel dilatation. The degree of epidermal changes related to water accumulation correlated well with clinical severity scores. HD-OCT features of irritant contact dermatitis The degree of dermal edema also corre- lated with clinical severity scores. The more severe the dermal edema, the less the blood vessel dilatation and superﬁcial dermal inﬂammatory inﬁltrate could be per- ceived Figs. 4, 5. Two groups of features describing inﬂammation could be determined. The ﬁrst group consisted of features linked to water accumulation: spongiosis, intraepidermal vesicle formation and dermal edema. The second group consisted of features related to cellular inﬁltrate: exocytosis and 123 123 Arch Dermatol Res (2015) 307:11–22 17 ch test scoring assisted patch test scoring at g The difference was not signiﬁcant between visual scoring and HD-OCT evaluation at the ﬁrst reading. HD-OCT assisted scoring at the second reading, however suggested that ﬁve irritant reactions could be re-classiﬁed as allergic reactions and two doubtful reactions as irritant reactions At second reading reactions Fig. 3 Visual patch test scoring graded and corresponding EF HD-OCT images 48 h after patch application: (a) ?? for Ni(II) and (b) IR for Cr(VI). Pronounced spongiosis of stratum granulosum and stratum spinosum is observed in both reaction types (yellow circles). No vesicle formation is noticed. Exocytosis in stratum granulosum and stratum spinosum is more pronounced in the ??-reaction (yellow arrows). Blood vessel dilatation and superﬁcial dermal inﬂammatory inﬁltrate is similar in both reaction types (magenta circles). Dermal edema is almost absent because dermal structures are visible at higher Z-values. Z-values = depth in lm of the en-face image Optimization of the visual patch test scoring by HD-OCT signiﬁcant between visual scoring and HD-OCT evaluation at the ﬁrst reading. HD-OCT assisted scoring at the second reading, however suggested that ﬁve irritant reactions could be re-classiﬁed as allergic reactions and two doubtful reactions as irritant reactions. At second reading, reactions Table 2 displays the HD-OCT assisted patch test scoring at the ﬁrst and second reading. The difference was not 12 Arch Dermatol Res (2015) 307:11–22 18 ically most often misclassiﬁed as IR instead ?) reactions. Although HD-OCT improved ing, the difference between visual scoring assisted scoring was not signiﬁcant. Discussion Differentiating ACD and ICD remains a challenge for clinicians and researchers [26]. A perfect golden standard ch test scoring g cross- (at different Z- images of Ni on graded (?) application. ermal edema is pared to ?? idermal visicle rved. Due to e visualization vessels and l inﬂammatory Z- n lm of the en- Fig. HD-OCT features of irritant contact dermatitis 4 Visual patch test scoring and corresponding cross- sectional and EF (at different Z- values) HD-OCT images of Ni (II) allergic reaction graded (?) 48 h after patch application. Spongiosis and dermal edema is more severe compared to ?? reactions. Intraepidermal visicle formation is observed. Due to dermal edema the visualization of dilated blood vessels and superﬁcial dermal inﬂammatory inﬁltrate is lesser. Z- values = depth in lm of the en- to Pd were clinically most often misclassiﬁed as IR instead of doubtful (??) reactions. Although HD-OCT improved patch test scoring, the difference between visual scoring and HD-OCT assisted scoring was not signiﬁcant. Discussion Differentiating ACD and ICD remains a challenge for clinicians and researchers [26]. A perfect golden standard to Pd were clinically most often misclassiﬁed as IR instead of doubtful (??) reactions. Although HD-OCT improved patch test scoring, the difference between visual scoring and HD-OCT assisted scoring was not signiﬁcant. Differentiating ACD and ICD remains a challenge for clinicians and researchers [26]. A perfect golden standard Differentiating ACD and ICD remains a challenge for clinicians and researchers [26]. A perfect golden standard 123 12 123 Arch Dermatol Res (2015) 307:11–22 19 ntiation between ACD and ICD does not ns can be clinically and histologically despite their different pathogenesis [17, two hallmarks of ICD are perturbation of hyperproliferation [9, 25]. The predominant ultrastructural change in the epidermis of acute ACD is spongiosis [20]. This paper suggests that the increased epidermal thick- ness (ET) observed in ICD at ﬁrst reading is a signiﬁcant ﬁnding useful in differentiating doubtful (??) ACD from test scoring CS and EF s) HD-OCT ergic ) 48 h after he more he more is (yellow ation exocytosis re severe e lesser the on and ﬂammatory erceived. Z- m of the en- Fig. 5 Visual patch test scoring and corresponding CS and EF (at different Z-values) HD-OCT images of Cr(VI) allergic reaction graded (??) 48 h after patch application. The more severe the reaction the more intense the spongiosis (yellow circle), vesicle formation (yellow arrow) and exocytosis (red circle). The more severe the dermal edema the lesser the blood vessel dilatation and superﬁcial dermal inﬂammatory inﬁltrate could be perceived. Z- values = depth in lm of the en- face image permitting differentiation between ACD and ICD does not exist. Both conditions can be clinically and histologically indistinguishable despite their different pathogenesis [17, 28, 35]. HD-OCT features of irritant contact dermatitis A previous study suggested that HD-OCT features of inﬂammatory skin conditions with epidermal alteration correlate well with dermatopathologic descriptors as deﬁned in RCM [7]. The morphological CS and EF fea- tures of acute spongiotic skin conditions imaged by HD- OCT in this study were very similar to those demon- strated for conventional OCT and RCM, respectively [11, 16]. Furthermore, this paper suggests that HD-OCT might be an useful and unique tool for non-invasive in vivo real-time three-dimensional ET measurements. In vivo measure- ments of ET have been systematically studied in healthy skin using RCM [27] and conventional OCT [12, 13, 18, 32]. Epidermal thickness measurement by RCM is based on at least one vertical (z-axis) stack of 21 EF images collected at 5 lm intervals form the skin surface to a depth of at least 100 lm. The ET corresponds to the distance from the ﬁrst living cells of the granular layer to the basal cells lying on the top of the dermal papillae. Although some epidermal properties vary by site, the most important limitation is the degree of within-site variation accounting for between 50 and 74 % of the total variation in ET observed. This variation was not due to measurement errors but corresponds to topographical irregularity. This ﬁne- scale variation limits the use of RCM for quantitative studies of epidermis [27]. Reﬂectance confocal microscopy has been used in non- invasive in vivo studies to demonstrate intraepidermal and superﬁcial dermal cellular changes and micro-architectural alterations in ACD and ICD. These features were later conﬁrmed by correlating them with histopathology [16]. Furthermore, the kinetics of ACD and ICD over time, as well as the individual ethnic susceptibility to ICD have also been examined by using RCM [1, 2]. The sensitivity and speciﬁcity of a series of 12 RCM criteria to differentiate ACD and ICD in reference to patch testing have been determined [3]. This study demonstrates that the degree of changes in some epidermal HD-OCT features of acute spongiotic skin conditions, such as spongiosis, vesicle formation and exocytosis, correlates well with clinical severity scores conﬁrming the ﬁndings of previous histopathological studies and RCM studies [1, 3, 36]. Furthermore, the degree of changes in dermal HD-OCT features correlates also with clinical severity scores. Dermal edema correlates with clinical severity scores which was clearly observed in the CS HD-OCT images. HD-OCT features of irritant contact dermatitis However, two hallmarks of ICD are perturbation of the skin barrier and the epidermal regenerative hyperproliferation [9, 25]. The predominant ultrastructural change in the epidermis of acute ACD is spongiosis [20]. This paper suggests that the increased epidermal thick- ness (ET) observed in ICD at ﬁrst reading is a signiﬁcant ﬁnding useful in differentiating doubtful (??) ACD from (IR) ICD reactions, following exposure to transition metals. hyperproliferation [9, 25]. The predominant ultrastructural change in the epidermis of acute ACD is spongiosis [20]. This paper suggests that the increased epidermal thick- ness (ET) observed in ICD at ﬁrst reading is a signiﬁcant ﬁnding useful in differentiating doubtful (??) ACD from (IR) ICD reactions, following exposure to transition metals. permitting differentiation between ACD and ICD does not exist. hyperproliferation [9, 25]. The predominant ultrastructural change in the epidermis of acute ACD is spongiosis [20]. Both conditions can be clinically and histologically indistinguishable despite their different pathogenesis [17, 28, 35]. However, two hallmarks of ICD are perturbation of the skin barrier and the epidermal regenerative 12 Arch Dermatol Res (2015) 307:11–22 20 The increased ET is probably the result of acute regener- ative hyperproliferation and disturbed differentiation expressed by parakeratosis. [9, 28] Normal values how- ever, have to be established for reference when direct comparisons are not made in an experimental set-up. The increased ET in the case of irritant reactions has already been documented quantitatively by RCM, conventional OCT and high-frequency ultrasound [1, 23, 34] and qual- itatively by immunohistochemical ﬁndings [22, 23]. Con- ventional OCT has been used for the quantiﬁcation and monitoring of the epidermal and dermal changes in irritant contact dermatitis. Repeated measurements were per- formed in healthy volunteers after experimental induction of irritant contact dermatitis. A thickening of the epidermis was a constant ﬁnding and could be monitored over time [34]. determined ET measurement can be performed on CS HD- OCT imaging with higher precision than conventional OCT because of the higher axial resolution of the former (3 lm compared to the 7.5 lm axial resolution of OCT). In a recent paper, HD-OCT measurement of ET in relatively thin lesions appears to create valid results compared with routine histopathology indicating that this method could be well suited to skin monitoring purposes and therapy out- come assessments [14]. 123 References References 1. Astner S, Gonzalez E, Cheung AC, Rius-Diaz F, Mihm MC, Doukas A, Gonzalez S (2005) The kinetics of allergic and irritant contact dermatitis in vivo—a non-invasive evaluation. J Invest Dermatol 124:351–359 2. Astner S, Burnett N, Cheung AC, Rius-Diaz F, Doukas AG, Gonzalez S, Gonzalez E (2006) The impact of skin color on the susceptibility to irritant contact dermatitis, a non-invasive eval- uation. J Am Acad Dermatol 54:458–465 We found an optimization of the visual patch test scoring by HD-OCT assessment although not signiﬁcant. A possible explanation could be the experience in visual patch test scoring. Dermatologists not so familiar with patch test reading could be assisted by HD-OCT to increase their diagnostic accuracy. The qualitative and quantitative differences found between the reactions may imply that earlier readings of patch tests may be possible if supple- mented by HD-OCT. HD-OCT assisted patch test scoring could be very useful when it is difﬁcult to assess erythema; e.g. when dealing with patients with Fitzpatrick phototype IV or higher [2]. Additional studies are essential to validate these suggestions. 3. Astner S, Ulrich M (2012) Spongiotic dermatitis. In: Hofmann- Wellenhof R, Pellacani G, Malvehy J, Soyer HP (eds) Reﬂec- tance confocal microscopy for skin diseases, 1st edn. Springer- Verlag, Berlin Heidelberg, pp 381–389 4. Boone M, Jemec GB, Del Marmol V (2012) High-deﬁnition optical coherence tomography enables visualization of individual cells in healthy skin, comparison to reﬂectance confocal microscopy. Exp Dermatol 21:740–744 5. Boone M, Norrenberg S, Jemec GB, Del Marmol V (2012) Imaging of basal cell carcinoma by high-deﬁnition optical coherence tomography. Histomorphologic correlation. A pilot study. Br J Dermatol 167:856–864 6. Boone M, Norrenberg S, Jemec GB, Del Marmol V (2013) Imaging actinic keratosis by high-deﬁnition optical coherence tomography. Histomorphologic correlation. A pilot study. Exp Dermatol 22:93–97 Some important drawbacks need to be mentioned. Firstly an obligate irritant agent has not been included into the test substances because the increased ET in the case of irritant reactions has already been documented previously by other authors [1, 22, 23, 34]. In addition, the use of transition metals was thought to reﬂect a real-world situa- tion better. Secondly the visual patch test scoring as well as the HD-OCT assisted patch test scoring was performed only by one observer. References However, as mentioned in the method section on three different spots in between adnexal epithelium and at the suprapapillary plate, ET measure- ment has been repeated three times The average of the nine measurements was calculated corresponding to the mean ET. 7. Boone M, Norrenberg S, Jemec GB, Del Marmol V (2013) High- deﬁnition optical coherence tomography, adapted algorithmic method for pattern analysis of inﬂammatory skin diseases. A pilot study. Arch Derm Res 305:283–297 8. Ermolli M, Menne´ C, Pozzi G, Serra MA, Clerici LA (2001) Nickel, cobalt and chromium-induced cytotoxicity and intracel- lular accumulation in human hacat keratinocytes. Toxicology 159:23–31 9. Fartasch M (1997) Ultrastructure of the epidermal barrier after irritation. Microsc Res Tech 37:193–199 10. Frosch JP, John SW (2006) Clinical aspects of irritant contact dermatitis. In: Frosch JP, Menne´ T, Lepoittevin JP (eds) contact dermatitis, 4th edn. Springer-Verlag, Berlin Heidelberg, pp 255–294 11. Gambichler T, Moussa G, Sand M, Sand D, Orlikov A, Altmeyer P, Hoffmann K (2005) Correlation between clinical scoring of allergic patch test reactions and optical coherence tomography. J Biomed Opt 10(6):064030 In conclusion, HD-OCT may be considered as a unique tool for in vivo, non-invasive and real time three-dimen- sional epidermal thickness measurements. Epidermal thickness measurements by HD-OCT help to differentiate IR from doubtful (??) reactions in patch testing and may be performed repeatably in real time. Selected HD-OCT features corresponded well with severity of visual scoring. These features might help to quantify the degree of inﬂammation in inﬂammatory skin conditions. HD-OCT might help in optimizing visual patch test scoring in some situations. 12. Gambichler T, Boms S, Stu¨cker M, Kreuter A, Moussa G, Sand M, Altmeyer P, Hoffmann K (2006) Epidermal thickness asses- sed by optical coherence tomography and routine histology: preliminary results of method comparison. J Eur Acad Dermatol Venereol 20(7):791–795 13. Gambichler T, Matip R, Moussa G, Altmeyer P, Hoffmann K (2006) In vivo data of epidermal thickness evaluated by optical coherence tomography: effects of age, gender, skin type and anatomic site. J Dermatol Sci 44:145–152 14. Gambichler T, Valavanis K, Plura I, Georgas D, Kampilafkos, Stu¨cker M (2014) In vivo determination of epidermal thickness using high-deﬁnition optical coherence tomography. Br J Der- matol 170:737–739 15. Gawkrodger DJ, Lewis FM, Shah M (2000) Contact sensitivity to nickel and other metals in jewerl reactions. J Am Acad Dermatol 43:31–36 HD-OCT features of irritant contact dermatitis The more severe the dermal edema, the less the blood vessel dilatation and superﬁcial dermal inﬂammatory inﬁltrate could be perceived. This inﬂammatory inﬁltrate in HD-OCT is very similar in ICD and (??) or (?) ACD reactions which is in agreement with the literature [21]. Conventional optical coherence tomography is an interesting tool for ET measurements. Epidermal thickness is determined in the OCT image (B-scan) on the computer screen using the integrated measure tool (ruler). ET is manually measured in the OCT image from the skin sur- face reﬂection (entrance signal) to the ﬁrst well-demarcated change of reﬂectance intensity as expressed in a more signal-poor zone [13]. Local positioning of the dermo- epidermal junction between observers was often different. Therefore, a tendency to overestimate the thickness of the epidermis was noticed [12]. The overall irritative/allergic reactions ratio was not signiﬁcantly different between visual scoring and HD-OCT evaluation. At second reading, reactions to Pd were most often misclassiﬁed as IR instead of doubtful (??) reactions. The morphology of patch test reactions can vary with different metals. Some allergens such as Pd report to show a delay in the development of positive tests [15]. This could probably explain why Pd ‘‘behaves’’ as Cr/Co at ﬁrst reading with high irritative/allergic reactions ratio and as Ni at second reading with low irritative/allergic reactions High-deﬁnition optical coherence tomography offers some practical advantages in ET measurement by provid- ing three micrometer lateral and axial resolution data in the 3D micro-anatomical context [4–7]. EF HD-OCT imaging permits to deﬁne with precision both the position of the junction between stratum corneum and granular layer and the position of the basal cell layer in contact with the dermo-epidermal junction. Once both positions have been 123 Arch Dermatol Res (2015) 307:11–22 21 ratio. The allergic reactions to Pd are signiﬁcantly more present at second reading in this study. At ﬁrst reading we probably observe indirectly the stimulation of innate immunity by Pd [24]. As the strength of the innate inﬂammatory response is much higher for Ni than for Pd, the threshold for activation of the adaptive immune system is eventually much higher for Pd [15]. As a result, it takes more time before an allergic reaction to Pd develops. Conﬂict of interest None declared. 16. Gonzalez S, Gonzalez E, White WM, Rajadhyaksha M, Anderson RR (1999) Allergic contact dermatitis, correlation of in vivo confocal imaging to routine histology. J Am Acad Dermatol 40:708–713 Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. 12 3 22 Arch Dermatol Res (2015) 307:11–22 28. Roediger B, Weninger W (2011) How nickel turns on innate immune cells. Immunol Cell Biol 89:1–2 17. 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https://openalex.org/W2615145500	https://hal.archives-ouvertes.fr/hal-02425105/file/Abdou_FrontCellInfectMicrobiol_2017_PubliRegA.pdf	English	null	RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity	Frontiers in cellular and infection microbiology	2,017	cc-by	19,599	To cite this version: Elias Abdou, María Jiménez de Bagüés, Ignacio Martínez-Abadía, Safia Ouahrani-Bettache, Véronique Pantesco, et al.. RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity. Frontiers in Cellular and Infection Microbiology, 2017, 7, pp.186. 10.3389/fcimb.2017.00186. hal-02425105 RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicityi Elias Abdou, María Jiménez de Bagüés, Ignacio Martínez-Abadía, Safia Ouahrani-Bettache, Véronique Pantesco, Alessandra Occhialini, Sascha Al Dahouk, Stephan Köhler, Véronique Jubier-Maurin HAL Id: hal-02425105 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. ORIGINAL RESEARCH published: 18 May 2017 doi: 10.3389/fcimb.2017.00186 ORIGINAL RESEARCH published: 18 May 2017 doi: 10.3389/fcimb.2017.00186 RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity Elias Abdou 1, María P. Jiménez de Bagüés 2, Ignacio Martínez-Abadía 1, Saﬁa Ouahrani-Bettache 1, Véronique Pantesco 3, Alessandra Occhialini 1, Sascha Al Dahouk 4, Stephan Köhler 1 and Véronique Jubier-Maurin 1* Elias Abdou 1, María P. Jiménez de Bagüés 2, Ignacio Martínez-Abadía 1, Saﬁa Ouahrani-Bettache 1, Véronique Pantesco 3, Alessandra Occhialini 1, Sascha Al Dahouk 4, Stephan Köhler 1 and Véronique Jubier-Maurin 1* 1 Institut de Recherche en Infectiologie de Montpellier UMR9004, Centre National de la Recherche Scientiﬁque, Université de Montpellier, Montpellier, France, 2 Unidad de Tecnología en Producción y Sanidad Animal, Centro de Investigación y Tecnología Agroalimentaria, Instituto Agroalimentario de Aragón (CITA-Universidad de Zaragoza), Zaragoza, Spain, 3 Institut de Médecine Régénératrice et Biothérapie—U1183 Institut National de la Santé et de la Recherche Médicale, Montpellier, France, 4 Department of Biological Safety, German Federal Institute for Risk Assessment, Berlin, Germany Edited by: Matthew C. Wolfgang, University of North Carolina at Chapel Hill, USA For aerobic human pathogens, adaptation to hypoxia is a critical factor for the establishment of persistent infections, as oxygen availability is low inside the host. The two-component system RegB/A of Brucella suis plays a central role in the control of respiratory systems adapted to oxygen deﬁciency, and in persistence in vivo. Using an original “in vitro model of persistence” consisting in gradual oxygen depletion, we compared transcriptomes and proteomes of wild-type and 1regA strains to identify the RegA-regulon potentially involved in the set-up of persistence. Consecutive to oxygen consumption resulting in growth arrest, 12% of the genes in B. suis were potentially controlled directly or indirectly by RegA, among which numerous transcriptional regulators were up-regulated. In contrast, genes or proteins involved in envelope biogenesis and in cellular division were repressed, suggesting a possible role for RegA in the set-up of a non-proliferative persistence state. Importantly, the greatest number of the RegA-repressed genes and proteins, including aceA encoding the functional IsoCitrate Lyase (ICL), were involved in energy production. A potential consequence of this RegA impact may be the slowing-down of the central metabolism as B. suis progressively enters into persistence. Moreover, ICL is an essential determinant of pathogenesis and long-term interactions with the host, as demonstrated by the strict dependence of B. suis on ICL activity for multiplication and persistence during in vivo infection. RegA regulates gene or protein expression of all functional groups, which is why RegA is a key regulator of B. suis in adaptation to oxygen depletion. This function may contribute to the constraint of bacterial growth, typical of chronic infection. Reviewed by: Clayton Caswell, Virginia Tech, USA Roy Martin Roop II, East Carolina University, USA Correspondence: Véronique Jubier-Maurin veronique.maurin@cpbs.cnrs.fr Received: 24 March 2017 Accepted: 28 April 2017 Published: 18 May 2017 Reviewed by: Clayton Caswell, Virginia Tech, USA Roy Martin Roop II, East Carolina University, USA Correspondence: Véronique Jubier-Maurin veronique.maurin@cpbs.cnrs.fr Received: 24 March 2017 Accepted: 28 April 2017 Published: 18 May 2017 INTRODUCTION abortus, and signal transmission were comprehensively demonstrated (Carrica et al., 2013). In B. suis and B. abortus, identical RegB/A or PrrB/A regulate the expression of nirK encoding the nitrite reductase, the two operons cyd and cco encoding the bd and cbb3 oxidases, respectively, and fnrN under diﬀerent conditions of oxygenation (Abdou et al., 2013; Carrica et al., 2013). B. suis Oxygen deprivation has a major impact on the physiological state adopted by obligate aerobic human bacterial pathogens to maintain long-term interactions with their host organisms. Chronic infections represent a critical health problem since persistent bacteria are or become resistant to the clinically used antibiotics. This is exempliﬁed by Mycobacterium tuberculosis and the opportunistic pathogen Pseudomonas aeruginosa, and has been shown more recently for Brucella suis, our model of interest. The intracellular pathogen M. tuberculosis induces its dormancy regulon under hypoxia, via the two-component system DosST/R (Boshoﬀand Barry, 2005; Honaker et al., 2009; Boon and Dick, 2012), to become able to persist in vitro (Voskuil et al., 2003), and evidences ﬂexibility of its energy metabolism during mouse lung infection (Shi et al., 2005). P. aeruginosa shelters in nutrient- and oxygen-limited bioﬁlms that allow the bacteria to persist in chronic infections. This bacterium modiﬁes the transcriptional program of its metabolic pathways during the time course of infection in cystic ﬁbrosis (CF) patients up to the stage of long-term persistence, to adapt to the physiological conditions of CF lung including denitrifying anaerobiosis (Palmer et al., 2007; Hoboth et al., 2009). Another characteristic of P. aeruginosa lung infections is the continuous recruitment of polymorphonuclear neutrophils (PMNs) (Jensen et al., 2007). The response regulator of the two-component system RoxS/R, functional homolog of the redox-responsive system RegB/A ﬁrst discovered in Rhodobacter species, plays a crucial role in their trans-epithelial migration (Hurley et al., 2010). The regulon of this system may still be underestimated in P. aeruginosa, since it was characterized under aerobic conditions (Kawakami et al., 2010). RegB/A has representatives in many alphaproteobacteria (Elsen et al., 2004; Wu and Bauer, 2008), among which Brucella spp. belong to the group of bacteria possessing a system with the highest similarity to that of Rhodobacter capsulatus and Rhodobacter sphaeroides. Brucella is a facultative intracellular bacterium responsible for brucellosis, a zoonosis that aﬀects livestock, causing abortion and sterility in animals, but also transmissible to humans provoking a debilitating febrile disease known as Malta fever. INTRODUCTION characterized by nutritional paucity and low oxygen tension (Köhler et al., 2002). Without adequate treatment, Brucella can establish chronic infections with focal complications such as sacroiliitis, the most frequent osteoarticular involvement (Turan et al., 2011), endocarditis or neurobrucellosis (Ariza et al., 2001; Sohn et al., 2003). At the chronic stage, bacteria can be found in hypoxic organs within granulomas or abscesses where anoxic conditions predominate. Therefore, the capacity of this aerobic microorganism to adapt and persist under oxygen deﬁciency could be of major interest for novel therapeutic approaches. Indeed, our previous studies demonstrated that Brucella suis, one of the Brucella species most pathogenic for humans, expresses systems allowing this adaptation. On one hand, the oxidative respiration can use two high-oxygen-aﬃnity terminal oxidases, the cbb3-type cytochrome c and the bd ubiquinol oxidases, on the other hand, the complete denitriﬁcation pathway enables utilization of nitrogen oxides as alternate electron acceptors in an anaerobic or microaerobic respiration process (Loisel- Meyer et al., 2005; Haine et al., 2006). Both respiratory systems are crucial for virulence and/or persistence in vivo. Lack of the cbb3-type oxidase or of its transcriptional activator FnrN (Loisel-Meyer et al., 2005), a potential direct oxygen sensor, caused strong attenuation of B. suis during the chronic phase of infection in oxygen-deﬁcient organs of mice (Jiménez de Bagüés et al., 2007; Abdou et al., 2013). Replication of B. suis and Brucella melitensis strains devoid of nitric oxide reductase, or of its regulator NnrA in the latter, was altered within activated macrophages due to the defective detoxiﬁcation of cellular NO, and in a murine model of in vivo infection (Haine et al., 2006; Loisel-Meyer et al., 2006). Moreover, activity of NtrYX as a redox sensor two-component system involved in oxygen sensing and in regulation of the denitriﬁcation enzymes was described in Brucella abortus (Carrica et al., 2012). Recently, we showed the central role of the two-component system RegB/A in the coordinated control of oxidative respiration and denitriﬁcation in B. suis (Abdou et al., 2013). Extensively studied, this redox sensing system also regulates the respiratory systems as well as many metabolic pathways of Rhodobacter species (Wu and Bauer, 2008). In response to hypoxic conditions, a signal is transmitted, via the redox state of the quinone pool and the cytosolic cysteine, to the sensor histidine kinase RegB. Redox sensor function of PrrB/A, the RegB/A ortholog in B. Citation: Citation: Abdou E, Jiménez de Bagüés MP, Martínez-Abadía I, Ouahrani-Bettache S, Pantesco V, Occhialini A, Al Dahouk S, Köhler S and Jubier-Maurin V (2017) RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity. Front. Cell. Infect. Microbiol. 7:186. doi: 10.3389/fcimb.2017.00186 Citation: Abdou E, Jiménez de Bagüés MP, Martínez-Abadía I, Ouahrani-Bettache S, Pantesco V, Occhialini A, Al Dahouk S, Köhler S and Jubier-Maurin V (2017) RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity. Front. Cell. Infect. Microbiol. 7:186. doi: 10.3389/fcimb.2017.00186 May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Abdou et al. Role of RegA in Brucella suis Persistence Oxygen-dependent activation of two-component systems that control persistence regulons, shared by several aerobic human pathogens, has not been studied in Brucella sp. before. This work therefore contributes signiﬁcantly to the unraveling of persistence mechanisms in this important zoonotic pathogen. Keywords: Brucella, two-component system, RegA, oxygen, persistence, isocitrate lyase, infection, energy metabolism Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org INTRODUCTION suis long-term survival, isocitrate lyase (ICL), the ﬁrst enzyme of the glyoxylate shunt, was assessed by analysis of the corresponding mutant, using our model of persistence. The role of ICL in B. suis-host interaction was evaluated during mice infection. INTRODUCTION Brucellosis is considered as a major cause of economic loss and a re-emerging disease whose impact could increase since the pathogenic potential of the six recently described Brucella species for livestock and humans is yet unknown (Pappas, 2010; Scholz et al., 2016). Brucella possesses a type IV secretion system (T4SS) encoded by the virB operon (O’Callaghan et al., 1999) and responsible for the set-up of the replicative niche (Celli, 2006), May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 2 Role of RegA in Brucella suis Persistence Abdou et al. biotin (0.1 µg), MgSO4(H2O)7 (20 mg), MnSO4H2O (0.1 mg), FeSO4(H2O)7 (0.1 mg)]. RegA is required for bacterial growth or survival under oxygen deﬁciency and, more importantly, is a critical determinant of pathogenesis, as a 1regA strain is unable to trigger a chronic infection in mice (Abdou et al., 2013), suggesting a key role in the establishment of the state of persistence. The present study was undertaken to identify and characterize the RegA-dependent genes potentially involved in the set-up of the persistence state in B. suis. Because large-scale expression analyses are technically diﬃcult to implement in vivo, particularly at the persistence phase when bacteremia is becoming extremely low (Abdou et al., 2013), we developed a novel experimental model in Brucella called “in vitro model of persistence.” This model allowed evaluation of the impact of regA inactivation during long-lasting in vitro persistence of B. suis. Transcriptomic and proteomic approaches were performed to compare wild-type and 1regA mutant strains at the time point where anaerobic conditions become established. In R. sphaeroides, such analyses revealed that PrrA (RegA) regulates as a “master controller” directly or indirectly ∼25% of the genes (Eraso et al., 2008). In the case of B. suis, we demonstrated that under low oxygen tension, the bacteria reduce their basal metabolism as growth decreases or stops and activate glycolysis and denitriﬁcation to maintain energy production (Al Dahouk et al., 2009). According to these results, we correctly predicted numerous systems under the control of RegA and potentially needed for persistence, which will contribute to give an insight into the mechanisms enabling B. suis to set up persistence. Assays were performed to biologically validate the impact of the response regulator on the activity of enzymes involved in energy production. As a signiﬁcant candidate among other factors potentially essential for in vitro B. In vitro Model of Persistence The model was adapted from the Wayne’s model of M. tuberculosis persistence (Wayne and Sohaskey, 2001). The method consists in growing a bacterial suspension in airtight tubes (Sterilin, UK) containing GMM, respecting a head space ratio of 0.5, with slow stirring for continuous homogenization. Ten milliliters-precultures from the stored bacteria (see above) were grown in GMM to an OD600 = 0.7. Precultures were then harvested and diluted 1:30 in fresh GMM to reach a bacterial density of ∼2 × 108 CFU (colony forming units)/ml. Twenty milliliters of the dilution were distributed in tubes of 30 ml. Bacteria were incubated at 37◦C with slow agitation (130 RPM) to ensure homogenization. An additional control tube with an anaerobic indicator strip was prepared to detect the establishment of anaerobic conditions. At each time point, the number of viable bacteria within the corresponding tube was determined by plating serial dilutions on TS agar, and bacteria were collected for RNA extraction. Bacterial Adaptation to GMM Bacterial Adaptation to GMM Fresh colonies of B. suis strains were inoculated in 10 ml of GMM at an initial optical density (OD) of 0.2 and grown at 37◦C, with continuous agitation (160 RPM), for 8 days until cultures reached an OD ∼0.8. Cultures were then diluted (1:4 or 1:5) and subcultured in 10 ml of GMM for 2 days to obtain an OD = 0.8. These subcultures were supplemented with 20% glycerol and stored at –80◦C for further use. Bacterial Strains and Media The Brucella reference strain used in this study, B. suis 1330 (ATCC 23444), and derived mutants were grown in Tryptic Soy (TS) at 37◦C under BSL3 containment according to national laws and under control of the French National Agency for Drugs and Health Products Security (ANSM). Ultra-competent Escherichia coli DH5α (Invitrogen, Carlsbad, CA, U.S.A.) were used for cloning, and plasmid production after culture in Luria Bertani (LB) broth. For strains carrying a kanamycin resistance gene, 50 µg ml−1 antibiotic was used. The knock-out 1regA mutant and its complemented strain were constructed previously (Abdou et al., 2013). For persistence assays, B. suis strains were adapted and grown in Gerhardt’s minimal medium (GMM) (Gerhardt and Wilson, 1948), prepared as follows (per liter): glycerol (30 g), dipotassium phosphate (10 g), sodium chloride (7.5 g), lactic acid 85% (5.8 g), sodium thiosulfate (0.1 g), L-glutamic acid (1.5 g), and supplemented with vitaminic compounds [thiamine (0.2 mg), nicotinic acid (0.2 mg), pantothenic acid (0.04 mg), Construction of the B. suis aceA Mutant and of Its Complemented Strain p aceA (BR1614) mutant was constructed from an initial PCR product cloned into pUC18, comprising the complete coding sequence of aceA with 5′- and 3′-ﬂanking sequences of 550 and 800 bp, respectively. Sequences speciﬁc to restriction enzymes SphI and XbaI were included at the 5′-ends (underlined) of the forward (GCGGCATGCTCGCCGATTTCCACCAGTTCC) and reverse (GCTCTAGAAGCGAACTCAAACTGCGAACG) primers used, respectively. After introduction of the deletion (HincII+NaeI: 866 bp), the 1.2-kb blunted kanamycin resistance gene (kanr) was inserted, the recombinant plasmid was delivered to B. suis, and 1aceA B. suis clones were selected and veriﬁed as previously described (Abdou et al., 2013). The 1aceA strain of B. suis was complemented in trans with the native aceA gene produced from the initial pUC18-clone ampliﬁed with the initial forward primer (see above) and the Comp BR1614 reverse primer CGCGAGCTCGGTACTGGTCCTCCTGGTTC including the SacI restriction site (underlined). The native PCR product was cloned as a 1,407 bp SmaI (85 bp upstream the intiation codon) + SacI fragment into the replicative plasmid pBBR1MCS, under the control of the lacZ promoter. Microarrays Hybridations Speciﬁc B. suis 1330 arrays ((A4325-00-01), Roche Nimblegen, France) consisted of glass slides on which 19 distinct 60-mer oligonucleotides speciﬁc to each of the 3 271 open reading frames of B. suis were spotted in triplicates. All the microarray experiments were performed at the “GeT-Biopuces” platform (Toulouse, France). By using the One Color DNA Labeling Kit (Roche NimbleGen) and according to the manufacturer’s procedures, 1 µg of ds cDNA was labeled by random priming with 100 units of Klenow fragment and Cy3 random primers. After precipitation by isopropanol, 3 µg of Cy3-labeled cDNAs were added to the hybridization mix (Roche NimbleGen) for each microarray in the Hybridization System of Roche NimbleGen, during 17 h at 42◦C. After washing with the Wash Buﬀer Kit (Roche NimbleGen), the slides were automatically scanned with the MS200 Microarray Scanner (TECAN, Männedorf, Switzerland). Images were analyzed with the NimbleScan V 2.6 software. A RMA (Robust Multi-array Average) analysis was performed to normalize background-subtracted raw data. RT-qPCR Analysis q y Five hundred nanograms of the RNA samples used for microarray hybridizations were reverse-transcribed as reported in Abdou et al. (2013), and quantitative PCR experiments (RT-qPCR) were performed using the Light Cycler 480 with SYBR green chemistry (Roche). Primers (Sigma Genosys) were designed using Primer 3 Software (Table S5). Aliquots of cDNA samples were diluted 20- and 2,000-fold to quantify expression of the selected mRNAs and of the constitutively expressed 16S rRNA, respectively, the latter being used to normalize expression values. For the three biological replicates, normalized threshold cycles 1Ct from averaged three technical replicates were used for calculating the fold change using the 11Ct method (Table S3) and the relative fold change (WT/1regA) = 2−11Ct. BR0756, characterized by constant expression in both strains, was also used as reference gene and provided the same results as those obtained with 16S rRNA gene for ampliﬁcation of the target genes BR1614, BRA0703 and BRA0299. For comparison with the RT- qPCR results, microarray data were expressed as the mean log2 values of the hybridization ratio (WT/1regA) (Table S3). Double Stranded cDNA Synthesis y Double-stranded (ds) cDNAs were prepared according to an adapted protocol of SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, UK). Brieﬂy, 10 µg of each denatured RNA sample were reverse-transcribed with random primers (252 pmoles), 0.5 mM dNTP and 400 units of SuperScript III reverse transcriptase, at 45◦C overnight. The second strand was synthesized from the integral ﬁrst-strand reaction according to the manufacturer’s instructions. RNAse clean-up was performed with 4 µg of RNase A, at 37◦C for 10 min. ds cDNAs were puriﬁed with the QIAquick PCR puriﬁcation kit (Qiagen, USA), their concentration was measured with NanoDrop (170–240 ng/µl) and their quality was determined by using the Agilent 2100 bioanalyzer. COG Functional Group Analysis cultures, to stop de novo RNA synthesis and to prevent its degradation. Puriﬁed RNAs were controlled quantitatively and qualitatively, and for absence of contaminating DNA in 0.5 µg samples as previously described (Abdou et al., 2013). p y The COG (cluster of orthologous groups) classiﬁcation of CDS of the B. suis genome was based on the database of Comprehensive Microbial Resource (CMR) or on that of Genoscope found at http://www.genoscope.cns.fr/agc/microscope/mage/. All genes induced or repressed by RegA in the wild-type strain, having respectively a ratio WT/1regA mean value of ≥2-fold or ≤0.5- fold, were classiﬁed according to the functional category to which they belong. Microarrays Statistical Analyses y y Brieﬂy, the two groups of three 1regA (R) samples and three wild-type (W) samples data were compared by using Signiﬁcance Analysis of Microarrays (SAM) tool (Tusher et al., 2001). A two-class unpaired supervised analysis was computed with SAM between the R group and the W group, applying the Wilcoxon statistic test and a setting of 300 permutations. SAM generated a list of signiﬁcant genes with a fold change ≥2 and a false discovery rate (FDR) ≤5 (Figure S1). In silico Search for RegA-Binding Sequences Sequences We used the consensus sequence (C/T)-(G/C)-C-G-G-(C/G)- N(0−10)-G-(T/A)-C-(G/A)-(C/A) (Mao et al., 2005) of the DNA sites recognized by RegA/PrrA from R. sphaeroides and R. capsulatus to perform a manual search for potential RegA- binding sites upstream (1 kb upstream of the initiation codon) of 15 B. suis genes previously validated as being induced by RegA. Only motifs having at least one half-site 100% homologous to that of R. sphaeroides were taken into account. 44 potential RegA binding motifs were detected and used to generate 11 Positional Weight Matrices (PWM) corresponding to each possible spacer length N(0−10). These PWM were validated in our set of 15 B. suis sequences by using the MAST program (Bailey and Gribskov, 1998), which allowed to retrieve most of the 44 motifs initially found. Using these 11 PWM, putative RegA-binding motifs were searched for in DNA sequences either 1 kb upstream or within 200 bp upstream and downstream of the start codon of RegA- dependent individual genes or of the ﬁrst gene in each operon. The structures of operons were obtained from the literature or from predictions based on the transcription directions of the genes and their intergenic sequence lengths as indicated in DOOR (Mao et al., 2014) and BioCyc databases (Romero and Karp, 2004). The MAST program was used to scan the target sequences to search for the motifs. Isolation of RNA from B. suis At each time point, bacterial samples were recovered after addition of 2 ml ethanol/phenol solution (9:1) to 18 ml of the May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 3 Abdou et al. Role of RegA in Brucella suis Persistence DIGE (Differential in Gel Electrophoresis) Labeling g g In the ICPL (Isotope Coded Protein Labeling) experiment, each biological replicate was analyzed separately as technical duplicate, using the quadruplex ICPL experimental setup according to the manufacturer’s procedures. WT replicates were labeled with ICPL0 and ICPL6, and 1regA with ICPL4 and ICPL10. One hundred micrograms of total protein were used for each ICPL labeling reaction, performed according to the manufacturer’s instructions (SERVA Electrophoresis GmbH, Germany). The enzymatic cleavage utilized trypsin (sequencing grade, porcine, SERVA Electrophoresis) and endoproteinase Glu-C (MS grade, Protea Biosciences, Inc.), with an enzyme to substrate ratio of 1:50 and 1:75, respectively. After cleavage, the peptides were acidiﬁed to 1% formic acid for subsequent mass spectrometry analysis. The protein to dye ratio was 50 µg of total protein to 400 pmol dye. The internal standard was prepared by mixing equal amounts of all samples and was labeled with Cy2. For each Cy3 and Cy5 dye, 50 µg of total protein of wild-type and 1regA B. suis strain were used, respectively. A dye swap was performed for the second biological replicate. The labeling was performed as reported in Al Dahouk et al. (2009). Data Availability Statement The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus in the MIAME-compliant format and are accessible through GEO Series accession number GSE87538 (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE87538). May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 4 Role of RegA in Brucella suis Persistence Abdou et al. Image Analysis Protein spots were detected using the Diﬀerential In-gel Analysis mode of the DeCyder software (GE Healthcare, version 6.5.1.1). The spot detection was done estimating 5,000 spots, in the Biological Variation Analysis mode allowing inter-gel matching on the basis of the in-gel standards (Cy2) (Al Dahouk et al., 2009). Preparation of Protein Samples for Proteome Analyses the positive reﬂector mode (detection range 700–4,500 Da). For tandem mass spectrometry, the same instrument was used and fragmentation was based on Collision-Induced Dissociation (CID). The raw spectra were processed with the Data Explorer software (version 4.9, AB Sciex) or the GPS Explorer software (version 3.6, AB Sciex). The measured monoisotopic peptide masses and the tandem MS spectra were combined and compared to all sequences of B. suis in a customized database using the software Mascot (Table S7). The database consisted of the combination of four fasta ﬁles (NC_004310, NC_004311, NC_010167, NC_010169). Furthermore, all sequences were randomized and added to the database for evaluation of false positive rate. Triplicates of eight culture samples (20 ml) of each B. suis WT and 1regA strains have been maintained under conditions of persistence (see above) for 90 h. Bacteria from the three independent cultures were harvested and lysed in ice-cold phosphate-buﬀered saline (PBS), containing 10% trichlororacetic acid. The precipitated bacterial proteins were washed twice with acetone prior to air-drying. Proteins were resuspended and sonicated in the buﬀer previously described (Al Dahouk et al., 2009). After centrifugation, the protein concentration of each supernatant was determined with a Bradford assay and adjusted to 5 mg/ml. Protein Identiﬁcation by Mass Spectrometry Using LC-ESI-MS/MS p y g For nano-LC-ESI (Electrospray ionization)-MS/MS, peptides were separated on an analytical column (C18, 25 cm length, 35◦C) with a linear gradient of 5–50% solution B (A: 0.1% formic acid, B: 80% ACN, and 0.1% formic acid). Mass spectrometry was performed on a linear ion trap mass spectrometer (LTQ Orbitrap Velos, Thermo Scientiﬁc) operating in positive polarity mode online coupled to the nano-LC system. The MS method consisted of a cycle combining one full MS scan (Mass range: 300–1,500 m/z) with 10 data-dependent MS/MS events (CID; 35% collision energy). Splitting of the three biological replicates in separate MS runs allowed further statistical data analysis. 2D Gel Electrophoresis and Image Acquisition The Cy2-, Cy3-, and Cy5-labeled samples were pooled and, after addition of DTT and pharmalytes 3–10, immediately loaded onto the IPG strips to be separated on 2D-DIGE gels (one for each biological replicate) with a non-linear pH gradient of 3– 10. Isoelectric Focusing was performed as previously described (Al Dahouk et al., 2009). Second dimension separation was then carried out on hand-made 13%-acrylamide gels using a SDS- glycine-Tris buﬀer. The 2D-DIGE gels were scanned with an Ettan DIGE Imager at a resolution of 100 µm. Infection and Growth of B. suis Strains in the BALB/c Murine Model the average and standard deviation of all three biological data were calculated using all four combinations of the wild-type to regA ratio. A one-sided T-Test was calculated using these ratios compared to the ICPL10/ICPL4 and ICPL6/0 that represent the technical variation applying an equal variance of the data (Table S6). Methods relative to proteome analyses are available at http:// www.toplab.de/Downloads.html. Survival of wild-type, 1aceA and the complemented 1aceA strains was measured in 7-weeks-old female BALB/c mice inoculated intraperitoneally with 105 CFU of either strain. On days 2, 7, 14, 28, 56, and 84, spleens and livers of six mice per B. suis strain were aseptically removed. After homogenization of the whole organ in PBS, bacterial counts were determined by plating serial dilutions on TS agar supplemented with antibiotics, when needed. Measurement of In vitro Sensitivity to Superoxide Production Triplicates of bacterial cultures were harvested at day 3 of our in vitro model of persistence, and adjusted to a density of ∼107 CFU/ml in PBS. O− 2 was produced during the xanthine oxidase reaction in cell suspensions, according to a procedure, adapted in Hanna et al. (2013). At speciﬁc time points after initiating the reaction, the number of surviving bacteria was determined. The means from three independent platings of each bacterial cell suspension were calculated, and the data obtained were expressed as log10 CFU/ml at each sampling time, ± standard deviations. Ethics Statement Mice experiments were conducted following the guidelines from the Federation of Laboratory Animal Science Associations (FELASA) which adheres to the European Directive 2010/63/EU on the protection of animals used for scientiﬁc purposes. The protocol was approved by the Centro de Investigación y Tecnología Agroalimentaria (CITA) ethical animal experiment committee (protocol 2010-01). Bacterial Lysates Preparation For in vitro analyses, Student’s t-test was applied to the two sets of the three independent experiments to be compared. Variations were considered to be statistically signiﬁcant at P-value ≤0.05. In mice experiments, statistical comparisons between parental strain and the aceA mutant were performed by a one-way ANOVA and by the Fisher’s protected least signiﬁcant diﬀerences (PLSD) tests. P-values ≤0.05 were considered signiﬁcant. Eighty milliliters of bacterial cultures were harvested, then washed with ice-cold PBS, and bacterial pellets were resuspended in 1,200 µl of ice-cold 50 mM imidazole buﬀer pH = 6.8 supplemented with 1 mM DTT. After sonication of bacterial suspensions, the supernatant was obtained by centrifugation for 3 min at 13,000 RPM and ﬁltered (0.2 µm) (Millex, France). The lysates were conserved at −20◦C before further use. RegA Is required for Survival in an In vitro Model of Persistence To assess the activity of nitrite and nitric oxide reductases, NO− 2 and NO−(spontaneously transformed to NO2) consumption was determined by measuring the concentration of nitrite in 50 µl of each B. suis lysates supplemented with 10 mM NO2 in a ﬁnal volume of 200 µl. The reaction was performed under anaerobiosis generated by GENbox packs (oxygen concentration < 0.1%) (bioMérieux, Marcy l’Etoile, France). After 30 min, the nitrite concentration was measured in 100 µl of the samples added to 100 µl of Griess reagent (Loisel-Meyer et al., 2006). Our “in vitro model of persistence” consisted of progressive oxygen depletion consecutive to bacterial growth in minimal medium cultures. Several important parameters had to be adjusted during the development of the model, such as the inoculum dose (2 × 108 CFU/ml) and the head space ratio (0.5). Addition of 5 mM ammonium sulfate enhanced persistence of the wild-type strain. During the ﬁrst day of the experiment (Figure 1), replication of both B. suis wild-type (WT) and 1regA strains resulted in the consumption of oxygen, which was evidenced by the following decrease of the replication rate (day 1 to day 2). A slight diﬀerence between the two strains appeared around day 3 when bacterial counts declined, indicating the onset of anaerobiosis, which was achieved at day 4 as detected by a complete color change of the indicator strip. After day 7, WT counts were found unchanged until the end of the experiment (day 24), indicating that the bacteria entered a non- proliferative persistence phase. In contrast, 1regA showed a signiﬁcant decrease in CFU numbers from day 7 to day 9, and bacterial counts were 1.5-log lower (P < 0.005) than those of the WT beyond day 15 (Figure 1): 1regA was therefore strongly aﬀected during the persistence phase. Aside a slight delay in the decrease of bacterial counts at days 4 and 9, probably linked to regA overexpression, the survival phenotypes of complemented 1regA and WT were not signiﬁcantly diﬀerent (Figure 1). These Protein Identiﬁcation by Peptide Mass Fingerprinting and MALDI-TOF/TOF-MS ICPL Quantiﬁcation and Database Queries The raw data were converted to mzXML format using a software tool from the Trans-Proteomic Pipeline. Peak detection, deconvolution, deisotoping, and quantiﬁcation of the peaks were done using ICPL-ESIQuant. The quadruplet detection was performed for each run separately. Four separate database queries were done using the Mascot software as in 2D-DIGE analysis (see above). For each biological replicate, about 165 proteins were found for which the quantiﬁcation was based on at least two diﬀerent quadruplets and had a coeﬃcient of variance below 40%. Furthermore, the ratios between the technical duplicates were very similar. The threshold for a regulated protein was set to 1.5 and 0.66-fold. For statistical evaluation of the biological data, In order to have suﬃcient material for the mass spectrometry analysis, a preparative gel was prepared (1,000 µg protein load), and stained with Coomassie Blue. Spots, that showed either a quantitative regulation or a statistical signiﬁcance (P < 0.05), but also those that depicted a strong regulation in two biological replicates but not in the third, were selected for MS identiﬁcation. The spots were manually excised and destained, then in-gel trypsin digestion and peptides enrichment were performed as described elsewhere (Al Dahouk et al., 2009). MALDI-TOF mass spectra were acquired using the 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (AB Sciex) operating in May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 5 Role of RegA in Brucella suis Persistence Abdou et al. Isocitrate Lyase Assay Abundance of all class M proteins and most of those belonging to J, E, and G categories (translation, amino acid, and carbohydrate metabolism, respectively), detected as being RegA-controlled, was reduced (Table S2). Taken together, transcriptome and proteome data conﬁrmed that RegA acts mainly as a repressor, consistent with growth arrest and establishment of persistence. FIGURE 1 \| Bacterial growth of strains in the “in vitro model of persistence.” B. suis 1330 wild-type (•), 1regA mutant (⃝), and complemented 1regA (C-1regA) (▼) strains were grown in minimal medium supplemented with 5 mM (NH4)2SO4 for 24 days. At day 1, 2, 3, 4, 7, 9, 15, 18, and 24, triplicate tubes were removed for determining viable bacteria counts. The experiment was reproduced independently three times, in triplicates each. This ﬁgure represents data means ± standard deviation of one of these experiments. GURE 1 \| Bacterial growth of strains in the “in vitro model of FIGURE 1 \| Bacterial growth of strains in the “in vitro model of persistence.” B. suis 1330 wild-type (•), 1regA mutant (⃝), and complemented 1regA (C-1regA) (▼) strains were grown in minimal medium supplemented with 5 mM (NH4)2SO4 for 24 days. At day 1, 2, 3, 4, 7, 9, 15, 18, and 24, triplicate tubes were removed for determining viable bacteria counts. The experiment was reproduced independently three times, in triplicates each. This ﬁgure represents data means ± standard deviation of one of these experiments. Genetic validation of a selection of 62 diﬀerentially expressed genes representing most of the COG functional categories, was performed by quantitative PCR (RT-qPCR) (Table S3). Only seven genes were not validated, because of opposite (BR1781 and BR1043) or below threshold (≤1.5) (BR0111, BR1017, BR1358, BR1648, and BRA0066) fold-change values. It has to be noticed that BR1358 (ureC-2) and BRA0066 (virB4) belong to operons which were validated by RT-qPCR targeting other genes (Table S3). Forty-six genes showed a \|−11Ct\|≥1 (representing a fold change ≥2 or ≤0.5) (Table S3), and 9 other genes were also found adequately regulated but with a slightly lower rate, between −0.74 and −0.97 (Table S3). Namely, 55 genes (88.7%) having a signiﬁcant fold change were validated, as both microarrays and RT-qPCR approaches conﬁrmed identical patterns of RegA regulation. The transcriptome analysis identiﬁed 447 genes, of which 396 are potentially regulated by RegA, and therefore we can postulate that ∼12% of the 3,271 genes of B. Isocitrate Lyase Assay suis may be under the control of RegA. results indicated that regA is required to allow optimal B. suis survival in our model of in vitro persistence. Differential Expression during In vitro Persistence of B. suis According to Figure 1, genes potentially involved in the establishment of the persistent state were expected to be expressed after the initial phase of bacterial growth (day 2) and before the detection of phenotypic diﬀerences between the strains studied (day 4). Expression of RegA-dependent genes speciﬁcally induced under microaerobiosis or anaerobiosis (fnrN, ccoN, and nirK) (Abdou et al., 2013) or participating in nucleic acids metabolism (ada and rpoZ) was therefore quantiﬁed during the ﬁrst 4 days. Accordingly, the whole-genome microarray analysis was performed with three independent wild-type and 1regA cultures harvested at day 3, when anaerobic conditions become established but the viability of both strains was still rather similar. 447 genes were detected with a variation level of the hybridization intensities superior to 2, indicating a potential regulation by RegA (Table S1). In the wild-type strain, 203 (45%) and 244 (55%) genes were found potentially induced and repressed, respectively. Whole proteomes were compared using the 2D-DIGE and ICPL techniques to quantify proteins prepared from subsequent triplicate cultures of both strains at 90 h. Most strikingly, 30% (28) and 70% (66) of the RegA-dependent proteins increased and decreased in concentration, respectively (Table S2). Isocitrate Lyase Assay y y Activity of ICL was evaluated in crude lysates according to a method adapted from the Sigma Aldrich protocol developed for puriﬁed ICL. Glyoxylate produced by the enzyme from isocitrate was detected in the presence of phenylhydrazine, by measurement of increasing absorbance at 324 nm due to accumulation of the glyoxylic acid phenylhydrazone product. Brieﬂy, 20 µl of bacterial lysate were incubated at 30◦C in 1 ml containing 25 µl imidazole 1 M, 33 µl MgCl2 150 mM, 100 µl EDTA 10 mM, 33 µl phenylhydrazine 50 mM. After 30 s, 80 µl of isocitrate 250 mM were added to the reaction, and absorbance was recorded for 330 s. Activity of ICL in lysates was calculated by subtraction of the background values obtained in a reaction without isocitrate. May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 6 Role of RegA in Brucella suis Persistence Abdou et al. FIGURE 1 \| Bacterial growth of strains in the “in vitro model of persistence.” B. suis 1330 wild-type (•), 1regA mutant (⃝), and complemented 1regA (C-1regA) (▼) strains were grown in minimal medium supplemented with 5 mM (NH4)2SO4 for 24 days. At day 1, 2, 3, 4, 7, 9, 15, 18, and 24, triplicate tubes were removed for determining viable bacteria counts. The experiment was reproduced independently three times, in triplicates each. This ﬁgure represents data means ± standard deviation of one of these experiments. encoding proteins with unknown functions (34%) was detected. Taking into account genes with annotated functions only, the functional categories K: transcription (12.2%), M: cell envelope biogenesis (11.9%), and C: energy production and conversion (16.7%) were the most frequently represented (Table S1). A large number of genes involved in transcription were induced by RegA (11.6% of all class K genes), whereas 14 and 17.5% of genes involved in bacterial envelope biogenesis (M) and in energy metabolism (C), respectively, were repressed (Figure 2). Moreover, all the diﬀerentially expressed genes belonging to class D (cell division and chromosome partitioning) were found down-regulated in the WT strain (∼8% of total category D genes). Eight of the nine genes belonging to intracellular traﬃcking and secretion (class U) were part of the virB operon and were down-regulated in the WT strain (Figure 2). Importantly, protein quantiﬁcations obtained by 2D-DIGE and ICPL (Table S2) conﬁrmed the transcriptome data concerning class C genes. Possible Function of RegA as both Direct and Indirect Regulator J, Translation, ribosomal structure, and biogenesis; K, Transcription; L, DNA replication, recombination, and repair; D, Cell division and chromosome partitioning; O, Post-translational modiﬁcation, protein turnover, chaperones; M, Cell envelope biogenesis, outer membrane; V, Defense mechanisms; U, Intracellular trafﬁcking, secretion, and vesicular transport; N, Cell motility and secretion; P, Inorganic ion transport and metabolism; T, Signal transduction mechanisms; C, Energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme metabolism; I, Lipid metabolism; Q, Secondary metabolites biosynthesis, transport, and catabolism; R, General function prediction only; S, Function unknown. conﬁrmed a strong positive impact of B. suis RegA on ntrYX expression in our model. hypothesized that RegA activity in B. suis may be mediated through binding to highly conserved DNA sequences. The R. sphaeroides RegA-binding sequence is present upstream of the gene in the case of gene induction (possibly more than 900 bp from the putative initiation codon; Mao et al., 2005) or at its 5′- end vicinity, in the case of gene repression (Eraso et al., 2008). PWM (Positional Weight Matrices) (Material and Methods) were used to scan genes validated as being regulated by RegA using RT- qPCR (Table S3). Only putative RegA-binding motifs detected with the MAST program in DNA sequences located up to 1 kb upstream or close to the putative initiation codon (±150 bp) of induced or repressed genes were considered (Table S3). Detection of only one regulator, MucR, by the ICPL proteome analysis (Table S2) could be explained by transitory expression, as for fnrN used as a sentinel gene for determination of the time-point chosen for transcriptome analysis, and/or low-level expression of transcription factors. Hypoxia may therefore be considered as another stress factor potentially able to aﬀect MucR expression, in addition to osmotic stress (Mirabella et al., 2013) and nutrient starvation (Al Dahouk et al., 2013). MucR is known to activate a ferrous iron transporter in B. abortus (Caswell et al., 2013; Elhassanny et al., 2013). Interestingly, RegA was found to induce two operons, BR1344/1347 and BRA0675/0678 encoding iron compound ABC transporters (Table S1) and two proteins (Table S2), Dps, a ferritin-like protein (Calhoun and Kwon, 2011), and bacterioferritin, all of the P class (inorganic ion transport and metabolism). Despite the fact that our proteome and transcriptome analyses did not detect pairs of corresponding proteins and genes, both approaches yielded coinciding results. Possible Function of RegA as both Direct and Indirect Regulator The production proﬁles of MucR and both Dps and bacterioferritin proteins were strictly parallel to those previously determined under long-term harsh nutrient starvation of B. suis (Al Dahouk et al., 2013). These proteins could allow iron storage and homeostasis to contribute to long-term viability in media containing the same low ferrous iron concentrations. The RegA-dependent induction of MucR and of operons or proteins participating in iron uptake and storage in B. suis may therefore be linked. Interestingly, all the induced genes possess potential RegA- binding sites in the regulatory regions, whereas sites in a position susceptible to impair correct transcription initiation were detected in only 64% of the repressed genes, regulators excluded (Table S3). This indicated that indirect action of RegA may be more frequent in the case of gene repression. Possible Function of RegA as both Direct and Indirect Regulator Strikingly, among the diﬀerentially expressed genes involved in transcription (K class) and induced by RegA (78%), all of them except two (i.e., 26/28) encode transcriptional regulators (Table S1). Hence, RegA can exert its regulatory function also indirectly by positively interacting with expression of genes encoding regulatory proteins which are implicated in transcription of genes under RegA control. Since RegA of R. capsulatus, R. sphaeroides (PrrA), B. suis, B. melitensis (Elsen et al., 2004), and B. abortus (PrrA) (Wu and Bauer, 2008) share about 70% sequence identity and identical putative HTH (Helix-Turn-Helix) DNA-binding domains, we According to the COGs classiﬁcation (Material and Methods), diﬀerentially expressed genes were distributed over all functional groups (Figure 2). A very strong representation of genes May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 7 Abdou et al. Role of RegA in Brucella suis Persistence FIGURE 2 \| Distribution of B. suis genes under RegA control in COG functional categories. A total of 447 genes were differentially expressed at day 3 of our in vitro model of persistence. Numbers of up-regulated (blue bars) and down-regulated (red bars) genes in the wild-type strain refer to the presence of a functional regA. J, Translation, ribosomal structure, and biogenesis; K, Transcription; L, DNA replication, recombination, and repair; D, Cell division and chromosome partitioning; O, Post-translational modiﬁcation, protein turnover, chaperones; M, Cell envelope biogenesis, outer membrane; V, Defense mechanisms; U, Intracellular trafﬁcking, secretion, and vesicular transport; N, Cell motility and secretion; P, Inorganic ion transport and metabolism; T, Signal transduction mechanisms; C, Energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme metabolism; I, Lipid metabolism; Q, Secondary metabolites biosynthesis, transport, and catabolism; R, General function prediction only; S, Function unknown. FIGURE 2 \| Distribution of B. suis genes under RegA control in COG functional categories. A total of 447 genes were differentially expressed at day 3 of our in vitro model of persistence. Numbers of up-regulated (blue bars) and down-regulated (red bars) genes in the wild-type strain refer to the presence of a functional regA. Possible Function of RegA as both Direct and Indirect Regulator J, Translation, ribosomal structure, and biogenesis; K, Transcription; L, DNA replication, recombination, and repair; D, Cell division and chromosome partitioning; O, Post-translational modiﬁcation, protein turnover, chaperones; M, Cell envelope biogenesis, outer membrane; V, Defense mechanisms; U, Intracellular trafﬁcking, secretion, and vesicular transport; N, Cell motility and secretion; P, Inorganic ion transport and metabolism; T, Signal transduction mechanisms; C, Energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme metabolism; I, Lipid metabolism; Q, Secondary metabolites biosynthesis, transport, and catabolism; R, General function prediction only; S, Function unknown. FIGURE 2 \| Distribution of B. suis genes under RegA control in COG functional categories. A total of 447 genes were differentially expressed at day 3 of our in vitro model of persistence. Numbers of up-regulated (blue bars) and down-regulated (red bars) genes in the wild-type strain refer to the presence of a functional regA. J, Translation, ribosomal structure, and biogenesis; K, Transcription; L, DNA replication, recombination, and repair; D, Cell division and chromosome partitioning; O, Post-translational modiﬁcation, protein turnover, chaperones; M, Cell envelope biogenesis, outer membrane; V, Defense mechanisms; U, Intracellular trafﬁcking, secretion, and vesicular transport; N, Cell motility and secretion; P, Inorganic ion transport and metabolism; T, Signal transduction mechanisms; C, Energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme metabolism; I, Lipid metabolism; Q, Secondary metabolites biosynthesis, transport, and catabolism; R, General function prediction only; S, Function unknown. FIGURE 2 \| Distribution of B. suis genes under RegA control in COG functional categories. A total of 447 genes were differentially expressed at day 3 of our in vitro model of persistence. Numbers of up-regulated (blue bars) and down-regulated (red bars) genes in the wild-type strain refer to the presence of a functional regA. Up-Regulation by RegA in the In vitro Model of Persistence The K class (transcription) possesses the highest number of up- regulated genes (Figure 2), the vast majority of which encodes transcriptional regulators of diﬀerent pathways (Table S1) or of two-component systems, such as BR0604 (feuP)/BR0605 (feuQ) (Figure 3) (Tables S1, S3). RegA induced BR1118 (ntrB) and BR1119 (nifR3) (Figure 3) of the ntrBC operon, encoding the two-component system involved in regulation of nitrogen metabolism and depending on glutamine availability (Ronneau et al., 2016). Since the downstream operon ntrYX is also regulated by PrrA (RegA) in B. abortus (Carrica et al., 2013), we compared ntrY (BR1116) expression levels in the WT and 1regA strains by RT-qPCR. The 3.7-fold change (−11Ct) obtained (Figure 3) All eight RegA-induced genes involved in DNA metabolism (class L) encode enzymes participating in DNA recombination and repair [recombinases or transposases (Table S1)], possibly to counteract potential DNA-damage occurring in bacteria exposed to low oxygen levels. Similarly, two up-regulated genes (BRA0607 May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 8 Role of RegA in Brucella suis Persistence Abdou et al. (RegB) and thereby of activated PrrA (RegA) and its target genes (Oh et al., 2001). The functional class of energy production (C) (see below) and of envelope biogenesis (M) contain the highest percentages of RegA-down-regulated genes with deﬁned annotations (Figure 2), both having a very high proportion of repressed RegA-dependent genes (37/49 and 27/35, respectively) (Table S1). Five of the six genes belonging to an operon involved in LPS biosynthesis were repressed by RegA: BR0518, BR0519/0520 of class G producing the O-antigen export system, BR0521/0522 (Table S3, Figure 4A) encoding perosamine synthase and GDP-mannose 4,6-dehydratase (Table S1). Proteome data were perfectly consistent since all identiﬁed and BR0672) of the nucleotide metabolism (class F) produce “house-cleaning” hydrolases of the MutT/nudix family (Table S1, Figure 3) that target damaged metabolites accumulated during stress, for example mutagenic forms of dGTP (Bessman et al., 1996). The majority of the RegA-induced genes involved in carbohydrate (class G), or amino acid transport and metabolism (class E) encode ABC transporters for sugars, such as BRA0995/0996 with high aﬃnity for D-ribose (Lopilato et al., 1984), or for peptides and amino acids, respectively (Tables S1, S3, Figure 3). Genes and Proteins Down-Regulated by RegA in the In vitro Model of Persistence Genes and Proteins Down-Regulated by RegA in the In vitro Model of Persistence The predicted individual gene (Romero and Karp, 2004; Mao et al., 2014) regB (BR0133) encoding the sensor histidine kinase was repressed in the WT strain (Figure 4A). This suggests that RegA (BR0137) could exert a negative feedback on regB, as reported for the homologs in R. sphaeroides, where autoregulation prevents excessive production of PrrB FIGURE 4 \| Validation of down-regulation by RegA. (A) Fold-change of RegA-down-regulated genes are expressed as log2-values of normalized microarray data (white bars) and of RT-qPCR (−11Ct) (black bars). Each quantitative PCR was performed in technical triplicates of three independent samples. Genes represented are selected from the following functional groups K, transcription; L, DNA replication, recombination, and repair; D, Cell division and chromosome partitioning; E, amino acid transport and metabolism; M, Cell envelope biogenesis; P, Inorganic ion transport and metabolism; T, Signal transduction mechanisms. (BR1242: rpoC, BR0001: dnaA, BR1434: mraY, BR1475: omp28, BR0522: gmd, BR1359: ureE-2, BR1388: ilvH, BRA0703: sodC, BR0133: regB). (B) SodC activity on day 3 in the in vitro model of persistence. Enumeration of the surviving bacteria after 60 min of exposure to O− 2 generated by the xanthine oxidase reaction of B. suis wild-type (dark gray bars), 1regA mutant (light gray bars), and complemented regA mutant (medium gray bars) strains. Standard deviations are reported for the means of three independent experiments. FIGURE 4 \| Validation of down-regulation by RegA. (A) Fold-change of FIGURE 3 \| Fold-change of RegA-up-regulated genes calculated from normalized microarray data and RT-qPCR. Increased expression levels in wild-type strain with respect to the 1regA mutant are expressed as log2-values of the normalized hybridization ratios (white bars) compared to those obtained by RT-qPCR (−11Ct) (black bars). Each qPCR was performed in technical triplicates. Genes represented are selected from the following functional groups. K, Transcription; J, Translation, ribosomal structure, and biogenesis; T, Signal transduction mechanisms; G, carbohydrate transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme metabolism. (BR0604: feuP, BR1119: nifR3, BR1118: ntrB, BR1116: ntrY, BRA0996: rbsB-3, BR0672: Mut/nudix, BR0757: hemB). BR1116 showed a hybridization ratio below threshold (1.6). The mean ± standard deviations (error bars) of microarrays and RT-qPCR data were calculated from three independent experiments. FIGURE 4 \| Validation of down-regulation by RegA. (A) Fold-change of FIGURE 4 \| Validation of down-regulation by RegA. Genes and Proteins Down-Regulated by RegA in the In vitro Model of Persistence (A) Fold-change of RegA-down-regulated genes are expressed as log2-values of normalized microarray data (white bars) and of RT-qPCR (−11Ct) (black bars). Each quantitative PCR was performed in technical triplicates of three independent samples. Genes represented are selected from the following functional groups K, transcription; L, DNA replication, recombination, and repair; D, Cell division and chromosome partitioning; E, amino acid transport and metabolism; M, Cell envelope biogenesis; P, Inorganic ion transport and metabolism; T, Signal transduction mechanisms. (BR1242: rpoC, BR0001: dnaA, BR1434: mraY, BR1475: omp28, BR0522: gmd, BR1359: ureE-2, BR1388: ilvH, BRA0703: sodC, BR0133: regB). (B) SodC activity on day 3 in the in vitro model of persistence. Enumeration of the surviving bacteria after 60 min of exposure to O− 2 generated by the xanthine oxidase reaction of B. suis wild-type (dark gray bars), 1regA mutant (light gray bars), and complemented regA mutant (medium gray bars) strains. Standard deviations are reported for the means of three independent experiments. FIGURE 4 \| Validation of down-regulation by RegA. (A) Fold-change of RegA-down-regulated genes are expressed as log2-values of normalized microarray data (white bars) and of RT-qPCR (−11Ct) (black bars). Each quantitative PCR was performed in technical triplicates of three independent samples. Genes represented are selected from the following functional groups K, transcription; L, DNA replication, recombination, and repair; D, Cell division and chromosome partitioning; E, amino acid transport and metabolism; M, Cell envelope biogenesis; P, Inorganic ion transport and metabolism; T, Signal transduction mechanisms. (BR1242: rpoC, BR0001: dnaA, BR1434: mraY, BR1475: omp28, BR0522: gmd, BR1359: ureE-2, BR1388: ilvH, BRA0703: sodC, BR0133: regB). (B) SodC activity on day 3 in the in vitro model of persistence. Enumeration of the surviving bacteria after 60 min of exposure to O− 2 generated by the xanthine oxidase reaction of B. suis wild-type (dark gray bars), 1regA mutant (light gray bars), and complemented regA mutant (medium gray bars) strains. Standard deviations are reported for the means of three independent experiments. FIGURE 3 \| Fold-change of RegA-up-regulated genes calculated from normalized microarray data and RT-qPCR. Increased expression levels in wild-type strain with respect to the 1regA mutant are expressed as log2-values of the normalized hybridization ratios (white bars) compared to those obtained by RT-qPCR (−11Ct) (black bars). Each qPCR was performed in technical triplicates. Genes represented are selected from the following functional groups. RegA Coordinates Bacterial Adaptation to Low Oxygen Levels yg In the in vitro model of persistence, ∼15% (37/244) and 5.9% (12/203) of the respectively down- and up-regulated genes were identiﬁed as being involved in energy production and conversion (class C) (Table S1). The oxygen-dependent regulator fnrN (BR0654), cydD (BRA0508) and cydC (BRA0509) (Table S1) of the cyd operon encoding the bd ubiquinol oxidase of high aﬃnity for oxygen were very strongly induced by RegA, as shown previously (Abdou et al., 2013). One and ﬁve potential RegA- binding sites were accurately detected in cydD and fnrN upstream sequences, respectively (Table S3). RT-qPCR results (Table S3, Figure 5A) proved a fnrN induction fold-change similar to that obtained under direct anaerobiosis, thus indicating that oxygen concentration was diminishing toward anoxic conditions at day 3 of the in vitro model of persistence. We also conﬁrmed that RegA induced expression of both ubiquinol and cytochrome c oxidases under these conditions (Abdou et al., 2013). In fact, strong induction of cytochrome bc1 components (complex III of the respiratory pathway; operon BR1543/1541) (Tables S1–S3), of cytochrome c itself (BR0039), as well as of genes participating in its biogenesis (BR0096 and BR0607/0608, category O) (Tables S1, S3), indicated that B. suis may use an electron transport chain terminating with a cytochrome c oxidase. Nevertheless, the only cytochrome c oxidase under strong positive control of RegA was the aa3-type (BR0467 and BR0468) (Tables S1, S3), a quite unexpected ﬁnding given that it possesses a lower oxygen aﬃnity than the cbb3-type (Ekici et al., 2012). The operon encoding the latter (BR0363/0360) was, however, also well-expressed in the WT strain (microarray data, GEO accession number GSE87538). The aa3-type oxidase of B. suis could be active under our conditions, in accordance with detection of the cytochromes a and a3 only in late-log phase B. abortus (Rest and Robertson, 1975). It was suggested that its counterpart in P. aeruginosa participates in energy preservation, given the highly In class E (amino acid transport and metabolism), 25 genes were found to be down-regulated by RegA. One of the four detected genes belonging to the ure-2 operon was validated by RT-qPCR (Tables S1, S3, Figure 4A), they encode urease accessory proteins responsible for urea and nickel transport in B. abortus (Sangari et al., 2010). Bacteria in a neutral medium may not require elevated expression of the urea transporter, necessary for the acid-dependent induction of urease activity in B. abortus. SodC Is Down-Regulated by RegA under In Vitro Depletion of Oxygen The inorganic ion transport and metabolism category (P) comprises 9 genes down-regulated in the WT strain (Table S1). Notably, we detected BRA0703 (sodC) encoding a Cu, Zn superoxide dismutase, conﬁrmed by RT-qPCR (Figure 4A), and the catalase (BRA0355) (Table S2), both participating in detoxiﬁcation of oxidative compounds. Activity of the superoxide dismutase was estimated by the quantiﬁcation of bacterial survival to artiﬁcially produced superoxide anions, which showed that survival of 1regA was 100-fold higher (P < 0.001) than that of WT (Figure 4B). Survival of the mutant decreased following complementation with the intact regA gene and was identical to that of the WT strain (Figure 4B). These results corroborated the repression of sodC expression by RegA at day 3 of our model of persistence, which reﬂects the lack of oxygen resulting in a reduced production of reactive oxygen intermediates. RT-qPCR-validation (Table S3) conﬁrmed that, under our conditions, RegA repressed the virB operon (all virB genes, except virB7-9, and virB12) (Table S1) and its activator, vjbR. Identiﬁcation of a putative RegA-binding site 90 bp downstream of the initiation codon of vjbR but not at the 5′ ends of virB1 and virB2 (Table S3) suggested indirect regulation of the virB operon, possibly via direct repression of vjbR. Transcriptome and proteome analyses converged to the observation of a RegA-dependent decrease of transcriptional activity. In fact, ICPL experiments (Table S2) validated down- regulation of the RNA polymerase subunit beta’ (rpoC, BR1242) (Table S1, Figure 4A), and both proteomic methods (Table S2) also detected reduced abundance of the catalytic subunit beta. The class J (translation)-derived proteins were predominantly down-regulated (Table S2), while this category was underrepresented in the transcriptome data. We conﬁrmed by RT-qPCR the RegA-dependent down-regulation of genes BR0001 (dnaA) and BR1105 playing a role in DNA replication (class L), and of the three genes involved in cell division (class D) (Tables S1, S3, Figure 4A). Genes and Proteins Down-Regulated by RegA in the In vitro Model of Persistence K, Transcription; J, Translation, ribosomal structure, and biogenesis; T, Signal transduction mechanisms; G, carbohydrate transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme metabolism. (BR0604: feuP, BR1119: nifR3, BR1118: ntrB, BR1116: ntrY, BRA0996: rbsB-3, BR0672: Mut/nudix, BR0757: hemB). BR1116 showed a hybridization ratio below threshold (1.6). The mean ± standard deviations (error bars) of microarrays and RT-qPCR data were calculated from three independent experiments. FIGURE 3 \| Fold-change of RegA-up-regulated genes calculated from normalized microarray data and RT-qPCR. Increased expression levels in wild-type strain with respect to the 1regA mutant are expressed as log2-values of the normalized hybridization ratios (white bars) compared to those obtained by RT-qPCR (−11Ct) (black bars). Each qPCR was performed in technical triplicates. Genes represented are selected from the following functional groups. K, Transcription; J, Translation, ribosomal structure, and biogenesis; T, Signal transduction mechanisms; G, carbohydrate transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme metabolism. (BR0604: feuP, BR1119: nifR3, BR1118: ntrB, BR1116: ntrY, BRA0996: rbsB-3, BR0672: Mut/nudix, BR0757: hemB). BR1116 showed a hybridization ratio below threshold (1.6). The mean ± standard deviations (error bars) of microarrays and RT-qPCR data were calculated from three independent experiments. May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 9 Abdou et al. Role of RegA in Brucella suis Persistence conditions, in contrast to what could be expected during growth in a minimal medium devoid of amino acid sources. class M proteins (Table S2) were down-regulated, BR0522 included. MurB (Table S2) and four genes of the murEF-mraY- murD-ftsW operon were found to be repressed by RegA (Tables S1, S3, Figure 4A), as well as ddlA (BR1271) and BR1172. These results conﬁrmed that peptidoglycan biosynthesis was down-regulated by RegA during establishment of persistence. Three major outer membrane proteins, Omp25, Omp28, Omp31, together with the corresponding genes of the latter two [BR1475 (Figure 4A) and BR1622 (Table S1)], were also under negative RegA control. Omp31 is a porin and its down- regulation could indicate a RegA-dependent protection of the bacterium. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org RegA Coordinates Bacterial Adaptation to Low Oxygen Levels Conversely, RegA may participate in the basal urease activity, exclusively produced from the ure-1 operon in both B. suis and B. abortus (Bandara et al., 2007; Sangari et al., 2007), as indicated by the higher production of the subunit gamma (UreA-1) (Table S2) in the WT strain. Numerous genes (Table S1) and enzymes (Table S2) belonging to various amino acids biosynthesis pathways were found under the negative control of RegA. The superpathway of isoleucine, leucine and valine synthesis was identiﬁed at both the transcript and protein levels, validated by RT-qPCR targeting BR1388 (ilvH) (Figure 4A). Genes involved in biosynthesis of histidine, lysine or methionine and enzymes participating in arginine or serine metabolism were also down-regulated by RegA. BR0765 (glyA) and its product responsible for transformation of serine into glycine were found coherently regulated (Tables S1, S2). Repression of genes or proteins involved indicated that B. suis limited utilization of the relevant pathways under anaerobic May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 10 Abdou et al. Role of RegA in Brucella suis Persistence FIGURE 5 \| Validation of differentially expressed genes involved in adaptation to low oxygen levels. (A) Expression ratios of RegA-dependent genes are shown as log2-values of the normalized hybridization ratios (white bars) and of RT-qPCR (−11Ct) (black bars). Each quantitative PCR was performed in technical triplicates of three independent samples. (BR0654: fnrN, BRA0508: cydD, BR0809: nuoH, BRA0299: narG, BRA0260: nirK, BRA0248: norC, BRA0275: nosZ). (B) Nitrite and nitric oxide consumption in wild-type (black bars), 1regA mutant (gray bars), and complemented regA mutant (dark gray bars) after 30 min under anaerobiosis. The mean ± standard deviations (error bars) of three independent experiments are shown. eﬃcient proton translocation by the studied aa3-type oxidases (Kawakami et al., 2010). However, the nuoA-N operon encoding NADH dehydrogenase (complex I of the respiratory chain) was repressed (Table S1, Figure 5A), and one potential RegA-binding site was accurately detected in the vicinity of the initiation codon (Table S3). This possibly reﬂected low residual activity of the respiratory chain, with energy production suﬃcient for persistence. Genes BRA0299, BRA0260, BRA0248, and BRA0275 encoding catalytic subunits (except BRA0248) of the four reductases Nar, Nir, Nor, and Nos involved in denitriﬁcation, were detected as being repressed (Figure 5A), as well as additional genes belonging to the four operons (Table S1). RegA Coordinates Bacterial Adaptation to Low Oxygen Levels (A) Expression ratios of RegA-dependent genes are shown as log2-values of the normalized hybridization ratios (white bars) and of RT-qPCR (−11Ct) (black bars). Each quantitative PCR was performed in technical triplicates of three independent samples. (BR0654: fnrN, BRA0508: cydD, BR0809: nuoH, BRA0299: narG, BRA0260: nirK, BRA0248: norC, BRA0275: nosZ). (B) Nitrite and nitric oxide consumption in wild-type (black bars), 1regA mutant (gray bars), and complemented regA mutant (dark gray bars) after 30 min under anaerobiosis. The mean ± standard deviations (error bars) of three independent experiments are shown. FIGURE 5 \| Validation of differentially expressed genes involved in adaptation to low oxygen levels. (A) Expression ratios of RegA-dependent RegA Coordinates Bacterial Adaptation to Low Oxygen Levels In agreement, consumption of NO2/NO correlated with the respective nitrite and NO reductase activities in lysates of strains obtained at day 3, since 1regA eliminated 6.6 mM ± 0.1 mM of nitrites, instead of only 3.8 mM ± 0.05 mM used by the WT and complemented strains (Figure 5B). nnrA, encoding the activator of nir, nor, and nos (Haine et al., 2006; Carrica et al., 2013), was also found adequately regulated (Table S3). Potential RegA-binding sites were found in relevant positions of nnrA, but also of each of the four operons (Table S3), indicating that down-regulation of the three aforementioned operons could be direct or possibly acting via repression of nnrA. Unexpectedly, these results were opposed to RegA-mediated nirK induction observed under direct anaerobic denitrifying conditions (Abdou et al., 2013). Interestingly, they also indicate that the same nirK gene can be under positive or negative control by RegA, as reported in R. sphaeroides (Laratta et al., 2002), probably to ensure the best adaptive response depending on the availability of metabolites and on experimental conditions. In fact, the initial (day 1) RegA-dependent nirK-induction in the WT strain, a probable adaptation to hypoxia, reversed to repression at day 2 until at least day 4. This might be associated with lack of nitrate in the medium and the use of an alternative electron acceptor such as fumarate (see below). At the protein level, RegA-dependent repression of the beta- subunit of the ATP-synthase (Table S2) also indicated reduced energy production in the WT strain entering the persistence phase. These data lead us to the conclusion that RegA is a key player in eﬃcient adaptation of Brucella to oxygen deﬁciency. FIGURE 5 \| Validation of differentially expressed genes involved in adaptation to low oxygen levels. (A) Expression ratios of RegA-dependent genes are shown as log2-values of the normalized hybridization ratios (white bars) and of RT-qPCR (−11Ct) (black bars). Each quantitative PCR was performed in technical triplicates of three independent samples. (BR0654: fnrN, BRA0508: cydD, BR0809: nuoH, BRA0299: narG, BRA0260: nirK, BRA0248: norC, BRA0275: nosZ). (B) Nitrite and nitric oxide consumption in wild-type (black bars), 1regA mutant (gray bars), and complemented regA mutant (dark gray bars) after 30 min under anaerobiosis. The mean ± standard deviations (error bars) of three independent experiments are shown. FIGURE 5 \| Validation of differentially expressed genes involved in adaptation to low oxygen levels. RegA Contributes Signiﬁcantly to the Regulation of Energy Metabolism Remarkably, 75.5 (37/49) and 76.5 (13/17)%, respectively, of the RegA-dependent genes and proteins belonging to the COG category C (energy production and conversion) were found to be repressed. This incited us to measure transcription levels of numerous genes engaged in central carbon metabolism (Table S3, Figure 6A). Interestingly, the genes and proteins involved in glycolysis and tricarboxylic acid (TCA) cycle were all down-regulated in the WT (Figure 6B), except for the triosephosphate isomerase TpiA1 (Table S2) and fumB-encoded fumarate hydratase, the latter being strongly induced by RegA (Figure 6A). The relative contradiction to our previous study, suggesting that low oxygen conditions applied on cultures in rich medium can activate glycolysis (Al Dahouk et al., 2009), is most likely caused by diﬀerences in medium and conditions selected. The strong repression of the genes encoding each subunit of the glycerol-3-phosphate ABC transporter (Tables S1, S3) may be the consequence of a presumably fair level of glycerol import from the medium (30 g/L) (see discussion), possibly phosphorylated by the glycerol kinase (BRA0433). Glycerol-3-P May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 11 Abdou et al. Role of RegA in Brucella suis Persistence FIGURE 6 \| RegA-dependent regulation of pathways involved in energy production. (A) Fold-change of differentially expressed genes (BR0961: fumB, BR1614: aceA, BR1729: pgk, BR1127: aceF, BR1148: gltA, and BR1923: sucA) are represented as the log2-values of hybridization ratios (white bars) and of the RT-qPCR (−11Ct) (black bars). Each quantitative PCR was performed in technical triplicates of three independent samples. The mean ± standard deviations (error bars) of the three independent experiments are shown. (B) Representation of the pathways under the control of RegA. The crossed out third step of glycolysis indicates the lack of the corresponding enzyme phosphofructokinase. Dashed arrows represent the shunted reactions when the glyoxylate bypass is active. RegA–dependent proteins (in blue) and genes validated by RT-qPCR (in red) or not tested (underlined gray) are shown. The red box represents the only gene up-regulated by RegA. BR1781 (pyc) and BR1017 (maeB), not validated, were omitted for clarity. (C) ICL activity in crude lysates of the wild-type (WT) (dark gray bar), 1regA mutant (1regA) (gray bar) and complemented 1regA mutant (C−1regA) (dashed gray bar) strains was measured as described in Methods. Means and standard deviations of three independent experiments are reported. FIGURE 6 \| RegA-dependent regulation of pathways involved in energy production. RegA Contributes Signiﬁcantly to the Regulation of Energy Metabolism (A) Fold-change of differentially expressed genes (BR0961: fumB, BR1614: aceA, BR1729: pgk, BR1127: aceF, BR1148: gltA, and BR1923: sucA) are represented as the log2-values of hybridization ratios (white bars) and of the RT-qPCR (−11Ct) (black bars). Each quantitative PCR was performed in technical triplicates of three independent samples. The mean ± standard deviations (error bars) of the three independent experiments are shown. (B) Representation of the pathways under the control of RegA. The crossed out third step of glycolysis indicates the lack of the corresponding enzyme phosphofructokinase. Dashed arrows represent the shunted reactions when the glyoxylate bypass is active. RegA–dependent proteins (in blue) and genes validated by RT-qPCR (in red) or not tested (underlined gray) are shown. The red box represents the only gene up-regulated by RegA. BR1781 (pyc) and BR1017 (maeB), not validated, were omitted for clarity. (C) ICL activity in crude lysates of the wild-type (WT) (dark gray bar), 1regA mutant (1regA) (gray bar) and complemented 1regA mutant (C−1regA) (dashed gray bar) strains was measured as described in Methods. Means and standard deviations of three independent experiments are reported. may then be used by a speciﬁc dehydrogenase (BR1889, gpsA) to produce dihydroxyacetone-phosphate (DHAP), the substrate of TpiA1 (Figure 6B) whose induction may therefore be in line with repression of the glycerol-3P transporter. Instead, slight induction of this glycerol-3P transporter by anaerobiosis under denitrifying conditions and by nutrient starvation may be linked to the lack of glycerol in medium used in our previous analyses (Al Dahouk et al., 2009, 2013). Nevertheless, our studies concur with a possible use of glycerol as carbon source by B. suis under both conditions of oxygen deﬁciency. solely via the PPC, were repressed by RegA. This is in contrast with the induction of the large majority of genes encoding sugar ABC transporters, possibly reﬂecting the lack of substrates in the medium. Moreover, xfp (BRA0385) producing phosphoketolase (Tables S1, S3) and transketolase (Tkt, BR1727) (Table S2), were found negatively controlled by RegA, conﬁrming the down-regulation of enzymes of the PPC. Three enzymes, phosphoenolpyruvate carboxykinase (PckA), pyruvate phosphate dikinase (PpdK), and L-lactate dehydrogenase (L-Ldh), which catalyze reactions potentially supplying phosphoenolpyruvate or pyruvate for gluconeogenesis and for amino acids synthesis (Figure 6B), were also repressed by RegA (Table S2). Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org RegA Contributes Signiﬁcantly to the Regulation of Energy Metabolism coli, this anaerobic enzyme produces fumarate used as an alternative electron acceptor under anoxia. The behavior of WT, 1aceA and complemented 1aceA strains was similar during infection of both resting or IFNγ- and LPS- activated J774A.1 murine macrophage-like cells (Loisel-Meyer et al., 2006), under standard and microaerobic conditions. A possible role of aceA in pathogenesis in vivo was then assessed in a murine model of infection. In contrast to the WT strain, 1aceA did not display bacterial growth during the acute phase (day 7), a phenotype possibly correlated to the lower oxygenation inside the target organs than in the ﬁrst step of our in vitro model (Jiménez de Bagüés et al., 2007). In the spleen, after a persistence phase (days 14–56) parallel to that of the WT, elimination of the mutant was accentuated during the last 4 weeks of infection, resulting in a decrease of 2.3 logs in bacterial counts (P < 0.05), and two animals cleared infection (Figure 7B). The aceA mutant was more attenuated in the liver where it was eliminated shortly after colonization (Figure 7B), resulting in a higher diﬀerence in viable counts between the two strains than in the spleen at day 14 [2.5 logs (P < 0.001) vs. 1.8 log (P < 0.001)]. The mutant was unable to establish a chronic infection and was totally eliminated at 12 weeks post-infection (Figure 7B). The stronger impact of ICL loss in the liver is in line with the lower oxygen concentration within this organ, potentially aggravated by the formation of granuloma during chronic infection (Abdou et al., 2013). Another non-exclusive hypothesis could be an increased requirement of ICL activity consecutive to the possible use of fatty acids present in the liver. Complementation of the mutant strain resulted in partial restoration of the parental phenotype in the spleen only, with intermediate growth in the acute phase but survival similar to that of the mutant starting at day 56 (Figure 7B). Having checked the presence of the plasmid ensuring complementation in bacteria recovered at day 28, we propose that early strong overexpression of aceA may result in over-activation of the glyoxylate pathway. High ICL activity could be detrimental to replication and persistence in the liver due to overproduction of succinate (Figure 6B), leading to succinate dehydrogenase dysfunction (stimulated or inhibited, depending on the TCA cycle direction) with a probable consecutive imbalance in reduced cofactors. RegA Contributes Signiﬁcantly to the Regulation of Energy Metabolism RT- qPCR validated repression of ﬁve out of nine genes encoding enzymes of the central metabolism (Table S3, Figure 6A), among which BR1729 (pgk) whose product, phosphoglycerate kinase, generates ATP. Corresponding genes [BR1127 (aceF), BR1126 (lpdA-1)] (Tables S1, S3, Figure 6A) and proteic components E2 and E3 (dihydrolipoamide dehydrogenase) of the pyruvate dehydrogenase complex as well as its E1 α-subunit (BR1129) (Table S2), and BR1128 (pdhB) encoding E1 βsubunit (Table S1) Increased production of TpiA1 isomerase could sustain synthesis of glyceraldehyde-3-P from DHAP (Figure 6B), possibly providing enough of this essential intermediate of glycolysis under our particular conditions. This might make the pentose phosphate cycle (PPC) dispensable, although essential for glycolysis in Brucella devoid of phosphofructokinase (Figure 6B). In agreement, ABC-transporter periplasmic binding proteins (BRA0858 and BRA1150) speciﬁc for erythritol and xylose (Table S2), whose respective metabolites erythrose-4- P and xylulose-5-P can be used in carbohydrate metabolism May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 12 Role of RegA in Brucella suis Persistence Abdou et al. were also identiﬁed as being repressed by RegA (Figure 6B). Moreover, down-regulation of genes BR1148 (gltA), BR1923 (sucA), and BR1922 (sucB), encoding the citrate synthase, ﬁrst enzyme of the TCA cycle, and the E1 and E2 components of the α-ketoglutarate dehydrogenase complex (Tables S1, S3), respectively, were further validated by proteome analyses, except E2 (Table S2, Figures 6A,B). Lower concentrations of the E3 component (dihydrolipoamide dehydrogenase; see above) may also contribute to a reduced activity of this enzymatic complex. Furthermore, ICL and malate synthase of the glyoxylate shunt (Figure 6B) were also identiﬁed by microarray analysis as being down-regulated in the WT strain. BR1614 (aceA), encoding ICL which catalyzes the production of glyoxylate and succinate (Figure 6B), was the most down-regulated gene, with a log2- value of the hybridization ratios of –4.8. Validation by RT- qPCR (−11Ct = −5.4) (Table S3, Figure 6A) showed that this gene is indeed expressed in the WT (ratio of mRNA concentrations [aceA/(16S × 100) = 0.5 ± 0.08], and its high level of expression in 1regA (ratio of mRNA concentrations [aceA/(16S × 100) = 20.1 ± 0.7] therefore led us to consider the aceA “repression” as an over-expression in the mutant. Proteome analyses conﬁrmed higher ICL production by this strain (Table S2). Isocitrate Lyase Is Essential for In Vitro Persistence and In Vivo Virulence of B. suis Isocitrate Lyase Is Essential for In Vitro Persistence and In Vivo Virulence of B. suis The role of ICL was evaluated by studying the phenotype of 1aceA in the “in vitro persistence model” either in the absence or in the presence of fatty acids, expected to stimulate the glyoxylate bypass in B. suis, as their oxidation results in high levels of acetyl-CoA (Figure 6B). After comparable growth followed by a drop in bacterial counts, supplementation with fatty acids, regardless their nature [sodium palmitate or palmitic acid (C16), capric acid (C8), or oleate (C18 saturated)] and concentration (0.05, 0.5, 2, or 5 mM), gave rise to an increasing defect in survival of 1aceA, ∼10-fold beyond day 3 (Figure 7A). Without fatty acids, the persistence of 1aceA shifted down between day 15 and day 25, indicating that conditions of the persistence model evolve with time. The complementation of 1aceA (C-aceA in Figure 7A) allowed the restoration of the WT phenotype, and of the enzymatic activity (see above and data not shown). These results conﬁrmed the loss of ICL activity as a direct consequence of aceA inactivation and the requirement of a functional ICL for optimal bacterial persistence under anoxia in the presence of low fatty acid concentrations. RegA Contributes Signiﬁcantly to the Regulation of Energy Metabolism At day 3 of our model, the 4-fold higher ICL activity in the 1regA strain (P < 0.001) (Figure 6C), and restoration of the wild-type level after complementation with the intact regA gene, conﬁrmed ICL over-production in the mutant. As a control, the 1aceA mutant (Material and Methods) showed no ICL activity in the presence or the absence of isocitrate. Interestingly, up-regulation of a single gene from the TCA cycle, BR0961 (fumB) encoding fumarate hydratase B, was conﬁrmed in the WT strain (Figures 6A,B). In E. coli, this anaerobic enzyme produces fumarate used as an alternative electron acceptor under anoxia. were also identiﬁed as being repressed by RegA (Figure 6B). Moreover, down-regulation of genes BR1148 (gltA), BR1923 (sucA), and BR1922 (sucB), encoding the citrate synthase, ﬁrst enzyme of the TCA cycle, and the E1 and E2 components of the α-ketoglutarate dehydrogenase complex (Tables S1, S3), respectively, were further validated by proteome analyses, except E2 (Table S2, Figures 6A,B). Lower concentrations of the E3 component (dihydrolipoamide dehydrogenase; see above) may also contribute to a reduced activity of this enzymatic complex. Furthermore, ICL and malate synthase of the glyoxylate shunt (Figure 6B) were also identiﬁed by microarray analysis as being down-regulated in the WT strain. BR1614 (aceA), encoding ICL which catalyzes the production of glyoxylate and succinate (Figure 6B), was the most down-regulated gene, with a log2- value of the hybridization ratios of –4.8. Validation by RT- qPCR (−11Ct = −5.4) (Table S3, Figure 6A) showed that this gene is indeed expressed in the WT (ratio of mRNA concentrations [aceA/(16S × 100) = 0.5 ± 0.08], and its high level of expression in 1regA (ratio of mRNA concentrations [aceA/(16S × 100) = 20.1 ± 0.7] therefore led us to consider the aceA “repression” as an over-expression in the mutant. Proteome analyses conﬁrmed higher ICL production by this strain (Table S2). At day 3 of our model, the 4-fold higher ICL activity in the 1regA strain (P < 0.001) (Figure 6C), and restoration of the wild-type level after complementation with the intact regA gene, conﬁrmed ICL over-production in the mutant. As a control, the 1aceA mutant (Material and Methods) showed no ICL activity in the presence or the absence of isocitrate. Interestingly, up-regulation of a single gene from the TCA cycle, BR0961 (fumB) encoding fumarate hydratase B, was conﬁrmed in the WT strain (Figures 6A,B). In E. RegA Contributes Signiﬁcantly to the Regulation of Energy Metabolism Moreover, succinate accumulation would prevent the complemented mutant from using fumarate as an alternative electron acceptor in the absence of oxygen (see discussion), which may result in elimination of the strain (Figure 7B). Global Regulation of the Oxygen-Dependent Setting Up of Persistence in Brucella suis suis 1330 wild-type (WT), 1aceA mutant and complemented 1aceA (C-1aceA) strains were grown for 24 days in minimal medium containing 5 mM (NH4)2SO4, supplemented (+ F. A.) or not with 0.05 mM sodium palmitate. At each time point, tubes were removed to determine viable bacteria counts. Means and standard deviations of three independent experiments are reported. (B) The above-cited strains were used to inoculate BALB/c mice intraperitoneally with 105 CFU and were recovered from spleens (left) and livers (right) at 2, 7, 14, 28, 56, and 84 days post-infection. Error bars represent the standard error of the mean. Results of statistical analysis are indicated by apostrophes (′P < 0.05; ′′ ′′′ 2013). This earlier result raised our interest in the role of RegA in setting up the persistence state of Brucella which has been addressed for the ﬁrst time in the present work. role analogous to that of its ortholog in Rhodobacter (Eraso et al., 2008),which makes the two-component system RegB/A a central regulatory system required for adaptation to oxygen depletion. This function may contribute to the constraint of bacterial growth, typical of chronic infection. In agreement, both transcriptome and proteome analyses evidenced the down- regulation of cell envelope biogenesis and cellular division. We have shown that survival of a B. suis strain devoid of RegA was strongly aﬀected in our model creating a gradual decrease in oxygen concentration, which correlates with its behavior observed in vivo (Abdou et al., 2013). In addition, stable viability of the B. suis WT strain over a long period makes it a suitable bacterial candidate for the study of in vitro long-term persistence. Based on this in vitro model, we conducted whole-genome microarray transcription proﬁling of B. suis, in conjunction with two diﬀerent proteome analyses, to identify the full RegA regulon (12% of the genes) potentially involved in the establishment of the persistence state. RegA of B. suis modulates expression of genes belonging to all functional groups, acting predominantly as a repressor under the conditions applied. As initially expected, RegA of B. suis may play a NtrY/X, a second redox regulatory two-component system (Carrica et al., 2012), contributes with PrrB/A (RegB/A) to the complex network which controls the respiratory systems in B. abortus (Carrica et al., 2013). The histidine kinase NtrY is required in combination with PrrB for optimal expression of all B. abortus denitriﬁcation genes. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Global Regulation of the Oxygen-Dependent Setting Up of Persistence in Brucella suis The RegB/A two-component system has been identiﬁed in other studies and in our previous work as a redox state sensor and a regulator of both the oxidative respiration and the denitriﬁcation pathways in Brucella. The sensor PrrB (RegB) of B. abortus (Carrica et al., 2013) and the transcriptional regulator RegA of B. suis were found dispensable within spleens of infected mice, whereas we discovered B. suis RegA as being essential for bacterial persistence within low-oxygenated livers (Abdou et al., May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 13 Role of RegA in Brucella suis Persistence Abdou et al. FIGURE 7 \| Impact of aceA on B. suis survival during in vitro persistence and mice infection. (A) B. suis 1330 wild-type (WT), 1aceA mutant and complemented 1aceA (C-1aceA) strains were grown for 24 days in minimal medium containing 5 mM (NH4)2SO4, supplemented (+ F. A.) or not with 0.05 mM sodium palmitate. At each time point, tubes were removed to determine viable bacteria counts. Means and standard deviations of three independent experiments are reported. (B) The above-cited strains were used to inoculate BALB/c mice intraperitoneally with 105 CFU and were recovered from spleens (left) and livers (right) at 2, 7, 14, 28, 56, and 84 days post-infection. Error bars represent the standard error of the mean. Results of statistical analysis are indicated by apostrophes (′P < 0.05; ′′P < 0.01; ′′′P < 0.001). FIGURE 7 \| Impact of aceA on B. suis survival during in vitro persistence and mice infection. (A) B. suis 1330 wild-type (WT), 1aceA mutant and complemented 1aceA (C-1aceA) strains were grown for 24 days in minimal medium containing 5 mM (NH4)2SO4, supplemented (+ F. A.) or not with 0.05 mM sodium palmitate. At each time point, tubes were removed to determine viable bacteria counts. Means and standard deviations of three independent experiments are reported. (B) The above-cited strains were used to inoculate BALB/c mice intraperitoneally with 105 CFU and were recovered from spleens (left) and livers (right) at 2, 7, 14, 28, 56, and 84 days post-infection. Error bars represent the standard error of the mean. Results of statistical analysis are indicated by apostrophes (′P < 0.05; ′′P < 0.01; ′′′P < 0.001). FIGURE 7 \| Impact of aceA on B. suis survival during in vitro persistence and mice infection. (A) B. Global Regulation of the Oxygen-Dependent Setting Up of Persistence in Brucella suis suis respiratory systems by RegA in the “in vitro model of persistence.” The dashed lines indicate a putative effect of the two-component system NtrY/NtrX on expression of the operons encoding enzymes of the denitriﬁcation pathway and the cbb3-type cytochrome c oxidase, postulated on the basis of previous knowledge of this system in B. abortus (Carrica et al., 2013); see the manuscript for further explanations. Positive or negative regulation is indicated. FIGURE 8 \| Regulation of B. suis respiratory systems by RegA in the “in vitro model of persistence.” The dashed lines indicate a putative effect of the two-component system NtrY/NtrX on expression of the operons encoding enzymes of the denitriﬁcation pathway and the cbb3-type cytochrome c oxidase, postulated on the basis of previous knowledge of this system in B. abortus (Carrica et al., 2013); see the manuscript for further explanations. Positive or negative regulation is indicated. RegA-dependent repression of the virB operon is in line with the assumption that the bacterium does not likely need to activate the T4SS, the central virulence determinant, for establishment of the persistent state. In addition, our model does not confront the bacterium with the acidic conditions necessary for in vitro activation of the virB operon, mimicking the intracellular signal that triggers its induction for bacterial adaptation to the host cell (Boschiroli et al., 2002). Thus, our study allowed the characterization of RegA as a novel repressor of virB in addition to BlxR/BabR (de Jong and Tsolis, 2012). This result suggests that oxygen limitation of the installed replicative niche may be a signal to turn oﬀvirB expression. Most of the RegA- controlled genes or proteins previously described as virulence factors were detected as repressed by RegA (67% cumulated, vjbR and virB genes excluded) (Table S4). For example, the negative impact on LPS biosynthesis probably mirrors the global repression of envelope biogenesis, associated with a lack of bacterial proliferation. In fact, the number of the RegA-repressed genes or proteins known as virulence factors is signiﬁcant (Table S4), because their function is normally required for intracellular replication. of the PPC required for production of nucleic acids were also found under the negative control of RegA. Induction of a ribose ABC transporter of high aﬃnity suggests that B. Global Regulation of the Oxygen-Dependent Setting Up of Persistence in Brucella suis Positive control of ntrY expression by PrrB was conﬁrmed in our present work by the observation of its RegA-dependent induction. However, these results are not consistent with the RegA-dependent repression of the denitriﬁcation genes, unless NtrY exerts a negative eﬀect on May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 14 Role of RegA in Brucella suis Persistence Abdou et al. FIGURE 8 \| Regulation of B. suis respiratory systems by RegA in the “in vitro model of persistence.” The dashed lines indicate a putative effect of the two-component system NtrY/NtrX on expression of the operons encoding enzymes of the denitriﬁcation pathway and the cbb3-type cytochrome c oxidase, postulated on the basis of previous knowledge of this system in B. abortus (Carrica et al., 2013); see the manuscript for further explanations. Positive or negative regulation is indicated. their expression under the conditions applied. Otherwise, their down-regulation would be independent of NtrYX in B. suis in this context, meaning that the consequences of RegA-dependent ntrY induction on bacterial physiology still remain to be elucidated. We postulate that regulation of the denitriﬁcation pathway in Brucella spp. is complex, as several regulators are involved, and species-speciﬁc mechanisms may exist. Accordingly, we propose a hierarchical regulation scheme of respiratory systems reﬂecting conditions at day 3 in the “in vitro model of persistence,” placing the two-component system RegB/A in a lead position (Figure 8). In fact, RegA-dependent regulation of other two-component systems and regulatory genes involved in a broad range of bacterial processes is highly suggestive of master regulation in the adaptation of B. suis physiology to an anaerobic environment. Induction of the transcriptional regulator MucR represents an outstanding example of such a major role for RegA, since MucR acts mainly as a repressor of Brucella genes involved in very diverse functions (Caswell et al., 2013; Mirabella et al., 2013). In the late exponential growth phase, mucR expression was shown to be under negative control of the regulator VjbR (Mirabella et al., 2013). The RegA-dependent induction of MucR in our model was therefore not surprising, as RegA was found to repress vjbR. FIGURE 8 \| Regulation of B. Global Regulation of the Oxygen-Dependent Setting Up of Persistence in Brucella suis However, Salmonella enterica serovar Typhimurium required its unique aceA gene to establish persistent, but not acute infection in vivo (Fang et al., 2005). Another trait common to these pathogens is the need of ICL for infection. Therefore, our work provides a solid base allowing further studies of the regulation and of the role of selected genes such as aceA in adaptation of B. suis to the speciﬁc environment associated with persistence. The observed patterns of RegA- dependent regulation of several metabolic pathways could be speciﬁc of the time point chosen in our “in vitro model of persistence,” which is a dynamic model (see results). Expected results should provide a better understanding of the common mechanisms involved in setting-up a persistence state, typical of the chronic phase of infection. Whereas ICL has been considered as a potential target for drugs active against M. tuberculosis (Sharma et al., 2013), clinical isolates of P. aeruginosa and other gram-negative pathogens (Fahnoe et al., 2012), this will not be applicable to all Brucella species of clinical relevance. Finally, similar to a possible alternative approach for the design of anti-tubercular molecules (Banerjee et al., 2016), targeting of the response regulator RegA for the development of a new therapeutic strategy interfering with the establishment of persistent infection would be of great interest. ( g ) Despite the increased production of ICL in intracellular B. suis (Al Dahouk et al., 2008), the aceA mutant was not attenuated in the same model of infection, possibly due to less drastic hypoxia in cell cultures than in vivo (Atkuri and Herzenberg, 2005). The crucial role of ICL for B. suis multiplication in the low- oxygenated environment of host organs gave further indications that fatty acids may act as a possible source of energy for B. suis. A dual function of B. suis ICL and its participation as a major virulence factor is also conceivable, as P. aeruginosa ICL has a positive eﬀect on type III secretion system expression in oxygen- limited cultures (Chung et al., 2016). The enzyme of P. aeruginosa has also been identiﬁed as a virulence factor in a rat model of chronic lung infections (Lindsey et al., 2008). Surprisingly, the role of ICL seems to markedly diﬀer in B. suis and B. abortus, as the enzyme is dispensable in the latter for virulence and persistence (Zúñiga-Ripa et al., 2014). Global Regulation of the Oxygen-Dependent Setting Up of Persistence in Brucella suis suis possesses dehydrogenases speciﬁc for glycerol-3-P (BR0200, glpD), L-lactate and NADH, eﬃcient electron donors for fumarate reduction in E. coli (Gennis and Valley, 1996), the latter two were however repressed by RegA (see results). While anaerobiosis becomes established, use of oxygen via a terminal cytochrome c oxidase may be shifting to the use of fumarate as an alternative electron acceptor, adapted to low energetic needs. We suggest that decreased expression of aceA in the WT strain may be related to increased expression of fumB, as a mean to limit succinate accumulation (Figure 6B) and, under our experimental conditions, may also be explained by the lack of fatty acids in the medium. of bacterial persistence. Activation of two-component systems that control regulons involved in the setting-up of a low oxygen-dependent non-replicative state is likely to be a widespread mechanism among aerobic human pathogens. The RoxS/R regulon, the homolog of RegB/A in P. aeruginosa (Elsen et al., 2004), includes genes necessary for respiratory function and maintenance of the redox balance, and for sugar and amino acid metabolism (Kawakami et al., 2010). The M. tuberculosis DosST/R regulon (Voskuil et al., 2003) is expressed under hypoxia and during in vitro dormancy established by bacterial adaptation to the gradual limitation of oxygen obtained with the “Wayne model” (Wayne and Sohaskey, 2001). This model was the most extensively used to study the dormant state of tubercle bacilli and was developed further, resulting in the application of our “in vitro model of persistence” to B. suis. A functional ICL presented an advantage for persistence in vitro in the presence of fatty acids, but not for aerobic growth, leading to the hypothesis that B. suis might be able to use them under these speciﬁc conditions. Demonstrating a strict need of ICL for both the acute and chronic phases of infection, B. suis behaves similarly to other intracellular bacterial species, since alteration of both M. tuberculosis icl1 and icl2 genes prevented growth during the ﬁrst stage of infection (Muñoz-Elías and McKinney, 2005; Blumenthal et al., 2010) and persistence during the chronic phase in mice (McKinney et al., 2000; Blumenthal et al., 2010). At the chronic stage, ICL together with enzymes of fatty acids degradation were induced in lungs (Schnappinger et al., 2003), ICL activity being essential for use of fatty acids as carbon source (Muñoz-Elías and McKinney, 2005). Global Regulation of the Oxygen-Dependent Setting Up of Persistence in Brucella suis suis, unable to produce suﬃcient ribose via the PPC, may attempt to import this precursor possibly for residual biosynthetic needs, such as DNA repair (see results) and/or speciﬁc RNA synthesis for adaptation. The RegA-dependent repression of RNA polymerase subunits and of genes involved in DNA replication and transcription is compatible with their residual activity in the WT strain (GEO accession number GSE87538). During persistence, the PPC may become less central in B. suis physiology, as restricted gluconeogenesis provides less PPC substrates, and potential use of the DHAP-pathway produces glycolysis intermediates (Figure 6B). In B. suis, glycerol may represent an important carbon source to partially support the energy metabolism needed for bacterial survival under conditions of persistence, despite RegA-dependent down-regulation of the downstream genes and proteins (Figure 6B). Although a glycerol uptake facilitator protein was not identiﬁed in Brucella, growth of B. abortus B19 with glycerol as the main carbon supply suggested that at least this strain uses a common transporter system for erythritol and glycerol (Sangari et al., 1996). The present results strongly suggested that B. suis underwent a RegA-controlled slowdown of gluconeogenesis and of amino acids and nucleic acids synthesis, which is consistent with growth arrest of bacteria entering persistence. Role of RegA in Adaptation of Metabolism Due to the lack of a carbohydrate source in the medium used in the “in vitro model of persistence,” bacteria have to produce pentoses for nucleic acids synthesis, and hexoses for envelope biogenesis during the ﬁrst multiplicative step. Several main components of the medium such as glycerol with lactate and glutamate can serve as substrates for gluconeogenesis (Figure 6B). The RegA-mediated repression of enzymes belonging to the corresponding pathways further leads to a reduced production of intermediates feeding amino acids biosynthesis pathways, whose numerous genes and proteins were also RegA-repressed. ABC transporters and enzymes speciﬁc Induction of fumB highlights the hypothesis that B. suis may use fumarate as an electron acceptor under anoxia, itself converted to succinate by succinate dehydrogenase (Al Dahouk et al., 2009) (Figure 6B). Our previous proteome data already suggested that B. suis may use the reductive branch of the Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org May 2017 \| Volume 7 \| Article 186 15 Role of RegA in Brucella suis Persistence Abdou et al. TCA cycle under anaerobiosis (Al Dahouk et al., 2009). B. Global Regulation of the Oxygen-Dependent Setting Up of Persistence in Brucella suis These ﬁndings can be correlated to the fact that ICL was not regulated during in vitro infection with B. abortus (Lamontagne et al., 2009), in contrast to B. suis (Al Dahouk et al., 2008). Moreover, their respective cydB mutants devoid of cytochrome bd oxidase with high aﬃnity for oxygen displayed opposite fates in mice (Endley et al., 2001; Jiménez de Bagüés et al., 2007). Important variations in metabolic routes may exist among Brucella strains, which may contribute to their diﬀerent phenotypes at the chronic stage of spleen infection in mice (Bosseray et al., 1982). AUTHOR CONTRIBUTIONS EA, VJ-M, MJdB, IM-A, and SO-B carried out the experiments. VP performed the microarray processing and the associated statistical analysis. VJ-M and EA conceived the study and achieved the interpretation of the data. SK, SAD, and AO contributed to conception of the study. EA and VJ-M were involved in drafting the manuscript and, SK, VJ-M, AO, and SAD revised it. All authors read and approved the ﬁnal manuscript. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org REFERENCES Bosseray, N., Plommet, M., and De Rycke, J. (1982). Development of Brucella abortus, Brucella melitensis and Brucella suis infection in mice to a chronic stage and cure. Ann. Rech. Vet. 13, 153–161. Abdou, E., Deredjian, A., Jiménez de Bagüés, M. P., Köhler, S., and Jubier-Maurin, V. (2013). 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FUNDING This work was supported by funds from the Centre National de la Recherche Scientiﬁque (CNRS), France, the National Institute for Agricultural and Food Research and Technology (INIA) in Spain (Grant RTA 2013-00065-C02- 01), and by grant 1329-485 from the Bundesinstitut für Risikobewertung (BfR), Germany. EA was supported by a The present study has shed light on the central role of RegA in control of metabolic pathways involved in establishment May 2017 \| Volume 7 \| Article 186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 16 Role of RegA in Brucella suis Persistence Abdou et al. design. We thank P. Bouhours for the preparation of bacterial media. fellowship from the Lebanese CNRS and IM-A received funds from Infectiopôle Sud. fellowship from the Lebanese CNRS and IM-A received funds from Infectiopôle Sud. ACKNOWLEDGMENTS The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fcimb. 2017.00186/full#supplementary-material We acknowledge F. Allayaud for helpful technical assistance in RT-qPCR and in ICL assay developing. We are grateful to Patrick Lemaire and Edwin Jacox for Positional Weight Matrices Patrick Lemaire and Edwin Jacox for Positional Weight Matrices REFERENCES The Brucella abortus Cu, Zn superoxide dismutase is required for optimal resistance to oxidative killing by murine macrophages and wild- type virulence in experimentally infected mice. Infect Immun. 73, 2873–2880. doi: 10.1128/IAI.73.5.2873-2880.2005 Lestrate, P., Dricot, A., Delrue, R.-M., Lambert, C., Martinelli, V., De Bolle, X., et al. (2003). Attenuated signature-tagged mutagenesis mutants of Brucella melitensis identiﬁed during the acute phase of infection in mice. Infect. 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https://openalex.org/W4225727261	https://www.preprints.org/manuscript/202112.0200/v1/download	English	null	Spatial Spillover Effects of Agricultural Transport Costs in Peru	Land	2,021	cc-by	10,799	Pedro Herrera-Catalán 1, Coro Chasco 2,3 and Máximo Torero 4 Pedro Herrera-Catalán 1, Coro Chasco 2,3 and Máximo Torero 4 1 Faculty of Economics and Business Administration, Universidad Autónoma de Madrid. C/ Francisco Tomás 1 Faculty of Economics and Business Administration, Universidad Autónoma de Madrid. C/ Francisco Tomás y Valiente 5 28049 Madrid Spain; pedro herrera@predoc uam es 1 Faculty of Economics and Business Administration, Universidad Autónoma de Madrid. C/ Francisco Tomás y Valiente, 5, 28049 Madrid, Spain; pedro.herrera@predoc.uam.es Faculty of Economics and Business Administration, Universidad Autónoma de Madrid. C/ Francisco Tomás y Valiente, 5, 28049 Madrid, Spain; pedro.herrera@predoc.uam.es y y Valiente, 5, 28049 Madrid, Spain; pedro.herrera@predoc.uam.es y , , , p ; p p 2 Department of Applied Economics, Universidad Autónoma de Madrid. C/ Francisco Tomás y Valiente, 5, 28049 Madrid, Spain; coro.chasco@uam.es 2 Department of Applied Economics, Universidad Autónoma de Madrid. C/ Francisco Tomás y Valiente, 5, 28049 Madrid, Spain; coro.chasco@uam.es p 3 Nebrija University. C/ Sta. Cruz de Marcenado, 27, 28015 Madrid 3 Nebrija University. C/ Sta. Cruz de Marcenado, 27, 28015 Madrid 4 Food and Agriculture Organization (F.A.O.), Room B532, Ciale delle Terme di Carcalla, 00153-Rome; max- imo.torerocullen@fao.org Ne ija U i e i y / a u e a e a o, , 0 a i 4 Food and Agriculture Organization (F.A.O.), Room B532, Ciale delle Terme di Carcalla, 00153-Rome; max- imo.torerocullen@fao.org * Correspondence: pedro.herrera@predoc.uam.es * Correspondence: pedro.herrera@predoc.uam.es Abstract: The role of agricultural transport costs in core-periphery structures has habitually been ignored in New Economic Geography (NEG) models. This is due to the convention of treating the agricultural good as the numéraire, thus implying that agricultural transportation costs are assumed to be zero in these models. For more than three decades, this has been the standard setting in spatial equilibrium analysis. The paper examines the effects of agricultural transport costs on the spatial organisation of regional structures in Peru. In doing so, the Krugman’s formulation of iceberg transport costs is modified to introduce the agricultural transport costs into the dynamic of the NEG models. We use exploratory spatial flow data analysis methods and non-spatial and spatial origin- destination flow models to explore how the regional spatial structure change when real transporta- tion data for agricultural goods is included into the iceberg transport costs formulation. We show that agricultural transport costs generate flows that are systematically associated with flows to or from nearby regions generating thus the emergence of spatial spillovers across Peruvian regions. Pedro Herrera-Catalán 1, Coro Chasco 2,3 and Máximo Torero 4 The results of the paper support the contention that NEG models have overshadowed the role of agricultural transport costs in determining the spatial configuration of economic activities. Keywords: Agricultural Transport Costs; New Economic Geography; Spatial Spillovers; Agglomer- ation Effects; Origin-Destination Flow Models Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/preprints202112.0200.v1 doi:10.20944/preprints202112.0200.v1 Article Spatial spillover effects of agricultural transport costs in Peru Article Keywords: Agricultural Transport Costs; New Economic Geography; Spatial Spillovers; Agglomer- ation Effects; Origin-Destination Flow Models Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/preprints202112.0200.v1 2 of 19 Samuelson’s iceberg transport costs. One assumption in that model was that the oxen pulling the loaded carts ate part of the grain being shipped to the destination market. The iceberg specification considers that the cost of moving goods involves the loss of some fraction of the product during shipment, that is, contemplates that a part of these goods melts away during the transport process. Samuelson’s iceberg transport costs. One assumption in that model was that the oxen pulling the loaded carts ate part of the grain being shipped to the destination market. The iceberg specification considers that the cost of moving goods involves the loss of some fraction of the product during shipment, that is, contemplates that a part of these goods melts away during the transport process. The iceberg transport cost formulation tacitly assumes a strictly convex curve with respect to distance, meaning that transport costs are larger as the distance from the origin increases. Nevertheless, this convex distance-costs structure triggers some critical draw- backs. On the one hand, it does not allow capturing the empirical fact that delivered prices of manufactured goods are concave with respect to distance [2-3]. On the other hand, the convex curve defined only by the manufacturing sector prevents studying the role of ag- ricultural transport costs in defining the spatial configuration of economic activity, espe- cially in developing countries where an important part of their inhabitants is devoted to agricultural activities [8]. The objective of this paper is to examine the effect of agricultural transport costs on the spatial organisation of regional structures in Peru. In doing so, the Krugman’s formu- lation of iceberg transport costs is modified to introduce the agricultural transport costs into the dynamic of the NEG models. We use exploratory spatial flow data analysis meth- ods and non-spatial and spatial econometric origin-destination techniques to examine how the regional spatial structure change when real transportation data for agricultural goods is included into the iceberg transport costs technology. The results of the paper show that agricultural transport costs generate flows that are systematically associated with flows to or from nearby regions generating thus the emer- gence of spatial spillovers in the agricultural sector across Peruvian regions. As noted by [2], the Krugman’s formulation of iceberg transport costs acts as a break and counter- weight to the emergence of agglomeration economies. 2. Theoretical setting We focus on Krugman’s formulation of iceberg transport costs to implement the em- pirical analysis. To this end, the iceberg transport costs function is modified to capture transportation costs in the agricultural sector, as in [9]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 The results of the paper confirm this conceptual conjecture and allow us to assert that NEG models have overshadowed the role of agricultural transport costs in determining the spatial distribution of economic activities. The remainder of this paper is organised as follows. Section 2 addresses the theoret- ical setting of the empirical methodology. Section 3 presents the exploratory spatial flow data analysis (ESFDA) methods for evaluating spatial autocorrelation in the variables of agricultural transport costs. Section 4 considers the spatial econometric origin-destination flow models used in the empirical analysis, and Section 5 presents the major results. Sec- tion 6 concludes. 1. Introduction [1] sets up the basic New Economic Geography (NEG) model as a one composed of a two-region, two-specific-factor (labour and land, or physical capital and labour) and two-sector/good (manufactured and agricultural goods). Whereas the manufactured goods are produced under increasing returns to scale and monopolistic competition, and they are traded with non-zero transport costs, the agricultural goods are produced under a technology of constant returns to scale, and they are traded costlessly. Under these convenient assumptions, by allowing for inter-regional labour mobility generated by real wage gaps across regions, one of the regions ends up as the industrial core, concentrating all the production of manufacture, while the other region transforms into the agricultural periphery, acting as the supplier/importer of agricultural/manufac- tured goods for/from the core, respectively. This is the general spatial equilibrium that has been the standard for more than three decades in NEG literature. Some scholars have observed that this binary spatial structure relies crucially on the way of modelling transportation cost of manufactured goods [2-4]. According to these authors, manufacturing transport costs have been frequently modelled under the Krugman’s function of iceberg transport technology, which follows [5]’s precepts. In ef- fect, [6] point out that the agricultural land-use model of [7] comprises the predecessor to 2.1. Agricultural transport costs model Suppose there is a producer in an origin location o and a consumer in a destination location d, with the geographic distance between the producer and consumer represented by Dod in kilometres. The price per ton of a good in locations o and d is Po and Pd, respec- tively; and the tonnage of a good in each location o and d is correspondingly symbolised by is Qo and Qd. The modelling logic of iceberg-type transport cost involves that only a fraction of the good shipped from o reaches its destination at d, that is, a part of the agri- cultural good ‘melts away’ in transit. The melting speed is supposed to be a linear function of the geographic distance between locations as in [1]. Thus, the iceberg transport technol- ogy for the agricultural good shipped from o to d is given by Equation (1): 𝑄௢= 𝑄ௗ. 𝑒𝑥𝑝(−𝜂. 𝐷௢ௗ) , (1) (1) 𝑄௢= 𝑄ௗ. 𝑒𝑥𝑝(−𝜂. 𝐷௢ௗ) , doi:10.20944/preprints202112.0200.v1 3 of 19 where η is an iceberg parameter of the share of the remaining tonnage of the shipped agricultural good, with η > 0. The price per ton of the agricultural good shipped from location o that must pay a consumer in location d (Pd), in relative terms, corresponds to Equation (2): 𝑃ௗ= 𝑃௢. 𝑄௢ 𝑄ௗ , (2) (2) Combining Equation (2) and Equation (1) the iceberg transport technology defined in Equation (3) stipulates that the price per ton of the agricultural good delivered in d is an increasing function of both components, the distance-decay parameter, and the geo- graphic distance between o and d. 𝜏௢ௗ= 𝑃ௗ 𝑃௢ = 𝑒𝑥𝑝(𝜂. 𝐷௢ௗ) , (3) (3) Equation (3) is useful to examine the relationship between agricultural transport costs and geographic distance. This equation also allows us to understand the convex na- ture of iceberg function for agricultural products. However, this expression is less con- venient when it comes to estimating agricultural transport costs by means of real data. That’s why, Equation (3) is formulated in relative terms, which complicates the measure- ment of accurate transport costs of equal agricultural goods between origin and destina- tion locations. In a real-world context, farmers usually use intercity truck freight transpor- tation to move their products between regions, where the fee paid by farmers is added to the price per ton of the agricultural good that enterprises pay in the destination location. 2.1. Agricultural transport costs model p p g g p p y To deal with the nature of real transportation data, transport costs for agricultural goods are specified as the difference between price per ton in origin and destination mar- kets o and d as follows: (4) 𝜏௢ௗ= 𝑃ௗ−𝑃௢= 𝑒𝑥𝑝(𝜂. 𝐷௢ௗ) + 𝑋𝑜𝛽𝑜+ 𝑋𝑑𝛽𝑑+ 𝑒𝑜𝑑 , (4) The first term, 𝑒𝑥𝑝(𝜂. 𝐷௢ௗ), represents the geographic distance as inferred in the Krugman’s formulation of iceberg transport costs, which is convex in distance. In the sec- ond term, 𝑋𝑜, 𝑋𝑑 denotes a set of explanatory variables in the origin o and destination d that define price gap for agricultural goods such as determinists of supply, demand, and efficiency, 𝛽𝑜, 𝛽𝑑, represent their corresponding vector of parameters to be estimated. Lastly, the third term, 𝑒𝑜𝑑, captures residual components of the price gap, such as the mark-up price over costs and product market regulation in the agricultural sector. 2.2. Empirical specification The factors that determine the spatial dynamics of agricultural transport costs are associated with geographical distance, explanatory variables, and the residual element as stated in Equation (4). Following [9], we estimate the relationship between agricultural transport costs and the supply, demand, and efficiency variables through a linear version of Equation (4), as stated in Equation (5): (5) 𝜏௢ௗ= 𝛼ଵ𝐷௢ௗ+ 𝛼ଶ𝐷௢ௗ ଶ+ 𝑋𝑜𝛽𝑜+ 𝑋𝑑𝛽𝑑+ 𝑒𝑜𝑑 , (5) where 𝛼ଵ, 𝛼ଶ represent unknown parameters. where 𝛼ଵ, 𝛼ଶ represent unknown parameters. where 𝛼ଵ, 𝛼ଶ represent unknown parameters. Moreover, because of the relevance of delivery time in transporting goods, as has been expressed in the specialised transport literature [10-11], the calculation of agricul- tural transport costs includes transportation time into the empirical model. Given 𝑑௞, the 4 of 19 length in kilometres of the kth segment of road, we estimate the time required to traverse this segment as in Equation (6): 𝑡௞= 𝑑௞ 𝑠௞ , (6) 𝑡௞= 𝑑௞ 𝑠௞ , (6) where 𝑠௞ represents the travel speed along segment k. The estimated shipping time of agricultural products between locations o and d is given by Equation (7): 𝑡௢ௗ= ෍𝑡௞ ௞ , (7) (7) where: k ∈ K is the set of road segments which define the fastest route between loca- tions o and d. The freight transportation costs for agricultural goods in dollars per ton, as expressed in Equation (5), corresponds to the minimum cost route joining any sending and arriving location using Equation (7). This cost is defined as the sum of the economic costs related to distance (fuel, toll, etc.) and time opportunity costs (salaries, insurance, etc.) in line with the methodology for calculation of transport costs proposed in [12]. We consider different origin-destination regions and various agricultural commodi- ties compiled into a single agricultural good to estimate the empirical model. Agricultural transport costs (ATC) results from the calculation of Equation (5) using the least cost path surface to connect each pair of regions using Geographic Information System (GIS). There- fore, the model for sending location o and arriving location d is approximated by the fol- lowing Equation (8): 𝐴𝑇𝐶௢ௗ= 𝛼଴+ 𝛼ଵ𝐷௢ௗ+ 𝛼ଶ𝐷௢ௗ ଶ+ 𝑋𝑜𝛽𝑜+ 𝑋𝑑𝛽𝑑+ 𝜀௢ௗ , (8) (8) where ATC is agricultural transport costs in dollars per ton/hour; ε is an error term indicating other unobservable effects on agricultural transport costs. 2.2. Empirical specification In the next section, exploratory spatial flow data analysis (ESFDA) is used to detect the existence of spatial autocorrelation –agglomeration economies– in the variable of ag- ricultural transport cost. Spatial autocorrelation can be considered at the origin, at the destination or as interaction between origin and destination. If present, this spatial effect should be then included in the estimation of the appropriate model. 3. Exploratory spatial flow data analysis In this section, we describe the exploratory spatial flow data analysis (ESFDA) meth- ods for evaluating agglomeration effects generated by agricultural transport costs. It must be said that only a few studies have found evidence for spatial spillovers and agglomera- tion effects in agricultural and rural settings. For example, [13] found that neighbourhood effects had a central role in determining where croplands expand, which led to agricul- tural expansion and landscape formation in Mato Grosso, Brazil. For the case of Spain, [14] found the presence of business agglomerations around rural areas in the Extremadura region. The authors propose that agglomeration economies be considered as an important element to explore endogenous development in low-density business environments. And [15] found a strong accumulation of agricultural innovation in Ireland explained by the joint effect of spatial concentration of the agricultural sector and regional knowledge spill- overs. 3.1. Agricultural transport costs in Peru This variable is approximated by the agricultural transport costs of the agricultural trade flows between each pair of Peruvian regions in 2013, which are calculated using the Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/preprints202112.0200.v1 6 of 19 As we can see, trade flows are more concentrated on the west side of the country, area that houses the main cities of Peru and where the Lima-region - the capital of the country - is located. Predominantly, regions trade with the Lima-region, where 35% of population lives and around 50% of the GDP is generated. Note that some regions do not trade with each other. This can be explained by an unprofitable trade position that occurs when transport costs and other costs related to trade exceed a determined threshold [16]. This threshold is determining by structural economic conditions associated with varia- tions on regional economic dynamics, price-demand relationship of agricultural commod- ities, and efficiency of road transport network, precisely our independent variables of in- terest. Operationally, the transport flow variable is presented in the form of a square matrix of order n-by-n, which can be organised in two ways: the origin regions (O) represented in rows, and the destination regions (D) in columns, as in Table 1a, or the other way around, as in Table 1b, where n is the number of regions. Table 1. Organisation of the data for the agricultural transport cost matrix: (a) Destination-based flows; (b) Origin-based flows. D1 D2 Dn O1 O2 … On O1 11 12 … 1n D1   … n O2 21 22 … 2n D2   … n … … … … … … … … … On n1 n2 … nn Dn n n … nn (a) (b) Table 1. Organisation of the data for the agricultural transport cost matrix: (a) Destination-based flows; (b) Origin-based flows. In both cases, each element ij is the cost of transporting agricultural goods from the origin region i to the destination region j, as approximated in Equation (8) where the val- ues of the main diagonal are the transport costs of agricultural products traded within each region (intraregional flows). In Table 1a, each column presents the values of the var- iable iD corresponding to the transport costs from each of the origin regions i to a specific destination region, D, whereas in Table 1b, variables Oj show the transport costs from a given origin region, O, to each of the destination regions j. 3.2. ESFDA of agricultural transport costs The ESFDA applied to flow data makes it possible to contrast the statistical signifi- cance of the effects of spatial autocorrelation both in the regions of origin and destination in an agricultural transport cost model. The ESFDA also allows knowing the distribution of the model variables to estimate, as well as their mathematical relationships and the possible effects of spatial autocorrelation [17]. We follow [18] to address the application of the ESFDA methods to implement the empirical analysis. We use the following spatial statistics: Moran's I test of flows, Moran's scatter diagram, and the Local Indicator of Spa- tial Autocorrelation (LISA) test of spatial autocorrelation. 3.2.1. Moran’s I test of global spatial autocorrelation of flows Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi doi:10.20944/preprints202112.0200.v1 5 of 19 [12]’ methodology. This methodology uses real transport network and distance and time features related to road transport for different road categories to estimate transport costs for agricultural goods based on the 2013 Agricultural Trade Flow Registry (ATFR). This database contains Origin-Destination (OD) data of agricultural commodity flows in Peru for the year 2013. For each Peruvian region, [12] considers OD flows above 10,964.80 tons per year. It was collected by the Peruvian Ministry of Agriculture and Irrigation. p y y y g g Quantitatively, agricultural transport costs in terms of dollar per ton/hour are calcu- lated by combining real freight costs and GIS analysis using road transport by truck, the most common transport mode for agricultural commodities in Peru. Figure 1 shows agri- cultural trade flows in terms of transport costs for agricultural goods for the 25 regions in Peru. For statistical purposes, the 2013 ATFR divides the Lima region into two regional areas: Lima Province and Metropolitan Lima and Callao (where the red star is located) to capture the agricultural trade flows between these two regional areas. Figure 1. Peru: agricultural trade flows in 2013. Figure 1. Peru: agricultural trade flows in 2013. 3.2.1. Moran’s I test of global spatial autocorrelation of flows The Moran's I test of spatial flows allows us to globally contrast the level of spatial autocorrelation existing in the variable of agricultural transport costs for the n regions that form the scope of analysis. This statistic is defined as the cross product of the flow variable, expressed in deviations to the mean and its corresponding spatial lag. The Moran's I sta- tistic can be expressed in two ways depending on the double organisation of the transport flow data (as shown in Table 1). On the one hand, Equation (9) presents the test for the n variables ID of agricultural transport costs, which serves to detect the existence of a linear relationship between the transport cost values of each origin region, i, that carries doi:10.20944/preprints202112.0200.v1 7 of 19 agricultural goods to a given destination region, D, and the mean values of this variable in the regions neighbouring each origin region, 𝜏̅௜஽. This statistic I test of destinations (ID) is expressed as follows: 𝐼஽= ∑∑𝑤௜௝𝑧௜஽∙𝑧௝஽𝑆଴ ⁄ ௝ ௜ ∑𝑧௜஽ ଶ 𝑛 ⁄ ௜ , (9) (9) where 𝑧௜஽= 𝜏௜஽−𝜏̅௜஽ and 𝑆଴= ∑∑𝑤௜௝ ௝ ௜ is the sum of all elements, 𝑤௜௝, of a W spa- tial weight matrix. W is an n-by-n square matrix of the Peruvian regions where each ele- ment, 𝑤௜௝, is Euclidean distance between centroids of the spatial units, considering the four nearest neighbouring regions, which is the average number of contiguous neighbours for the 25 Peruvian regions. This matrix was row-standardized, as recommended in [18]. Note that there are as many ID statistics as destination regions. On the other hand, in Equation (10), the Moran's I test is presented for the n variables Oj of transport cost from a given origin region to all destination regions. This test serves to detect the existence of a linear relationship between the transport cost values of a given origin region that distributes agricultural products to the rest of the destination regions and the mean value this variable in the regions neighbouring each destination region. This statistic is called I test of origins (IO) and is represented as follows: 𝐼ை= ∑∑𝑤௜௝𝑧ை௜∙𝑧ை௝𝑆଴ ⁄ ௝ ௜ ∑𝑧ை௝ ଶ 𝑛 ⁄ ௜ , (10) (10) where 𝑧ை௝= 𝜏ை௝−𝜏̅ை௝ and 𝜏̅ை௝ is the mean value of the transport cost in the regions neighbouring each destination region. Note that there are as many IO statistics as origin regions. 3.2.1. Moran’s I test of global spatial autocorrelation of flows The inferential process follows the permutational approach, obtaining a pseudo p- value for each n origin and n destination variables, which is used to identify the level of statistical significance of the test when evaluating agglomeration effects –at origin and destination regions– generated by agricultural transport costs. 3.2.2. Moran scatterplots of spatial flows 3.2.2. Moran scatterplots of spatial flows 3.2.2. Moran scatterplots of spatial flows A Moran scatter diagram approximates a scatter plot in which the transport flow variables for agricultural goods previously standardized, 𝑧௜஽ and 𝑧ை௝, are represented on the horizontal axis and their corresponding spatial lagged variables are represented on the vertical axis. The Moran scatter diagram can be divided into four categories. As the diagram focuses on the mean of both variables (the value 0), all values to the right/left of the mean value will be above/below zero and will be labelled as high/low, respectively. These four categories concern the four quadrants of the scatter plot. On the one hand, the upper-right and lower-left quadrants correspond to situations of positive spatial autocor- relation and are referred to as high-high (HH) and low-low (LL), respectively. On the other hand, the lower-right and upper-left quadrants correspond to situations of negative spatial autocorrelation and are denoted as high-low (HL) and low-high (LH), respectively. 3.2.3. LISA tests of local spatial autocorrelation of flows 3.2.3. LISA tests of local spatial autocorrelation of flows The LISA tests of local spatial autocorrelation of flows detects those values of the quadrants of the Moran scatter diagrams with statistically significant values to identify hot spots, cold spots, and atypical agricultural trade flows at each origin and destination. As with the Moran's I statistics, the LISA tests can be expressed in two ways. Equation (11) presents the test for the n variables ID of agricultural transport costs from each origin region i to a specific destination D, which serves to detect the existence of clusters or doi:10.20944/preprints202112.0200.v1 8 of 19 spatial agglomerations in the vicinity of the origin regions that carries agricultural goods to a certain destination region. This statistic is called IiD test of destinations: spatial agglomerations in the vicinity of the origin regions that carries agricultural goods to a certain destination region. This statistic is called IiD test of destinations: 𝐼௜஽= ∑𝑤௜௝𝑧௜஽∙𝑧௝஽ ௝ ∑𝑧௜஽ ଶ 𝑛 ⁄ ௜ , (11) (11) In Equation (12), the test is presented for the n variables Oj of agricultural transport cost from a given origin region O to all destination regions j. This test serves to detect the existence of clusters or spatial agglomerations in the vicinity of the destination regions that carries agricultural goods to a certain origin region. This statistic is called IOj test of origins: 𝐼ை௝= ∑𝑤௜௝𝑧௜ை∙𝑧௝ை ௝ ∑𝑧௜ை ଶ 𝑛 ⁄ ௜ , (12) (12) For the computation of the LISA test, we use the median instead of the mean to cal- culate the spatially lagged variables, as proposed by [18]. The LISA inferential process is similar to that followed for the global Moran index, except that in this case, the permuta- tion is carried out for each and every one of the observations, obtaining a pseudo p-value for the total N2 dyadic elements of Table 1, for 𝑁= 𝑛× 𝑛 regions. y g The calculations of the Moran I tests and the LISA tests for the origin and destination regions that are statistically significant are presented in Table 2. Table2. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/preprints202112.0200.v1 9 of 19 These results show the existence of both global and local positive spatial autocorre- lation of agricultural trade flows at the Peruvian regional level. It means that nearby re- gions have similar agricultural trade flows indicating thus spatial clustering. Figure 2 and Figure 3 graphically represent the results presented in Table 2. Both figures show that geographically close values of the agricultural trade flows tend to cluster with similar val- ues of the variable in a map, i.e., high values tend to be located close to high values, and low values are located close to low values. The stars and mean significant for p-value < 0.01 and a p-value < 0.05, respectively and MLC corresponds to Metropolitan Lima and El Callao. Figure 2a to Figure 2d show the clusters formed in destination regions created by an origin region. We observe the conformation of three clusters in the north part of the coun- try (Figure 2a to Figure 2c) and one cluster in the southern Peru (Figure 2d). The clusters originated by the regions of Piura, Cajamarca, and San Martín make up an agricultural cluster of intense commercial dynamism in the northern Peru. These regions trade through the Fernando Belaunde Terry Highway, which connects the jungle regions (San Martín) with the coastal regions (Piura, La Libertad, and Lambayeque), passing through the Cajamarca region, located in the sierra of the country. The export agricultural products from these regions (cocoa and coffee, primarily) are concentrated in the Piura region, which houses the port of Paita, the second busiest container port after El Callao port, lo- cated in the MLC region. Figure 2d shows the cluster created by the Ica region in the southern part of the country. This region mainly supplies the regions of Cusco, Junín, Lima, and MLC (Metropolitan Lima and El Callao region) with avocados, asparagus, and grapes, part of whose production is sent abroad through the Jorge Chávez International airport and El Callao port. (a) (b) (c) (d) Figure 2. Clusters in destination regions created by origin regions: (a) Origin: Piura; (b) Origin: Cajamarca; (c) Origin: San Martín; (d) Origin: Ica. Figure 3a to Figure 3d show the clusters in origin regions created by destination re- gions. These clusters are made up of regions that agglomerate to provide a particular re- gion with agricultural goods and related services. 3.2.3. LISA tests of local spatial autocorrelation of flows Results of the Moran I and LISA tests for origin and destination regions Clusters in origin-regions Clusters in destination-regions Destination- region Origin- region Moran I LISA Origin- region Destination- region Moran I LISA Arequipa 0.272* Cajamarca 0.184* Cusco 0.311* La Libertad 0.518* Puno 0.331* Lambayeque 0.664* Tacna 0.430 San Martín 0.175* Cajamarca 0.224* Ica 0.164 Ancash 0.060 Cusco 0.163* La Libertad 0.258* Junín 0.192 Lambayeque 0.039 Lima 0.086 MLC a 0.331 Ica 0.103* Piura 0.122 Apurímac 0.152 Cajamarca 0.013 Arequipa 0.605 La Libertad 0.243* Cusco 0.008 Huancavelica 0.233 Lambayeque 0.127 San Martín 0.129 Cajamarca 0.351* Cajamarca 0.354 San Martín 0.168 Lambayeque 0.182 Piura 0.447 Table2. Results of the Moran I and LISA tests for origin and destination regions Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 This is the case of the regions of Puno, No signif. HH LL LH HL No signif. HH LL LH HL No signif. HH LL LH HL No signif. HH LL LH HL (b) No signif. HH LL LH HL (a) No signif. HH LL LH HL (b) (a) (d) No signif. HH LL LH HL (c) No signif. HH LL LH HL (d) (c) Figure 2. Clusters in destination regions created by origin regions: (a) Origin: Piura; (b) Origin: Cajamarca; (c) Origin: San Martín; (d) Origin: Ica. Figure 3a to Figure 3d show the clusters in origin regions created by destination re- gions. These clusters are made up of regions that agglomerate to provide a particular re- gion with agricultural goods and related services. This is the case of the regions of Puno, Figure 3a to Figure 3d show the clusters in origin regions created by destination re- gions. These clusters are made up of regions that agglomerate to provide a particular re- gion with agricultural goods and related services. This is the case of the regions of Puno, 4. Econometric modelling In this section, we present the econometric origin-destination flow models and the explanatory variables used to examine the effects of agricultural transport costs on the spatial organisation of regional structures in Peru. Origin-destination flow models, like the one set up in Equation (8), use agricultural trade flow data to explain spatial interac- tions from a set of origin locations to a set of destination locations in the geographic do- main of Peru at the regional level. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/preprints202112.0200.v1 11 of 19 11 of 19 We can say that ESFDA results yield valuable insights about the clustering of agri- cultural trade flows that are suggestive of the diffusion of interregional trade. Certainly, the northern regions of the country (Figure 4a) have created a more intense spatial net- work for trade exchange of agricultural products than the southern regions (Figure 4b). This is mainly due to the influence exerted by the Paita port that operates as a centripetal force on agricultural export goods in the northern regions of the country. Our results are consistent with the findings of [19] that has been identified six main agricultural export corridors in Peru, three of which have been detected by ESFDA methods. y These ESFDA tools detect the existence of significant global and local spatial auto- correlation in the distribution of agricultural transport flows. That is, agricultural trade flows are not randomly distributed among the Peruvian regions but spatially clustered. These detected spatial trends and clusters must be tested and most probably explicitly specified in econometric models of agricultural transport flows in Peru, since they are also relevant explanatory variables that are explaining this phenomenon. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/preprints202112.0200.v1 10 of 19 10 of 19 Tacna, and Cusco, which supply the Arequipa region with agricultural products such as coffee, quinoa, and olives, both for local consumption and for export through the Matarani Port (Figure 3a). Other important cluster is found in the south-central part of the country (Figure 3b), where the regions of Arequipa, Cusco, Apurímac, and Huancavelica provide the region Ica with agricultural goods (onions, avocados, asparagus, paprika, and coffee), which are mostly concentrated for export through El Callao port. Tacna, and Cusco, which supply the Arequipa region with agricultural products such as coffee, quinoa, and olives, both for local consumption and for export through the Matarani Port (Figure 3a). Other important cluster is found in the south-central part of the country (Figure 3b), where the regions of Arequipa, Cusco, Apurímac, and Huancavelica provide the region Ica with agricultural goods (onions, avocados, asparagus, paprika, and coffee), which are mostly concentrated for export through El Callao port. (a) (b) (c) (d) Figure 3. Clusters in origin regions created by destination regions: (a) Destination: Arequipa; (b) Destination: Ica; (c) Destination: Cajamarca; (d) Destination: Lambayeque. No signif. HH LL LH HL No signif. HH LL LH HL No signif. HH LL LH HL No signif. HH LL LH HL (a) No signif. HH LL LH HL (b) No signif. HH LL LH HL (b) (a) (b) (d) No signif. HH LL LH HL (a) (c) No signif. HH LL LH HL (d) (c) Figure 3. Clusters in origin regions created by destination regions: (a) Destination: Arequipa; (b) Destination: Ica; (c) Destination: Cajamarca; (d) Destination: Lambayeque. As can see in Figure 4a and Figure 4b, agricultural trade flows create intense trade exchange networks both in two spatial clusters located in the northern and southern Peru. (a) (b) Figure 4. Origin-destination transaction networks for agricultural goods; (a) North network; (b) Central-South network. (a) (b) (b) (a) Figure 4. Origin-destination transaction networks for agricultural goods; (a) North network; (b) Central-South network. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 do doi:10.20944/preprints202112.0200.v1 12 of 19 network because a better-quality infrastructure should conduct to more trade in both ur- ban and agricultural areas ([24-25]). network because a better-quality infrastructure should conduct to more trade in both ur- ban and agricultural areas ([24-25]). g Hence, the origin-destination model of agricultural trade flows of Peru includes three independent variables:  Gross value added in each region, which measures regional economic dynamics. Since agricultural trade flows are directly proportional to the economic activity in regional economies, we would expect a positive relationship between these flows and the gross value added.  Consumer price index variation for agricultural products in each region, which ap- proximates the price-demand relationship of agricultural goods. Prices increment should reduce the demand for goods, and lead to reductions of agricultural trade flows, thus effects associated with consumer price index change should be negative with respect to trade flows.  Paved neighbourhood road length in each region measures road transport network efficiency. A better-quality infrastructure should conduct to more trade therefore, we would expect a positive relationship between trade flows and paved neighbourhood road length. g In addition, we use the distance and the squared distance between each pair of re- gions as extra explanatory variables to capture convexity-concavity patterns associated with real transportation data for agricultural goods. The summary statistics of dependent and independent variables of the origin-destination model are shown in Table 3. In addition, we use the distance and the squared distance between each pair of re- gions as extra explanatory variables to capture convexity-concavity patterns associated with real transportation data for agricultural goods. The summary statistics of dependent and independent variables of the origin-destination model are shown in Table 3. Table 3. Description of the dependent and independent variables Variable Description Units Mean Std Min Max Dependent variable Agricultural trade flows Agricultural transport costs between each pair of regions. Authors' calculations (see Subsection 3.2). Dollar per ton/hour 351,504.6 542,531.8 732.7 3,336,130.4 Independent variables Regional economic dynamics Gross value added in each region. National Institute of Statistics and Informatics (INEI) (Index 2007=100) 137.3 20.6 89.0 189.8 Price- demand relationships Consumer price index variation for agricultural goods in each region. INEI. Consumer Price Index 2.8 1.3 0.4 5.7 Road transport network efficiency Paved neighbourhood roads length in each region. Ministry of Transport and Communications (MTC). Kilometres 77.6 94.1 66.2 403.3 Spatial variable d Distance between each pair of regions. 4.1. Non-spatial origin-destination model The non-spatial version of origin-destination flow models assumes independence of flows and uses Ordinary Least Square (OLS) for its estimation. Taking into account [9]’s linear specification of the model of agricultural trade costs in Equation (8), this model can be formulated as follows: 𝑦= 𝑋𝛽+ 𝜀 , (13) 𝑋𝛽= 𝛼଴𝜄ே+ 𝑋𝑜𝛽𝑜+ 𝑋𝑑𝛽𝑑+ 𝛼ଵ𝐷௢ௗ+ 𝛼ଶ𝐷௢ௗ ଶ , (14) 𝑦= 𝑋𝛽+ 𝜀 , (13) (13) 𝑦= 𝑋𝛽+ 𝜀 , 𝑦 𝛽 ( ) 𝑋𝛽= 𝛼଴𝜄ே+ 𝑋𝑜𝛽𝑜+ 𝑋𝑑𝛽𝑑+ 𝛼ଵ𝐷௢ௗ+ 𝛼ଶ𝐷௢ௗ ଶ , (14) (14) 𝑋𝛽= 𝛼଴𝜄ே+ 𝑋𝑜𝛽𝑜+ 𝑋𝑑𝛽𝑑+ 𝛼ଵ𝐷௢ௗ+ 𝛼ଶ𝐷௢ௗ ଶ , (14) where y is an N by 1 vector of the natural log of agricultural trade flows (ATC, in Equation (8)); 𝜄ே is an N by 1 vector of ones with α0 representing a constant parameter term; 𝑋௢𝛽௢ and 𝑋ௗ𝛽ௗare respectively 𝑋௢= 𝜄௡⨂𝑋 and 𝑋ௗ= 𝜄௡⨂𝑋, where X is an n-by-n matrix of the natural log of independent variables for the n regions and ⨂ represents a Kronecker product, with 𝛽௢and 𝛽ௗ being their related scalar parameters; 𝐷௢ௗ is an N by 1 vector of the natural log of distance between each o, d pair of regions with α1 and α2 being the associated scalar parameters, and ε is the N by error term vector which is as- sumed 𝜀~𝑁(0, 𝜎ଶ𝐼ே), for 𝜎ଶ the constant variance of the error term and 𝐼ே an identity matrix of order N. The X matrix must contain the independent variables of the model, that is, the main determinants of agricultural trade flow variables. As stated in the literature, these are as- sociated with the supply, demand, and efficiency sides of the economy. On the supply side, trade flow variations are directly proportional to the regional economic dynamics since that a higher economic growth should conduct to more trade in the agricultural sec- tor because of a higher production of goods ([20-21]). On the demand side, a price-demand relationship variable involves a negative relation between trade flows and prices of agri- cultural goods: an increase in prices for goods should reduce its demand, which in turn would lead to a reduction of agricultural trade flows ([22-23]). Last, on the efficiency side, agricultural trade flows are directly proportional to the efficiency of road transport Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 do Authors' calculations based on data from INEI. Kilometres 720.1 413.7 56.1 1948.9 The parameters of the non-spatial models are estimated by OLS (see the main results in Table 4). The R-squared statistic was 0.26, and the distance variable has a negative and significant coefficient, and the squared distance variable coefficient is positive, as ex- pected. This last result implies that an increase in distance is associated with a decline in Table 3. Description of the dependent and independent variables The parameters of the non-spatial models are estimated by OLS (see the main results in Table 4). The R-squared statistic was 0.26, and the distance variable has a negative and significant coefficient, and the squared distance variable coefficient is positive, as ex- pected. This last result implies that an increase in distance is associated with a decline in The parameters of the non-spatial models are estimated by OLS (see the main results in Table 4). The R-squared statistic was 0.26, and the distance variable has a negative and significant coefficient, and the squared distance variable coefficient is positive, as ex- pected. This last result implies that an increase in distance is associated with a decline in doi:10.20944/preprints202112.0200.v1 13 of 19 13 of 19 agricultural transport costs, a finding that is inconsistent with the strictly convex curve of the Krugman iceberg specification, as shown in Figure 5a. (b) (a) 2.7230 2.7430 2.7630 2.7830 2.8030 2.8230 2.8430 2.8630 2.8830 2.9030 0 200 400 600 800 1,000 OLS estimation (a) (b) Figure 5. Estimation of the effect of distance on agricultural transport costs. 2.7230 2.7430 2.7630 2.7830 2.8030 2.8230 2.8430 2.8630 2.8830 2.9030 0 200 400 600 800 1,000 OLS estimation (b) (a) Figure 5. Estimation of the effect of distance on agricultural transport costs. However, as noted by many econometricians (e.g., [26-27]), the OLS estimation of the non-spatial origin-destination model is unsuitable for many spatial interaction phenom- ena (e.g., the agricultural transport flows), because it assumes independence among flows. Due to spatial interactions are operationalised through spatial autocorrelation in econo- metric models, if OLS is used in its presence, the resulting parameter estimates can suffer from potential bias and inefficiency [28]. 4.2. Spatial origin-destination model 4.2.1. Specification 4.2.1. Specification [26] extended the basic formulation of the origin-destination flow model to allow for spatial dependence by means of explicitly account for origin based, destination based, and origin-to-destination based neighbourhood influences. Origin-specific variables denote the ability of origin locations to create flows, whereas destination-specific variables repre- sent the attraction of destination locations. Origin-destination functions in an agricultural setting indicate how agricultural trade interactions are obstructed by geographical dimen- sions. Methodologically, origin based, destination based, and origin-to-destination based neighbourhood influences are accounted for using the spatial weight matrices Wo, Wd, and Ww, respectively. Hence, the spatial autoregressive (SAR) interaction model of agricultural transport flows is expressed as follows: 𝑦= 𝜌௢𝑊௢𝑦+ 𝜌ௗ𝑊ௗ𝑦+ 𝜌௪𝑊௪𝑦+ 𝑋𝛽+ 𝜀 (15) 𝑋𝛽= 𝛼଴𝜄ே+ 𝑋𝑜𝛽𝑜+ 𝑋𝑑𝛽𝑑+ 𝛼ଵ𝐷௢ௗ+ 𝛼ଶ𝐷௢ௗ ଶ , (16) (15) (16) The spatial origin-destination flow model comprises three N by 1 spatially lagged dependent variables of flows, which represent the strength of origin-based, destination- based, and origin-to-destination based spatial dependence each one with their scalar pa- rameters 𝜌௢, 𝜌ௗ, 𝜌௪, correspondingly. [26] uses the Kronecker product of the conventional spatial weight matrix (W) of order n by n, and an identity matrix (In) equal in size to that of the spatial weight matrix to describe spatial connectivity between the n regions. The doi:10.20944/preprints202112.0200.v1 14 of 19 14 of 19 origin spatial weight matrix (Wo), the destination spatial weight matrix (Wd), and the origin-to-destination spatial weight matrix (Ww), are obtained respectively as follows: origin spatial weight matrix (Wo), the destination spatial weight matrix (Wd), and the origin-to-destination spatial weight matrix (Ww), are obtained respectively as follows: 𝑊௢= 𝑊⨂𝐼௡ 𝑊ௗ= 𝐼௡⨂𝑊 𝑊௪= 𝑊⨂𝑊, (17) (17) The 𝑊௢ is an N-by-N row standardized spatial weight matrix used to capture origin- based spatial dependence, and analogously, 𝑊ௗ, is an N by N row standardized spatial weight matrix used to capture destination-based spatial dependence. Operationally, 𝑊௢𝑦 and 𝑊ௗ𝑦 reflect respectively the average of flows from neighbours of the origin to the destination and the average of flows from the origin to neighbours of the destination. Lastly, 𝑊௪, is an N-by-N row standardized spatial weight matrix defined as the product of 𝑊௢ and 𝑊ௗ, i.e., 𝑊௪, = 𝑊௢∙𝑊ௗ, which captures origin-to-destination spatial depend- ence and it represents the average of flows from neighbours of the origin to neighbours of the destination. * p-value<0.01; p-value<0.05; * p-value<0.1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/preprints202112.0200.v1 15 of 19 15 of 19 Estimates for the SAR interaction model are shown in Table 4. There is a high indi- cation of origin- and origin-to-destination-based spatial dependence. These results con- firm the effectiveness of the ESFDA for clusters in origin-regions. The coefficients 𝜌௢ and 𝜌௪ were 0.64, and 0.48, respectively, and the 99 percent credible intervals indicate they are different from zero. The destination-based spatial dependence captured by 𝜌ௗ = - 0.18 also was significant although proved to be the least influential for detecting spatial de- pendence patterns. The coefficient for the distance variable was significant and negative (-0.0007) and much closer to zero than the least squares estimate (-0.0085), as shown in Figure 4. It is a usual result for the estimate on distance to fall in significance when spa- tially lagged variables are included into a SAR interaction model as distance operates as a proxy for spatial dependence when it is not incorporated ([30-31]). 4.2.2. Estimation of the SAR origin-destination model [26] propose estimating this SAR origin-destination model by Maximum Likelihood (ML) and [27] set forth a Bayesian Markov chain Monte Carlo (MCMC) approach to esti- mate the SAR model. We use the Bayesian MCMC to estimate the spatial autoregressive (SAR) interaction model of agricultural flows. Lower and upper 0.05 and 0.95 credible intervals are reported based on the retained 3,000 Bayesian MCMC SAR draws. Bayesian MCMC SAR estimates were based on 5,500 draws with 2,500 omitted for start-up as de- fined in [29]. Table 4. Coefficient estimates for the non-spatial and spatial autoregressive models * p-value<0.01; p-value<0.05; * p-value<0.1 Variable Least-Squares Model Spatial Autoregressive Coefficient Coefficient Constant -13.5381 * -8.6443 consumer price index for agricultural goods -0.6002 -0.2285 paved neighbourhood roads 0.6885 * 0.3143 * regional gross value added 3.5698 * 1.4912 consumer price index for agricultural goods -0.3295 0.0579 paved neighbourhood roads 0.1188 0.0653 regional gross value added 0.0008 0.1286 log(distance) -0.0085 * -0.0007 log(distance2) 0.30·e-5 * 0.13·e-6 -0.1817 * 0.6435 * 0.4874 * Table 4. Coefficient estimates for the non-spatial and spatial autoregressive models Variable 5. Discussion on the effect estimates Effects estimates for the non-spatial and SAR interaction model are reported in Table 5. Table 5. Effects estimates for the non-spatial and spatial autoregressive interaction Variables Least-Squares Model Spatial Autoregressive Mean Median Std. Dev. Mean Median Std. Dev. Origin - consumer price index for agricultural goods 0.0637 0.0614 0.3079 -0.1532 -0.1316 1.0399 Origin - paved neighbourhood roads 0.0595 0.0596 0.0956 0.5969 0.4749 0.7727 Origin - regional gross value added 0.1681 0.1422 1.0593 2.5751 2.0815 5.1307 Destination - consumer price index for agricultural goods -0.2081 -0.2102 0.3007 -0.5848 -0.5369 1.1752 Destination - paved neighbourhood roads 0.2967 0.2960 0.1036 1.0031 0.8885 0.8138 Destination - regional gross value added 1.4238 1.4327 1.0543 4.6975 4.1212 5.5379 Intraregional - consumer price index for agricultural goods -0.0060 -0.0060 0.0180 -0.0207 -0.0186 0.0564 Intraregional - paved neighbourhood roads 0.0148 0.0149 0.0059 0.0453 0.0409 0.0352 Intraregional - regional gross value added 0.0663 0.0661 0.0628 0.2052 0.1807 0.2516 Network - consumer price index for agricultural goods -- -- -- -5.7770 -4.0858 19.0636 Network - paved neighbourhood roads -- -- -- 12.3269 9.3101 17.7252 Network - regional gross value added -- -- -- 56.3468 38.4989 109.0265 Total - consumer price index for agricultural goods -0.1504 -0.1511 0.4493 -6.5357 -4.7502 21.1928 Total - paved neighbourhood roads 0.3711 0.3721 0.1471 13.9721 10.7266 19.3182 Total - regional gross value added 1.6582 1.6515 1.5710 63.8246 44.3224 119.5158 The non-spatial regression estimates indicate that a 1 percent increase in consumer price index for agricultural goods at the origin region would lead to a 0.06 percent increase in the agricultural trade flows while a 1 percent increase in paved neighbourhood roads at the origin region would lead to a 0.05 percent increase in trade flows in the agricultural sector. Both estimates are close to zero and thus proved to have little influence in the Table 5. Effects estimates for the non-spatial and spatial autoregressive interaction The non-spatial regression estimates indicate that a 1 percent increase in consumer price index for agricultural goods at the origin region would lead to a 0.06 percent increase in the agricultural trade flows while a 1 percent increase in paved neighbourhood roads at the origin region would lead to a 0.05 percent increase in trade flows in the agricultural sector. 5.2. Spatial origin-destination model Agricultural trade flows exert different types of spatial interactions depending on the geographic domains in which they operate. Economic factors that lead to agricultural trade flows from any origin region to a specific destination may create analogous trade flows from neighbours of that origin to the same destination. Likewise, an analogous rea- soning holds for destination-specific determinants that constitute a centripetal force for destination locations. Socio-economic influences that cause a destination region to import agricultural goods from a specific origin region may bring about the neighbours of that destination to import similar agricultural products from that origin. Origin-to-destination interactions among regions reflect the average of flows from neighbours of the origin re- gion to neighbours of the destination region. Spatial interactions in agricultural settings are approximated by the total effects of changes in geographical characteristics on agricultural trade flows in both origin and des- tination regions. Following [29], these total effects (𝑇𝐸) can be decomposed into four ef- fects: origin effect (𝑂𝐸), destination effect (𝐷𝐸), intraregional effect (𝐼𝐸) and network effect (𝑁𝐸), which are estimated using a matrix of partial derivatives. For every of these four components (𝑂𝐸, 𝐷𝐸, 𝐼𝐸 and 𝑁𝐸), we calculate similar scalar summary measures as for the 𝑇𝐸, that quantifies the overall impacts of a change to the 𝑟th characteristic of all regions on origin-destination agricultural trade flows. With respect to the effects estimates for the SAR interaction model, we can see that the SAR interaction model attributes a greater impact to origin and destination effects generated by for regional economic dynamics. We find that a 1 percent increase in gross value added respectively produces a 2.57 and 4.69 percent increase in agricultural trade flows both at the origin and destination. Note that effects estimates show that destination- based dependence is more important than origin-based and OD-based dependence for interregional agricultural trade flows, which implies that destination explanatory varia- bles exert trade flow attraction to destination locations. Note that for origin effects, a 1 percent increase in consumer price index for agricul- tural goods leads to a 0.15 percent decrease in agricultural trade flows under SAR inter- action models, while the same increment would lead to 0.06 percent increase in agricul- tural trade flows under the case of least squares estimates. This contradictory result may be indicative of biased and inconsistent least squares estimates in the presence of spatial dependence. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/preprints202112.0200.v1 16 of 19 16 of 19 standard regression model. The impact of increasing regional gross value added at the origin and at the destination region by 1 percent would be to increase agricultural trade flows by 0.16 and 1.42 percent, respectively. Thus, supply factors influencing trade flow variations proved to be the most important in the non-spatial model. The intraregional impacts of increasing consumer price index for agricultural goods, paved neighbourhood roads or regional gross value added in the representative region are small and correspond to -0.0060, 0.0148, and 0.0663, respectively. This suggests that increasing the conditions that generate transport costs variations in the Peruvian agricul- tural sector would lead to more trade within the region, limiting the emergence of external economies of scale across regions. Total effects reflect the sum of the origin, destination, and intraregional impacts on agricultural trade flows in and out of regions as well as within regions arising from vari- ations in the independent variables of the model. The total effect for consumer price index assessed by increasing consumer price index for agricultural goods by 1 percent is a de- crease in agricultural trade flows (in and out of regions as well as within regions) by around 0.150 percent. The total effect of increasing regional gross value added and paved neighbourhood roads by 1 percent is a 1.6582 and a 0.3711 percent increase in agricultural trade flows (in and out of regions as well as within regions), correspondingly. 5. Discussion on the effect estimates Both estimates are close to zero and thus proved to have little influence in the The non-spatial regression estimates indicate that a 1 percent increase in consumer price index for agricultural goods at the origin region would lead to a 0.06 percent increase in the agricultural trade flows while a 1 percent increase in paved neighbourhood roads at the origin region would lead to a 0.05 percent increase in trade flows in the agricultural sector. Both estimates are close to zero and thus proved to have little influence in the Author Contributions: Conceptualization, P.H.-C. and M.T.; methodology, P.H.-C., C.C. and M.T.; software, P.H.-C., and C.C..; validation, P.H.-C.; formal analysis, P.H.-C. and C.C..; investigation, P.H.-C. and M.T.; resources, P.H.-C.; data curation, P.H.-C.; writing—original draft preparation, P.H.-C. and C.C..; writing—review and editing, P.H.-C., C.C. and M.T.; project administration, P.H.- Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/preprints202112.0200.v1 17 of 19 17 of 19 agricultural trade flows lead to more trade within the region, which would be an expla- nation of the low levels of trade across regions in Peru as reviewed in Section 3.1. Note that effect estimates for the three explanatory variables proved to be greater in the settings of the SAR interaction model than in the non-spatial model estimates, which is an indica- tive of the relevance of spatial dependence in origin-destination flow models. The great contribution of spatial interaction models resides in the quantification of network effects or spillover effects and contextually rich information it produces. Spillo- ver effects are interpreted as changes at one point in time to the changes in flows agglom- erated over the network required to reach at a new steady-state equilibrium over all re- gions ([29]). We find that a 1 percent increase in consumer price index for agricultural goods in the representative region would lead to a 5.77 percent (cumulative) decrease in agricultural trade flows throughout the network of regions. Likewise, the network effect for a 1 percent increase in paved neighbourhood roads in the traditional region leads to a 12.32 percent cumulative increase in agricultural trade flows throughout the region. The spillover effects related to the diffusion of regional gross value added through- out the network of regions proved to be the most influential in the spatial origin-destina- tion flow model. An increment of 1 percent in gross value added would lead to a 56.34 percent cumulative increase in agricultural trade flows across all regions. This result can be understood as the long-run increase generated by an increment of regional economic dynamics into agricultural trade flows cumulated over the network of 25 Peruvian re- gions. This cumulative effect over time would contribute to the configuration of a new steady-state equilibrium throughout the network of regions in Peru. Network externalities prove that flows are systematically associated with flows to or from nearby regions gen- erating thus the emergence of spillover effects in the agricultural sector across Peruvian regions. 5.2. Spatial origin-destination model Intraregional effects were small but different from zero for the three explanatory var- iables under study. These results suggest that variations in the determinants of References Transport costs, distance, and time: evidence from the Japanese Census of Logistics. IDE D Institute of Developing Economies, Japan External Trade Organization. j j rt costs, distance, and time: evidence from the Japanese Census of Logistics. IDE Discussion Papers 2010, 241, ing Economies, Japan External Trade Organization. 10. Evans, C.L.; Harrigan, J. Distance, time, and specialization: lean retailing in general equilibrium. Am Econ Rev 2005, 95(1), 292- 313. https://doi.org/10.1257/0002828053828590 p g 11. Djankov, S.; Freund, C.; Pham, C.S. Trading on Time. Rev Econ Stat 2010, 92(1), 166-173. https://doi.org/10.1162/rest.2009.11498 12 IFPRI Constructing a typology of rural communities of the Peruvian highlands using stochastic profit frontier estimation; International p g 11. Djankov, S.; Freund, C.; Pham, C.S. Trading on Time. Rev Econ Stat 2010, 92(1), 166-173. https://doi.org/10.1162/rest.2009.11498 12. IFPRI. Constructing a typology of rural communities of the Peruvian highlands using stochastic profit frontier estimation; International Food Policy Research Institute, Washington, U.S., 2009. 1. Djankov, S.; Freund, C.; Pham, C.S. Trading on T j , ; , ; , g , ( ), p g 12. IFPRI. Constructing a typology of rural communities of the Peruvian highlands using stochastic profit frontie Food Policy Research Institute, Washington, U.S., 2009. 12. IFPRI. Constructing a typology of rural communities of the Peruvian highlands using stochastic profit frontier estimation; International Food Policy Research Institute, Washington, U.S., 2009. 13. Richards, P. It’s not just where you farm; it’s whether your neighbor does too. How agglomeration eco agricultural landscapes. J Econ Geogr 2018, 18(1), 87-110. https://doi.org/10.1093/jeg/lbx009 just where you farm; it’s whether your neighbor does too. How agglomeration economies are shaping new pes. J Econ Geogr 2018, 18(1), 87-110. https://doi.org/10.1093/jeg/lbx009 14. Rangel-Preciado, J.; Parejo-Moruno, F.M.; Cruz-Hidalgo, E.; Castellano-Álvarez, F.J. Rural Districts and Business Agglomera- tions in Low-Density Business Environments. The Case of Extremadura (Spain). Land 2021, 10(3), 280. https://doi.org/10.3390/land10030280 k, A.; Cullinan, J.; Thorne, F. What drives innovation in the agricultural sector? A spatial analysis of rs. Land Use Policy 2016, 56(1), 238-250. https://doi.org/10.1016/j.landusepol.2016.04.032 15. Läpple, D.; Renwick, A.; Cullinan, J.; Thorne, F. What drives innovation in the agricultural secto knowledge spillovers. Land Use Policy 2016, 56(1), 238-250. https://doi.org/10.1016/j.landusepol.2016.04 16. Ranjan, P.; Tobias, J.L. Bayesian inference for the gravity model. J Appl Economet 2007, 22(4), 817-838. https://doi.org/10.1002/jae.926 17. Herrera-Catalán, P.; Chasco, C. Agglomeration on the periphery? Exploratory spatial analysis of agricultural transport flows in Peru. In Agglomeration Economies and rural Development; Parejo-Moruno, F.; Rangel- Preciado, J. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Krugman, P. Increasing returns and economic geography. J Polit Econ 1991, 99(3), 483-499. https://doi. 1. Krugman, P. Increasing returns and economic geography. J Polit Econ 1991, 99(3), 483-499. https://doi.org/10.1086/261763 2. McCann, P. Transport costs and new economic geography. J Econ Geogr 2005, 5(3), 305-318. https://doi.org/10.1093/jnlecg/lbh050 3. Fingleton, B.; McCann, P. 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All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by eS-MiData Project, grant number 447379001. Data Availability Statement: Data and scripts are available in the Spatial and Regional Economics research group (ECONRES) of the Universidad Autónoma de Madrid at https://econresuam.word- press.com/opendata. Acknowledgments: We are grateful to participants at the 15th PRSCO Summer Institute \| 4-6 July 2018, Lima, Peru; the 66th NARSC Conference \| 13-16 November 2019, Pittsburgh, Pennsylvania, EEUU, and the International Seminar on Agglomeration Economies and Rural Development \| 20- 21 May 2021, Universidad de Extremadura, Spain for their valuable comments to several versions of this paper. Likewise, we would like to thank James P LeSage for his kind provision of the MATLAB code to estimate the OLS and SAR models. 6. Conclusions Most NEG models have approached agglomeration process by assuming free transport of the agricultural good. This assumption however involves the theoretical shortcoming that the agricultural sector must be sufficiently large for dispersion to occur as an equilibrium outcome, which impedes the examination of the spatial spillover effects in agricultural settings. To the best of our knowledge, the empirical implications of this theoretical flaw has not been addressed until now. The issue is investigated in two dimensions. First, the Krugman’s formulation of ice- berg transport costs is reformulated to capture the agricultural transport costs. Second, ESDA methods and non-spatial and SAR interaction models are used to explore how the regional spatial structure change when agricultural trade flows are included into the ice- berg transport costs technology. The results of ESDA revealed the existence of agglomer- ation effects generated by agricultural transport costs, implying that prices of agricultural goods are concave with respect to distance. These results were confirmed by SAR interac- tion models, which support the notion that agricultural transport costs create flows are associated with flows to or from neighbouring regions causing the emergence of spatial spillovers across regions in Peru. The results of the paper allow us to confirm [2]’s assertation relative to that Krugman’s formulation of iceberg transport costs acts as a break and counterweight to the emergence of agglomeration economies ([32]). Our findings also support that NEG mod- els have overshadowed the role of agricultural transport costs in determining the spatial distribution of economic activities, especially in developing countries like Peru where an important part of their inhabitants is devoted to agricultural activities. Author Contributions: Conceptualization, P.H.-C. and M.T.; methodology, P.H.-C., C.C. and M.T.; software, P.H.-C., and C.C..; validation, P.H.-C.; formal analysis, P.H.-C. and C.C..; investigation, P.H.-C. and M.T.; resources, P.H.-C.; data curation, P.H.-C.; writing—original draft preparation, P.H.-C. and C.C..; writing—review and editing, P.H.-C., C.C. and M.T.; project administration, P.H.- Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/prepr Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 13 December 2021 doi:10.20944/preprints202112.0200.v1 19 of 19 22. McCalla, A.F. Impact of macroeconomic policies upon agricultural trade and international agricultural development. Am J Agr Econ 1982, 64(5), 861- 868. https://doi.org/10.2307/1240745 23. Diaz-Bonilla, E.; Robinson, S. Macroeconomics, macrosectoral policies, and agriculture in developing countries. In Handbook of Agricultural Economics, Pingali, P., Evenson, R. 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https://openalex.org/W3014450318	https://link.springer.com/content/pdf/10.1007/s10461-020-02840-2.pdf	English	null	Overlapping Transmission Networks of Early Syphilis and/or Newly HIV Diagnosed Gay, Bisexual and Other Men Who Have Sex with Men (MSM): Opportunities for Optimizing Public Health Interventions	AIDS and behavior	2,020	cc-by	8,536	Abstract Syphilis and HIV among gay, bisexual and other men who have sex with men (MSM) are syndemic suggesting current preven- tion strategies are not effective. Sex partner meeting places and their networks may yield effective and optimal interventions. From 2009 to 2017, 57 unique venues were reported by > 1 MSM and 7.0% (n = 4), 21.1% (n = 12) and 71.9% (n = 41) were classified as syphilis, HIV or co-diagnosed venues, respectively. Forty-nine venues were connected in one main network component with four online, co-diagnosis venues representing 51.6% of reports and the highest degree and eigenvector centralities. In a sub-analysis during a local syphilis epidemic, the proportion of venues connected in the main component increased 38.7% (61.5% to 86.4%); suggesting increasing overlap in syphilis and HIV transmission and density of the venue network structure over time. This network analysis may identify the optimal set of venues for tailored interventions. It also suggests increasing difficulty of interrupting network transmission through fragmentation. Keywords MSM · Syphilis · HIV · Sex partner meeting places · Venue co-affiliation networks Jacky M. Jennings1,6 · Carla Tilchin1 · Benjamin Meza2 · Christina Schumacher1 · Errol Fields1,3 · Carl Latkin3 · Anne Rompalo5 · Adena Greenbaum4 · Khalil G. Ghanem5 Published online: 1 April 2020 © The Author(s) 2020 Published online: 1 April 2020 © The Author(s) 2020 4 STI/HIV Program, Baltimore City Health Department, Baltimore, MD, USA 5 Department of Infectious Disease, Johns Hopkins School of Medicine, Baltimore, MD, USA 6 Department of Pediatrics, School of Medicine, The Johns Hopkins University Bayview Medical Center, 5200 Eastern Avenue, Mason F. Lord Bldg‑Center Towers, Suite 4200, Baltimore, MD 21224, USA AIDS and Behavior (2020) 24:2895–2905 https://doi.org/10.1007/s10461-020-02840-2 AIDS and Behavior (2020) 24:2895–2905 https://doi.org/10.1007/s10461-020-02840-2 ORIGINAL PAPER * Jacky M. Jennings jennings@jhmi.edu Overlapping Transmission Networks of Early Syphilis and/or Newly HIV Diagnosed Gay, Bisexual and Other Men Who Have Sex with Men (MSM): Opportunities for Optimizing Public Health Interventions Jacky M. Jennings1,6 · Carla Tilchin1 · Benjamin Meza2 · Christina Schumacher1 · Errol Fields1,3 · Carl Latkin3 Anne Rompalo5 · Adena Greenbaum4 · Khalil G Ghanem5 Jacky M. Jennings1,6 · Carla Tilchin1 · Benjamin Meza2 · Christina Schumacher1 · Errol Fields1,3 · Carl Latkin3 · Anne Rompalo5 · Adena Greenbaum4 · Khalil G. Ghanem5 Jacky M. Jennings1,6 · Carla Tilchin1 · Benjamin Meza2 · Christina Schumacher1 · Errol Fields1,3 · Carl Latkin3 · Anne Rompalo5 · Adena Greenbaum4 · Khalil G. Ghanem5 1 Center for Child and Community Health Research (CCHR), Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD, USA Introduction The current syphilis epidemic in the United States (U.S.) among gay, bisexual and other men who have sex with men (MSM) is closely linked to the HIV epidemic. In 2017, MSM accounted for 79.6% of all primary and secondary (P&S) syphilis cases among males and 57.9% of all P&S syphilis cases overall [1]. Approximately half of MSM with P&S syphilis were also living with HIV [2]. This syndemic of HIV and syphilis is particularly acute for Black MSM. In 2017, the National HIV Behavioral Surveillance found that HIV prevalence among Black MSM was 39% among the 23 selected U.S. cities [3]. The use of specific sex partner meeting places to disrupt network transmission is not new. Bathhouses were an impor- tant meeting place to find anonymous sex partners among MSM in San Francisco in the 1980s and were used to create sexual networks in which the first HIV/AIDS cases were traced and mapped [19]. More recently, at least one outbreak of syphilis was linked to seeking sex partners through an online chatroom [21]. Venues where early syphilis and/or newly HIV diagnosed MSM report recently meeting their sex partners are likely to be access points for other members of a syphilis and/or HIV transmission network. In addition, if there is substantial transmission overlap, dual strategies for syphilis and HIV prevention and treatment may be opti- mal and more cost-effective than separate disease-specific strategies. The syndemic is likely due in part to the fact that syphi- lis infection can facilitate the transmission and acquisition of HIV infection [4–6]. There is an estimated 2- to 5-fold increase in the risk of HIV acquisition among persons with syphilis [3, 7, 8]. In addition, syphilis may be an indicator of ongoing sexual risk behaviors and membership in a high transmission network for both syphilis and HIV. The syn- demic nature of HIV and syphilis suggests a need to better understand the network transmission dynamics of syphilis and HIV among MSM and specifically, Black MSM. In our recent work, we have taken an innovative approach to analyzing the sex partner meeting places of newly HIV diagnosed MSM [22], an approach utilized by a few other researchers as well [23–25]. We have been approaching the study of venues like the study of networks by mapping the network connections of venues through venue affiliation and venue co-affiliation networks. Introduction This is made possible by the fact that some individuals report more than one venue, which creates connectivity between venues. Venue co-affil- iation network analyses can reveal tightly connected venues where syphilis and/or HIV transmission may be occurring by evaluating metrics related to venue centrality (i.e. how many times the venue was nominated) and network density (i.e. how closely venues are connected to each other or how socially intertwined the venues are) [26, 27]. Measures of venue centrality and network density have been useful in studying HIV, STI, and tuberculosis transmission networks [28–31] and may be one approach to prioritizing venues for targeted control. Venue co-affiliation network analyses may lead to new control activities by explicitly recognizing and taking advantage of both the nominated venues themselves and the connections between venues (i.e. network structure), and optimizing activities across, for example, syphilis and HIV. i Traditional methods of syphilis (and later, HIV) con- trol were designed to address network transmission. Local health departments have traced the partners of patients with syphilis, a process called contact tracing, since the 1930s for the purpose of treatment and prevention [9]. While useful for disrupting network transmission, these methods have limitations for understanding transmission dynam- ics because missing links are common. Missing links may be due to casual or anonymous contacts with less reliable locating information [10], or mistrust of government institu- tions including the health department staff eliciting contact information [11]. Missing links may also be due to recency and recall bias, as demonstrated by inconsistent reporting of particular partners, self-reported forgetting and partners reported in some interview modes such as daily diaries and not in others [12–14]. It is therefore possible that contact tracing does not represent or even misrepresents the dynam- ics of the population and may miss high-risk transmission events and network connections [15–18]. The overall goal of this work is to inform innovative, data-driven public health control strategies for reducing STIs and HIV. Among MSM with early syphilis and/or a new HIV diagnosis, the objectives are to (1) characterize the connectivity of sex partner meeting places to assess venue co-affiliation network structure and examine transmission overlap; (2) in a sub-analysis, describe temporal changes in the co-affiliation network structure over a 5 year period when early syphilis cases increased by 40% among MSM. Resumeni La sífilis y el VIH entre hombres homosexuales, bisexuales y otros que tienen relaciones sexuales con hombres (HSH) son sindémicos, lo que sugieren que las estrategias de prevención actuales no son efectivas. Los lugares de encuentro de parejas sexuales y sus redes pueden producir intervenciones efectivas y óptimas. De 2009 a 2017, 57 lugares únicos fueron reportados por > 1 HSH y 7.0% (n = 4), 21.1% (n = 12) y 71.9% (n = 41) fueron clasificados como sífilis, VIH o lugares co- diagnosticados, respectivamente. Cuarenta y nueve lugares se conectaron en un componente principal de la red con cuatro lugares de co-diagnóstico en línea, que representan 51.6% de los informes y el más alto grado y centralidades de vector pro- pio. En un subanálisis durante una epidemia de sífilis local, la proporción de lugares conectados en el componente principal aumentó 38.7% (61.5% a 86.4%); lo que sugiere una mayor superposición en la transmisión de sífilis y VIH y la densidad de la estructura de la red de lugares a lo largo del tiempo. Este análisis de red puede identificar el conjunto óptimo de lugares para intervenciones a medida. También sugiere una creciente dificultad para interrumpir la transmisión de la red a través de la fragmentación. 4 STI/HIV Program, Baltimore City Health Department, Baltimore, MD, USA 5 Department of Infectious Disease, Johns Hopkins School of Medicine, Baltimore, MD, USA 6 Department of Pediatrics, School of Medicine, The Johns Hopkins University Bayview Medical Center, 5200 Eastern Avenue, Mason F. Lord Bldg‑Center Towers, Suite 4200, Baltimore, MD 21224, USA 4 STI/HIV Program, Baltimore City Health Department, Baltimore, MD, USA 1 Center for Child and Community Health Research (CCHR), Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD, USA 5 Department of Infectious Disease, Johns Hopkins School of Medicine, Baltimore, MD, USA 2 Department of General Internal Medicine, Johns Hopkins School of Medicine, Baltimore, MD, USA 3 Department of Health, Behavior and Society, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA (0121 3456789) 3 AIDS and Behavior (2020) 24:2895–2905 2896 (ART), testing and linkage to care/treatment for STIs and HIV, and messaging for undetectable equals untransmiss- able (U = U) campaigns and safer sex practices [15, 19, 20].i Setting Baltimore City, Maryland ranks among U.S. cities with the highest incidence of syphilis and HIV among MSM. In 2017, the rate of P&S syphilis was 3.6-fold higher than the national rate (34.2 vs. 9.5 per 100,000) [2]. Additionally, 69.6% (n = 268) of all reported early syphilis cases (P&S and early latent) were among MSM, among whom 82.2% (n = 220) were Black MSM (Unpublished results). From 2010 to 2014, early syphilis among MSM increased 40%, and this time period was selected for the sub-analysis in objective two [32]. In 2016, the Baltimore–Columbia–Tow- son metropolitan statistical area (MSA) ranked 14th in the nation for HIV prevalence and 8th in the nation for cumula- tive number of persons living with HIV ever classified as AIDS [33]. A specific sex partner meeting place was defined as a physical location that could be placed on a map or an online location that was a specific application or website. Reported places that were non-specific venues or regions, such as “bars,” “online” and “West Baltimore”, were not included. In order to build the venue co-affiliation networks, only ven- ues reported by more than one individual were included in the analysis. This study used routine public health surveil- lance information and was considered exempt from human subjects research by the Johns Hopkins Medicine IRB. Individual‑Level Measures This work was conducted through a public health-academic partnership between the STI/HIV prevention and control program of the Baltimore City Health Department (BCHD) and the Johns Hopkins University Center for Child and Community Health Research (CCHR). Reporting of syphi- lis and HIV to state and local health departments is legally mandated in Maryland. The BCHD refers all early syphi- lis and/or newly HV diagnosed individuals with a primary residential address within Baltimore City for partner ser- vices. An early syphilis case is defined as an individual with primary, secondary, or early latent syphilis as staged by a clinical provider or a BCHD disease intervention specialist (DIS). A new HIV diagnosed case is defined as an individual without a prior record of an HIV infection reported in the U.S. During routine partner services, in addition to collect- ing demographic, risk behavior, and sex partner informa- tion, the BCHD routinely collects information on sex partner meeting places (e.g., name of bar, club, app, website, market, park, school, or street corner). The information is elicited by a question to the individual, “Where did you meet your sex partners in the past 12 months?”. Summary statistics were generated to describe and compare the study population overall and by diagnosis (early syphilis only, new HIV diagnosis only, and co-diagnosed with syphi- lis and HIV) using chi-squared tests or t-tests, as appropri- ate. Descriptive statistics included demographics (e.g. age, race), sexual risk behavior (e.g. number of sex partners), and number and type of venues reported. In addition, we compared individuals who reported a sex partner meeting place (i.e. the study population) to those who did not report a sex partner meeting place to identify potential biases in the study population. Introduction We hypothesize that a majority of venues will be nominated An alternative or complement to contact tracing is to col- lect information on sex partner meeting places. Sex part- ner meeting places are venues such as a bar, club, internet application (app), website, market, park, school, or street corner where individuals meet sex partners forming sexual dyads and larger sexual networks. The rationale is that sex partner meeting places are less subject to the limitations of contact tracing and may be an effective means to access pri- ority populations for interventions such as providing access to pre-exposure prophylaxis (PrEP), antiretroviral therapy 3 3 3 2897 AIDS and Behavior (2020) 24:2895–2905 For this study, we used public health surveillance data from MSM with an early syphilis and/or new HIV diagnosis and reporting at least one sex partner meeting place from 2009 to 2017. Male cases were considered to be MSM if during the partner services interview, they self-identified as gay or bisexual or reported having sex with men. The recall for sexual risk behaviors (i.e. number of sex partners, commercial sex work) and drug use during partner ser- vices interviews was determined by the critical period for the diagnosis: 12 months for HIV diagnoses and 3, 6, and 12 months for primary, secondary, and early latent syphi- lis diagnoses, respectively. In this analysis, drug use was defined as self-report of injection drug use or any method of consumption of the following drugs—cocaine, heroin, and/ or methamphetamines.ii by both early syphilis and/or newly HIV diagnosed indi- viduals, and that a small number of venues with the great- est centrality measures will reveal a discrete set of venues that may be useful for public health control activities and/or interventions. We also hypothesize that the venue co-affilia- tion networks of early syphilis and/or newly HIV diagnosed MSM will become increasingly dense over the 5-year period during an increase in early syphilis cases among MSM. by both early syphilis and/or newly HIV diagnosed indi- viduals, and that a small number of venues with the great- est centrality measures will reveal a discrete set of venues that may be useful for public health control activities and/or interventions. We also hypothesize that the venue co-affilia- tion networks of early syphilis and/or newly HIV diagnosed MSM will become increasingly dense over the 5-year period during an increase in early syphilis cases among MSM. Network‑Level Measures To determine syphilis and HIV transmission overlap (objective one), we first described the connectivity of syphi- lis only and HIV only venues (i.e. single diagnosis venues) vs. co-diagnosis venues and then we compared the median degree centrality and median eigenvector centrality of single diagnosis venues vs. co-diagnosis venues using Mann–Whit- ney tests (i.e. a nonparametric test of difference). To describe the co-affiliation network, we calculated the pro- portion of venues by diagnoses and sexual risk behaviors (i.e. commercial sex work and drug use) of the individuals who nominated the venue and by venue typology. Two measures of venue centrality, degree centrality and eigenvector centrality, were calculated to evaluate the rela- tive prominence of specific venues in the venue co-affiliation network for objective one. Degree centrality was calculated as the number of ties (i.e. a tie is where two venues share a patron) the venue had to all other venues within the net- work [35]. A venue with a degree centrality of eight could have one tie each to eight unique venues, eight ties to one venue, or anything in between. Degree centrality measures the number of connections or potential transmission events a venue has in the network. f To assess changes in density and transmission overlap over time (objective two), we calculated the median degree centrality and median eigenvector centrality of the co-affil- iation network for each year. The percent change in median degree centrality and median eigenvector centrality of the co-affiliation network in 2010 was compared to 2014 using Mann–Whitney tests. We also calculated two additional net- work measures, connectedness and compactness, by year to assess changes in network density over time. Changes in connectedness and compactness were described using per- cent changes from 2010 to 2014. Eigenvector centrality was calculated as the number of ties the venue had within the network (i.e. degree central- ity) weighted by the degree centrality of each of the venues’ adjacent venues in the network [35]. For example, a venue with three ties to three venues that have no other ties in the network has a lower eigenvector centrality than a venue with three ties to three venues that themselves have two additional ties each. When applied to transmission dynamics, venues with a higher eigenvector centrality represent higher risk venues because they are more highly connected to other ven- ues that themselves are highly connected. Hypothesis Testing To characterize the connectivity of sex partner meeting places, we created a venue co-affiliation network of sex partner meeting places reported by at least two individuals over the entire study time period (objective one) from 2009 to 2017 and in a sub analysis, by each year (objective two) from 2010 to 2014. Venues were connected in the network, i.e. shared a tie, when they shared at least one patron. Network‑Level Measures Descriptive analyses and network analyses were per- formed using Stata Version 14 and UCINET Version 6.657. Network visualizations were built in R version 3.5.0 using the iGraph package [36]. For all analyses, statistical signifi- cance was determined by a p-value of < 0.05. Venue‑Level Measures Venues were classified by typology, diagnoses, and sexual risk behaviors. Venues were classified into six typologies: internet/app (i.e. online), bar/club, street/park/neighbor- hood, market/mall, or other [34]. Venues were also classi- fied based on the diagnoses of the individuals reporting the venue. Venues were classified as a “syphilis venue” if only individuals diagnosed with only early syphilis nominated the venue. Venues were classified as an “HIV venue” if only 1 3 2898 AIDS and Behavior (2020) 24:2895–2905 Compactness was calculated as the proportion of venue pairs that can reach each other by a path of any length (i.e. con- nectedness) weighted inversely by the lengths of the paths connecting the nodes [35]. When applied to transmission dynamics, compactness takes into account that disease will spread more quickly within networks with shorter distances between nodes (i.e. denser). individuals newly diagnosed with HIV nominated the venue. Venues were classified as an “syphilis and HIV venue” (i.e. co-diagnosis venue) through two definitions; (1) if both an individual with an early syphilis diagnosis and an individual with a new HIV diagnosis nominated the venue and/or (2) if at least one individual co-diagnosed with early syphilis and HIV, including individuals known to be HIV positive at the time of their early syphilis diagnosis, nominated the venue. Venues were also classified for some analyses as single diag- noses venues (i.e. syphilis venues and separately, HIV ven- ues) and co-diagnosis venues (as above). Venues were also classified based on the individual level sexual risk behaviors reported by individuals (i.e. compositional venue character- istics) who also nominated the venue including commercial sex work, any drug use, anonymous sex and condomless sex. 3 Study Population In the venue co-affiliation network of venues from 2009 to 2017, there were 57 unique venues and among these, 7.0% (n = 4) were classified as syphilis venues, 21.1% (n = 12) as HIV venues, and 71.9% (n = 41) as co- diagnosed venues. A majority of the venues were online venues (45.6%, n = 26) followed by bars or clubs (15.8%, n = 9). The remaining venues were streets, parks, or neigh- borhoods (10.5%, n = 6), markets or malls (8.8%, n = 5), or other (5.3%, n = 3). Almost half of all venues (42.1%, n = 24) had at least one report of commercial sex work, and almost half of all venues (42.1%, n = 24) had at least On average, individuals were 27 years old [interquartile range (IQR) 11.00], 79.1% were Black (n = 507), and 5.0% were Latino (n = 32). Study Population Two measures of network density, connectedness and compactness, were calculated to assess changes in the venue co-affiliation network density over time for objec- tive two. Connectedness was calculated as the proportion of venue pairs that could reach each other by a path of any length to all possible venue pairs in the network (i.e. the proportion of nodes in the main component) [35]. A net- work with seven nodes in one main component and three unconnected nodes (i.e. isolates) has a connectedness of 0.7. Between January 2009 and December 2017, 6175 individu- als diagnosed with early syphilis and/or a new HIV diag- nosis were reported to the BCHD. Among these 6175 indi- viduals, 77.1% (n = 4758) were males, among whom 61.4% (n = 2921) were MSM. Among MSM, 88.5% (n = 2585) received partner services interviews, of whom 24.8% (n = 641) reported at least one specific venue and were 1 3 AIDS and Behavior (2020) 24:2895–2905 2899 included in this analysis. MSM who reported a venue were not statistically different by age, race, ethnicity or diagnoses compared to MSM who did not report a venue (data not shown). reported any drug use, 53.8% (n = 345) reported anonymous sex, and 95.0% (n = 609) reported sex without a condom. The 641 individuals nominated 979 venues, of which 85.4% (n = 836) were nominations of venues reported by more than one MSM and were included in this analysis. The median number of venue reports per individual was 1 (IQR 7.00). Among these 641 individuals, 31.8% (n = 204) were diagnosed with early syphilis only, 42.3% (n = 271) were diagnosed with HIV only, and 25.9% (n = 166) were co-diag- nosed with early syphilis and new HIV (Table 1). Among those diagnosed with early syphilis only, 15.9% (n = 43) were diagnosed with primary syphilis, 43.2% (n = 117) were diagnosed with secondary syphilis, and 41.0% (n = 111) were diagnosed with early latent syphilis. Among those co- diagnosed with early syphilis and new HIV, 4.5% (n = 8) were diagnosed with primary syphilis, 30.7% (n = 51) were diagnosed with secondary syphilis, and 28.9% (n = 48) were diagnosed with early latent syphilis (data not shown). Objective one: characterize the connectivity of sex part- ner meeting places to assess venue co-affiliation network structure and examine syphilis and HIV transmission overlap. b The critical period for increased likelihood of transmission is defined as 12 months for and HIV diagnosis, and 3, 6, and 12 months for a pri- mary, secondary, and early latent syphilis diagnosis, respectively a Includes individuals known to be HIV positive at the time of their early syphilis diagnosisi n to be HIV positive at the time of their early syphilis diagnosisi c Includes reports of injection drug use or any method of consumption of the following drugs—cocaine, heroin, and/or methamphetamines Study Population There were no differences in age, race, ethnicity, or diagno- sis comparing 2010 to 2014 (data not shown). one report of drug use. The percent of reports of com- mercial sex work at each venue ranged from 0 to 100% (median 0%, IQR 13.33%). The percent of reports of any drug use at each venue ranged from 0 to 50% (median 0%, IQR 12.50%) A total of 94.7% (n = 54) of venues had at least one report of anonymous sex, and 100.0% (n = 57) of venues had at least one report of condomless sex. The percent of reports of anonymous sex at each venue ranged from 0 to 100% (median 60%, IQR 21.42%), and the per- cent of reports of condomless sex at each venue ranged from 50 to 100% (median 100%, IQR 5.41%). Of the four venues with the highest degree centralities and eigenvector centralities across the 5-year period, Face- book and Adam4Adam were nominated all 5 years, and Ins- tagram and Jack’d were first nominated in 2012. The degree centrality of these venues and tie strength between these venues increased annually from 2010 to 2014 (data depicted visually in Fig. 2). Overall, the median degree centrality was 4 (IQR 54.00). Four venues, Facebook, Adam4Adam, Jack’d, and Insta- gram, represented 51.6% of all unique venue reports. All four are online venues and were classified as co-diagnosed venues. These four venues had the highest degree centralities of 152, 101, 64, and 54, respectively. These four venues also had the highest eigenvector centralities of 1.00, 0.50, 0.27, and 0.22, respectively. The total number of venues nominated by more than one MSM increased 69.2% (n = 13 to 22, Fig. 2). The pro- portion of co-diagnosed venues decreased from 85% in 2010 to 73% in 2014. Comparing 2010 to 2014, the connectedness (i.e. proportion of venues connected in the main compo- nent) increased 57% from 0.47 to 0.74, and compactness (i.e. connectedness, weighted inversely by the length of the ties) increased 28% from 0.36 to 0.46 (Table 2). p y Of the 57 unique venues, 86.0% (n = 49) were con- nected in one main network component of which 6.1% of venues (n = 3) were syphilis venues, 20.4% (n = 10) were HIV venues, and 73.5% (n = 36) were co-diagnosed ven- ues. To assess transmission overlap, connectivity of single diagnosis venues (i.e. Study Population The median number of sex partners reported was 2 (IQR 50.00), 7.2% (n = 46) of individuals reported engagement in commercial sex work, 8.7% (n = 56) Table 1 Characteristics of gay, bisexual and other men who have sex with men (MSM) with early syphilis and/or a new HIV diagnosis and who reported at least one specific venue stratified by diagnosis, Baltimore City, 2009–2017 (n = 641) Characteristics Overall (n = 641) Early syphilis only diagnosis (n = 271) HIV only diagno- sis (n = 204) Early syphilis and HIV co-diagnosisa (n = 166) Demographics Age, median (IQR) 27 (11.00) 26 (10.00) 25 (9.00) 31 (16.00) Race Black, n (%) 507 (79.1) 216 (79.7) 160 (78.4) 131 (78.9) White, n (%) 97 (15.1) 39 (14.4) 30 (14.7) 28 (16.9) Other, n (%) 37 (5.8) 16 (5.9) 14 (6.9) 7 (4.2) Ethnicity, Latino, n (%) 32 (5.0) 13 (4.8) 12 (5.9) 7 (4.2) Sexual and drug behaviors Number of sex partners during critical periodb (n = 635), median (IQR) 2 (50.00) 2 (20.00) 2 (35.00) 2 (50.00) Commercial sex work, n (%) 46 (7.2) 18 (6.6) 17 (8.3) 11 (6.6) Any drug use, n (%)c 56 (8.7) 22 (8.1) 17 (8.3) 17 (10.2) Anonymous sex, n (%) 345 (53.8) 154 (56.8) 106 (52.0) 85 (51.2) Sex without a condom, n (%) 609 (95.0) 255 (94.1) 193 (94.6) 161 (97.0) Venue-related information Total number of venue nominations 836 335 (40.1) 293 (35.0) 208 (24.9) Median number of venue nominations (IQR) 1 (6.00) 1 (5.00) 1 (4.00) 1 (3.00) Online venues, n (%) 622 (74.4) 253 (75.5) 232 (79.2) 137 (65.9) Bars or clubs, n (%) 144 (17.2) 53 (15.8) 46 (15.7) 45 (21.6) Streets, parks or neighborhoods, n (%) 33 (4.0) 15 (4.5) 7 (2.4) 11 (5.3) Markets or malls, n (%) 27 (3.2) 9 (2.7) 5 (1.7) 13 (6.3) Other, n (%) 10 (1.2) 5 (1.5) 3 (1.0) 2 (1.0) Table 1 Characteristics of gay, bisexual and other men who have sex with men (MSM) with early syphilis and/or reported at least one specific venue stratified by diagnosis, Baltimore City, 2009–2017 (n = 641) xual and other men who have sex with men (MSM) with early syphilis and/or a new HIV diagnosis and who stratified by diagnosis, Baltimore City, 2009–2017 (n = 641) 1 1 3 3 2900 AIDS and Behavior (2020) 24:2895–2905 The median degree centrality increased 33% from 3 (IQR 4.00) in 2010 to 4 (IQR 13.00) in 2014 (Mann–Whitney Fishers p = 0.49), and the median eigenvector centrality decreased 78% from 0.14 (IQR 0.22) in 2010 to 0.03 (IQR 0.09) in 2014 (Mann–Whitney Fishers p = 0.09, Table 2). Study Population syphilis only and HIV only venues) and co-diagnoses venues were described, and centrality measures were compared between single and co-diagnosis venues. Among single diagnosis venues, 69.2% (n = 9) were directly connected to at least one co-diagnosis venue, 7.7% (n = 1) were indirectly connected (i.e. connected through another single-diagnosis venue) to a co-diagnosis venue, and 23.1% (n = 3) were not connected to any other venue (i.e. an isolate). Among co-diagnosis venues, 100% (n = 36), were directly connected to another co-diagnosis venue, and 2 co-diagnosed venues formed the only dyad (i.e. pair of venues disconnected from the main network component). 33% (n = 12) of co-diagnosis venues were directly connected to at least 1 single diagnosis venue, 52.8% (n = 19) were indirectly connected to a single diagnosis venue, and 8.3% (n = 3) were isolates.i Discussion The overall goal of this study was to inform innovative, data-driven public health prevention and control strategies for reducing STIs and HIV. The focus was on sex partner meeting places as an important means to access priority populations and their networks to reduce the transmission of syphilis and HIV. The approach utilizing venue co-affilia- tion analyses inherently recognizes that sex partner meeting places form a network of venues, i.e. individuals attend more than one venue, which may yield important information for the optimization of public health control strategies [22–25, 27]. Our approach is novel in that the design sought to address the syndemic of syphilis and HIV instead of treating each disease separately as is often the approach [37]. In addi- tion, we applied a novel methodology, venue co-affiliation network analyses, and the findings have important implica- tions for public health programs considering how best to allocate resources optimally for syphilis and HIV control [17, 26, 27]. Single diagnosis venues had significantly lower median degree centralities compared to co-diagnosed venues [3.5 (IQR 3.00), 5.0 (IQR 53.00), Mann–Whitney Fishers p < 0.01, respectively]. The median eigenvector central- ity was not significantly different between the two groups [0.00 (IQR 0.01), 0.01 (IQR 0.03), Mann–Whitney Fishers p = 0.15, respectively, Fig. 1].fi The median age of the study population of MSM was 27 years (IQR 11.00), and 79.1% were Black. These demo- graphics are notable because in Baltimore City, as well as other cities across the U.S., young Black MSM are a popula- tion that has continued to experience a high incidence and, in some places, an increasing incidence of syphilis and HIV [1–3]. Targeted and tailored control strategies are desper- ately needed for this population to reverse the epidemic. Objective two: describe temporal changes in the co-affil- iation network structure from 2010 to 2014. Over the 5-year period from 2010 to 2014 when early syphilis cases increased 40% among MSM, 408 MSM nomi- nated 54 unique venues. 3 AIDS and Behavior (2020) 24:2895–2905 2901 2901 AIDS and Behavior (2020) 24:2895 2905 Fig. 1 Co-affiliation network of sex partner meeting places (n = 57) reported more than once among men who has sex with men (MSM) with an early syphilis and/or a new HIV diagnosis (n = 641), Balti- more City, 2009–2017. Venues are linked if they have at least one shared case. Discussion The color of the node reflects the venue level diagno- sis: red is syphilis only nominations, blue is HIV only nominations, and black is co-diagnosed nominations. The size of the node reflects the degree centrality (i.e. a larger node represents more venue con- nections). The width of the line indicates tie strength (i.e. a wider tie reflects more shared cases between venues) (Color figure online) Fig. 1 Co-affiliation network of sex partner meeting places (n = 57) reported more than once among men who has sex with men (MSM) with an early syphilis and/or a new HIV diagnosis (n = 641), Balti- more City, 2009–2017. Venues are linked if they have at least one shared case. The color of the node reflects the venue level diagno- sis: red is syphilis only nominations, blue is HIV only nominations, and black is co-diagnosed nominations. The size of the node reflects the degree centrality (i.e. a larger node represents more venue con- nections). The width of the line indicates tie strength (i.e. a wider tie reflects more shared cases between venues) (Color figure online) The venue co-affiliation networks of early syphilis and/ or newly HIV diagnosed MSM suggest a high degree of overlap in the potential transmission of both syphilis and HIV transmission networks. Approximately 72% of venues were co-diagnosed venues. When comparing the median degree centrality of the venues, single diagnosed venues had a lower median degree than co-diagnosed venues dem- onstrating that these co-diagnosed venues are overall more central indicating that they have more shared patrons with other venues than single diagnosis venues. In addition, almost half of the venues were reported by individuals also reporting high sexual risk behaviors such as commercial sex work (41%) and drug use (41%). Four venues, Face- book, Adam4Adam, Jack’d, and Instagram, represented 51.6% of all unique venue reports and were all online, co-diagnosed venues. These venues also had the highest degree centrality and eigenvector centralities. These net- work measures suggest that these four venues had the most nominations and shared the most ties with other venues in the network, i.e. they were connected to the most venues that also had a high number of other venue ties. Discussion Other venue-based studies have identified a similar pattern of a dense core of venues, although most have identified a core with both physical and online venues [23, 24].i The four online core venues we identified may repre- sent the optimal set for tailored interventions. Interven- tions, for example, could include offering HIV/STI testing, sexual risk reduction behavioral interventions and/or PrEP through messaging on the apps. The data also suggest, however, that an intervention targeting each of the four 1 3 3 AIDS and Behavior (2020) 24:2895–2905 2902 Table 2 Measures of co-affiliation network structures (i.e. total num- ber of venues, degree centrality, eigenvector degree centrality) and density (i.e. connectedness, compactness) among sex partner meet- ing places (n = 32) reported more than once among gay, bisexual and other men who have sex with men (MSM) diagnosed with early syphilis and/or a new HIV diagnosis (n = 408), during a time period of increases in early syphilis cases among MSM, Baltimore City, 2010–2014 g p ( ) p g g y, Networks were created by linking venues that shared at least one case of early syphilis and/or HIV Year Number of venues reported by > 1 MSM Degree centrality of venues Eigenvector degree central- ity of venues Connectedness of network Compact- ness of network n Median (IQR) Median (IQR) Value Value 2010 13 3 (4.00) 0.14 (0.22) 0.47 0.36 2011 11 2 (6.00) 0.07 (0.27) 0.51 0.34 2012 13 2 (4.00) 0.04 (0.13) 0.58 0.38 2013 18 3 (7.00) 0.03 (0.07) 0.89 0.52 2014 22 4 (13.00) 0.03 (0.09) 0.74 0.46 % Change 2010–2014 + 69 + 33 − 79 + 57 + 28 Networks were created by linking venues that shared at least one case of early syphilis and/or HIV Fig. 2 Co-affiliation networks by year of sex partner meeting places (n = 54) reported more than once among men who have sex with men (MSM) diagnosed with an early syphilis and/or a new HIV diagnosis during a time period of increases in early syphilis cases among MSM, Baltimore City, 2010–2014 (n = 408). Venues are linked if they have at least one shared case. The color of the node reflects the venue level diagnosis: red is syphilis only nominations, blue is HIV only nomina- tions, and black is co-diagnosed nominations. The size of the node reflects the degree centrality (i.e. a larger node represents more venue connections). Discussion Missing information in the sur- veillance data did not allow for a thorough examination of individual risk information such as drug use, commercial sex work, unprotected sex, and co-infection, which if incorpo- rated may have helped to further elucidate the transmission potential of venues and assist in tailoring control strategies. In addition, we did not interview non-infected individuals to assess how their networks may differ. Another limitation was that we simplified the analyses by comparing single vs. co-diagnosis venues and this may have minimized some nuances related to the connectivity of the venues. Finally, there are different interpretations of what it means to be central in an affiliation network, and a related challenging aspect is that we assume one-mode relationships of patron co-memberships without having important information about the pattern of affiliation ties [38]. In addition, it is notable that the majority of nominated sex partner meeting places (53.1%) were online venues. Our prior work showed increasing trends in nominations of online sex partner meeting places, but also suggested that physical places remained important and highly con- nected to online spaces [22, 34]. Our current work sug- gests that sex partner meeting venues are dynamic and for MSM, may be increasingly online. In part this may also be due to other social forces. In Baltimore City, for example, a number of bars and clubs specifically tailored to MSM have closed in the past 5 years. This trend may in part be due to an increasing social acceptance of MSM with, for example, the legalization of same sex marriages in Maryland in 2013. It may also be due to the increasing availability and use of online venues particularly among youth who are increasingly digital natives. The dynamic nature suggests that patterns of sex partner meeting places are changing, and local health departments need to mod- ify current approaches and/or develop new strategies and interventions that include online approaches. Our analysis of temporal changes of the venue con- nectivity over a 5-year period of increasing early syphilis cases among MSM suggests increasing nomination, den- sity and connectivity of the co-affiliation network struc- ture over the time period. The network structure changes over time may also suggest higher transmission rates and increased difficulty of interrupting network transmission through fragmentation. The findings suggest a 69.2% increase in the number of venues that were reported by at least 2 MSM. Discussion The width of the line indicates tie strength (i.e. a wider tie reflects more shared cases between venues (Color figure online) Fig. 2 Co-affiliation networks by year of sex partner meeting places (n = 54) reported more than once among men who have sex with men (MSM) diagnosed with an early syphilis and/or a new HIV diagnosis during a time period of increases in early syphilis cases among MSM, Baltimore City, 2010–2014 (n = 408). Venues are linked if they have at least one shared case. The color of the node reflects the venue level diagnosis: red is syphilis only nominations, blue is HIV only nomina- tions, and black is co-diagnosed nominations. The size of the node reflects the degree centrality (i.e. a larger node represents more venue connections). The width of the line indicates tie strength (i.e. a wider tie reflects more shared cases between venues (Color figure online) 1 3 1 3 AIDS and Behavior (2020) 24:2895–2905 2903 The latter scenario in contrast, may present fewer challenges with fewer venues to choose from. venues may be redundant, and optimal interventions may be able to target one or two of the highly central venues and reach a majority of the venues and individuals. The broader network of venues also suggests that it may be important to additionally target venues that are less closely tied to this highly connected set of four venues. venues may be redundant, and optimal interventions may be able to target one or two of the highly central venues and reach a majority of the venues and individuals. The broader network of venues also suggests that it may be important to additionally target venues that are less closely tied to this highly connected set of four venues. There are important limitations to this study. Data were limited to venues reported by MSM with an early syphilis and/or new HIV diagnosis. The men reporting venues may be different than those not reporting venues, although our statistical testing did not suggest differences. In addition, the data reported on venues may be subject to recall bias and may not represent all venues MSM frequented to meet sex partners in the past 12 months. Also, because co-affili- ation network analysis is a tool to identify opportunities for social and sexual connection, these data are not necessarily a reflection of where direct connections occurred or where the infection was acquired. References 1. Centers for Disease Control and Prevention. STD facts—syphilis (detailed). 2019. https://www.cdc.gov/std/syphilis/stdfact-syphi lis-detailed.htm. Accessed 12 Aug 2019. 17. Rothenberg R, Narramore J. Commentary: the relevance of social network concepts to sexually transmitted disease control. Sex Transm Dis. 1996;23(1):24–9. 2. Centers for Disease Control and Prevention. 2017 Sexually trans- mitted disease surveillance. Centers for Disease Control and Prevention; 2018. https://www.cdc.gov/std/stats17/default.htm. Accessed 12 Aug 2019. 18. Woodhouse D, Rothenberg R, Potterat J, Darrow W, Muth S, Klovdahl A, et al. Mapping a social network of heterosexuals at high risk for HIV infection. AIDS. 1994;8(9):1331–6. 19. Doherty IA, Padian NS, Marlow C, Aral SO. Determinants and consequences of sexual networks as they affect the spread of sexu- ally transmitted infections. J Infect Dis. 2005;191(s1):S42–S54. 3. Centers for Disease Control and Prevention. HIV prevention through early detection and treatment of other sexually transmit- ted diseases—United States Recommendations of the Advisory Committee for HIV and STD Prevention. 1998. https://www.cdc. gov/mmwr/preview/mmwrhtml/00054174.htm. Accessed 12 Aug 2019. 20. Fichtenberg C, Ellen J. Moving from core groups to risk spaces. Sex Transm Dis. 2003;30(11):825–6. 21. Klausner JD, Wolf W, Fischer-Ponce L, Zolt I, Katz MH. Tracing a syphilis outbreak through cyberspace. JAMA. 2000;284(4):447–9.i 4. Jarzebowski W, Caumes E, Dupin N, Farhi D, Lascaux A-S, Piketty C, et al. Effect of early syphilis infection on plasma viral load and CD4 cell count in human immunodeficiency virus- infected men: results from the FHDH-ANRS CO4 cohort. Arch Intern Med. 2012;172(16):1237–43. 22. Brantley M, Schumacher C, Fields EL, Perin J, Safi AG, Ellen JM, et al. The network structure of sex partner meeting places reported by HIV-infected MSM: opportunities for HIV targeted control. Soc Sci Med. 2017;182:20–9.fi 23. Fujimoto K, Williams ML, Ross MW. Venue-based affiliation networks and HIV risk-taking behavior among male sex workers. Sex Transm Dis. 2013;40(6):453–8. 5. Buchacz K, Patel P, Taylor M, Kerndt PR, Byers RH, Holmberg SD, et al. Syphilis increases HIV viral load and decreases CD4 cell counts in HIV-infected patients with new syphilis infections. AIDS. 2004;18(15):2075–9. 24. Patel RR, Luke DA, Proctor EK, Powderly WG, Chan PA, Mayer KH, et al. Sex venue-based network analysis to identify HIV pre- vention dissemination targets for men who have sex with men. LGBT Health. 2018;5(1):78–85. 6. Fleming DT, Wasserheit JN. From epidemiological synergy to public health policy and practice: the contribution of other sexu- ally transmitted diseases to sexual transmission of HIV infection. Sex Transm Infect. 1999;75(1):3–17. 25. Discussion Funding This study was supported by Centers for Disease Control and Prevention Grants entitled Network Epidemiology of Syphilis Trans- mission (NEST, 1U01PS005171-01), the Thrive Program (PS15-1509), the STD Surveillance Network (PS13-1306), and the Minority HIV/ AIDS Research Initiative (1 U01 PS 005115). Funding This study was supported by Centers for Disease Control and Prevention Grants entitled Network Epidemiology of Syphilis Trans- mission (NEST, 1U01PS005171-01), the Thrive Program (PS15-1509), the STD Surveillance Network (PS13-1306), and the Minority HIV/ AIDS Research Initiative (1 U01 PS 005115). 10. Michaud JM, Ellen J, Johnson SM, Rompalo A. Responding to a community outbreak of syphilis by targeting sex partner meeting location: an example of a risk-space intervention. Sex Transm Dis. 2003;30(7):533–8. 11. Jackman K-M, Tilchin C, Grieb SM, Clark C, Benton-Denny J, Sawyer S, et al. “We are just a dollar and a number!” Minimizing medical research distrust and stigma among black MSM through more community-informed HIV/STI research approaches, Atlanta, Georgia; 2019. Conflict of interest The authors report no conflicts of interest. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. g 12. Brewer DD, Potterat JJ, Muth SQ, Malone PZ, Montoya P, Green DL, et al. Randomized trial of supplementary interviewing tech- niques to enhance recall of sexual partners in contact interviews*. Sex Transm Dis. 2005;32(3):189–93. 13. Brewer DD, Garrett SB. Evaluation of interviewing techniques to enhance recall of sexual and drug injection partners. Sex Transm Dis. 2001;28(11):666–77. 14. Brewer DD, Garrett SB, Rinaldi G. Patterns in the recall of sexual and drug injection partners. In: Levy J, Pescosolido B, editors. Social networks and health. Advances in medical sociology, vol. 8. Emerald Group Publishing Limited; 2002. p. 131–49. https:// doi.org/10.1016/S1057-6290(02)80024-9. Accessed 4 Feb 2020. 15. Day S, Ward H, Ison C, Bell G, Weber J. Sexual networks: the integration of social and genetic data. Soc Sci Med. 1998;47(12):1981–92. 16. Rothenberg RB, Sterk C, Toomey K, Potterat JJ, Johnson D, Schrader M, et al. Using social network and ethnographic tools to evaluate syphilis transmission. Sex Transm Dis. 1998;25(3):154–60. Discussion The proportion of venues connected in the main component as measured by connectedness increased 57% over the 5 years, suggesting an increasing density of venue connection over time and potentially increasing transmission potential over time. The increase in compact- ness of 28% over the 5 years is also crucial because it suggests that the paths between venues are shorter and therefore, transmission may be more efficient.l fi In summary, this approach is innovative as it harnesses the potential power of routinely collected public health sur- veillance data, may be more reliable than traditional con- tact tracing sexual network data, and importantly, focuses on the inherent networks of sex partner meeting places to identify transmission networks and overlapping transmis- sion networks of syphilis and/or HIV [26]. Leveraging this information may help to increase the efficiency of syphilis and HIV prevention and control strategies and maximize the use of public health funds. The highly nominated places and highly connected venues (i.e. high degree centrality and eigenvector centrality) may help direct local programs to places for prioritized outreach and linkage to care to reduce ongoing transmission, but the less nominated and connected venues may also be important since interventions would reach them in a time lagged way or not at all if they are disconnected. Future research should seek to develop, test and evaluate control strategies based on these findings to determine whether these control strategies result in the iden- tification of more syphilis and HIV positives, specifically those individuals who are infectious, and whether ultimately the strategies result in future declines in syphilis and HIV transmission. fi These results may reflect either an increasing number or use of venues for MSM to meet sex partners, or alternatively a condensing of venues for MSM to meet sex partners, there- fore increasing the likelihood that a venue is nominated by more than one individual. These two scenarios—increasing number or use versus condensing of venues—are very differ- ent phenomena and each has different implications for trans- mission and intervention. The former scenario may result in greater challenges in zeroing in on the transmission sources. 1 3 3 AIDS and Behavior (2020) 24:2895–2905 2904 9. Parran T. Shadow on the land: syphilis. New York: Reynal and Hitchcock; 1937. References Chan PA, Crowley C, Rose JS, Kershaw T, Tributino A, Mont- gomery MC, et al. A network analysis of sexually transmitted diseases and online hookup sites among men who have sex with men. Sex Transm Dis. 2018;45(7):462–8. 7. Pathela P, Braunstein SL, Blank S, Shepard C, Schillinger JA. The high risk of an HIV diagnosis following a diagnosis of syphilis: a population-level analysis of New York City men. Clin Infect Dis. 2015;61(2):281–7. 26. Frost SDW. Using sexual affiliation networks to describe the sexual structure of a population. Sex Transm Infect. 2007;83(Suppl_1):i37–i42.fi 8. Tilchin C, Schumacher CM, Psoter KJ, Humes E, Muvva R, Chaulk P, et al. Human immunodeficiency virus diagnosis after a syphilis, gonorrhea, or repeat diagnosis among males includ- ing non-men who have sex with men: what is the incidence? Sex Transm Dis. 2019;46(4):271–7. 27. Borgatti SP, Halgin DS. Analyzing affiliation networks. In: The SAGE handbook of social network analysis. London: SAGE Pub- lications Ltd; 2014. p. 417–33. https://methods.sagepub.com/ book/the-sage-handbook-of-social-network-analysis/n28.xml. Accessed 12 Aug 2019. 1 3 AIDS and Behavior (2020) 24:2895–2905 2905 33. Centers for Disease Control and Prevention. HIV Surveillance Report 2016, vol 28. Centers for Disease Control and Prevention; 2017. p. 125.i 28. Oster AM, Wejnert C, Mena LA, Elmore K, Fisher H, Heffelfinger JD. Network analysis among HIV-infected young black men who have sex with men demonstrates high connectedness around few venues. Sex Transm Dis. 2013;40(3):206–12. 34. Jennings JM, Reilly ML, Perin J, Schumacher C, Sharma M, Safi AG, et al. Sex partner meeting places over time among newly HIV diagnosed men who have sex with men (MSM) in Baltimore, Maryland. Sex Transm Dis. 2015;42(10):549–53. 29. Hurt CB, Beagle S, Leone PA, Sugarbaker A, Pike E, Kuruc J, et al. Investigating a sexual network of black men who have sex with men: implications for transmission and prevention of HIV infection in the United States. J Acquir Immune Defic Syndr 1999. 2012;61(4):515–21. g Maryland. Sex Transm Dis. 2015;42(10):549–53. 35. Borgatti SP, Everett MG, Johnson JC. Analyzing social networks. London: SAGE; 2013. 30. Klovdahl AS, Graviss EA, Yaganehdoost A, Ross MW, Wanger A, Adams GJ, et al. Networks and tuberculosis: an undetected community outbreak involving public places. Soc Sci Med. 2001;52(5):681–94. 36. Csardi G, Nepusz T. The Igraph software package for complex network research. InterJournal Complex Syst. 2006:1695. https ://igraph.org. 37. Syndemics and the biosocial conception of health. Lancet. https://www.thelancet.com/journals/lancet/article/PIIS0140- 6736(17)30003-X/fulltext. Accessed 4 Feb 2020. 31. References Cook VJ, Sun SJ, Tapia J, Muth SQ, Argüello DF, Lewis BL, et al. Transmission network analysis in tuberculosis contact investiga- tions. J Infect Dis. 2007;196(10):1517–27. 38. Faust K. Centrality in affiliation networks. Soc Netw. 1997;19(2):157–91. 32. Schumacher CM, Fields E, Chandran A, Heidari O, Kingon Y, Chaulk P, et al. Investigation of early syphilis trends among men who have sex with men to identify gaps in screening and case-finding in Baltimore City, Maryland. Sex Transm Dis. 2018;45(2):69–74. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3 1 3 3
https://openalex.org/W2899996968	https://europepmc.org/articles/pmc6307803?pdf=render	English	null	MiR-520b promotes the progression of non-small cell lung cancer through activating Hedgehog pathway	Journal of Cellular and Molecular Medicine	2,018	cc-by	6,320	Received: 10 July 2018 \| Accepted: 20 August 2018 DOI: 10.1111/jcmm.13909 Received: 10 July 2018 \| Accepted: 20 August 2018 DOI: 10.1111/jcmm.13909 DOI: 10.1111/jcmm.13909 Xiaoming Liu1* \| Jichun Liu1* \| Xuekang Zhang2 \| Yuben Tong2 \| Xin Gan3 1Department of Cardiothoracic Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China Abstract Although the non‐small cell lung cancer (NSCLC) is one of the most malignant tumours worldwide, the mechanisms controlling NSCLC tumourigenesis remain unclear. Here, we find that the expression of miR‐520b is up‐regulated in NSCLC samples. Further studies have revealed that miR‐520b promotes the proliferation and metastasis of NSCLC cells. In addition, miR‐520b activates Hedgehog (Hh) path- way. Inhibitor of Hh pathway could relieve the oncogenic effect of miR‐520b upon NSCLC cells. Mechanistically, we demonstrate that miR‐520b directly targets SPOP 3′‐UTR and decreases SPOP expression, culminating in GLI2/3 stabilization and Hh pathway hyperactivation. Collectively, our findings unveil that miR‐520b promotes NSCLC tumourigenesis through SPOP‐GLI2/3 axis and provide miR‐520b as a poten- tial diagnostic biomarker and therapeutic target for NSCLC. 2Department of Anesthesiology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China 3Department of Respiration, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China Correspondence Xin Gan, Department of Respiration, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China. Email: ganxin006@126.com These authors contributed equally to this work. wileyonlinelibrary.com/journal/jcmm \| 205 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 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. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. J Cell Mol Med. 2019;23:205–215. wileyonlinelibrary.com/journal/jcmm \| 205 J Cell Mol Med. 2019;23:205–215. Funding information g Jiangxi Provincial Natural Science Foundation of China, Grant/Award Number: 2012ZBBG70008; Jiangxi Provincial Education Department Support Program, Grant/Award Number: GJJ60139; Major Project of Jiangxi Natural Science Foundation, Grant/Award Number: 20161ACB20012; National Natural Science Foundation of China, Grant/Award Number: 81660009 K E Y W O R D S Hh pathway, miR-520b, NSCLC, SPOP 2.2 \| In vitro tumorigenicity assay Cell proliferation was assessed using MTT assay. After distinct treatments, log‐phase cells were seeded into 96‐well plates. After additional 48 hours, 10 mL MTT (5 mg/mL) was added into each well, followed by incubation for 4 hours before discarding the supernatants. Washing the cells with PBS for three times and add- ing 100 mL DMSO in each well to dissolve crystals for 10 min- utes. The absorbance on 490 nm was measured using microplate reader. The cell invasion assay was performed with Biocoat Matrigel Invasion Chambers. After indicated treatments, equivalent cells were seeded on top of a thick layer of Matrigel in tran- swell inserts and cultured for 24 hours. Invasive cells were washed with PBS, fixed with 70% ethanol for 15 minutes and stained with 2% crystal violet. The invasive cells were counted under a micro- scope. In this study, we found that the expression of miR‐520b was apparently up‐regulated in NSCLC samples, and miR‐520b levels were positively correlated with Hh pathway activities. In NSCLC cells, treated with miR‐520b mimic promoted cell proliferation and migration, while miR‐520b inhibitor treatment showed an opposite effect. Via bioinformatics and biochemical analyses, we revealed that miR‐520b suppressed SPOP expression through direct targeting its 3′‐UTR region. In addition, SPOP decreased in NSCLC samples and negative correlated with patient survival. Knockout of SPOP pro- moted NSCLC cell proliferation and metastasis, which could not be attenuated by miR‐520b, suggesting that miR‐520b promotes NSCLC progression through SPOP. Finally, we demonstrated that SPOP inhibited NSCLC tumourigenesis through ubiquitinating and destabi- lizing GLI2/3. Taken together, our findings show that miR‐520b pro- motes NSCLC tumourigenesis via SPOP‐GLI2/3 axis, thus providing miR‐520b as a potential diagnostic biomarker and therapeutic target for NSCLC. Cell migration was tested using wound healing assay. After indi- cated treatments, equivalent cells were seeded into 6‐well plates with 1% FBS. One yellow pipette tip was used to make a straight scratch. The width of wound was measured at 48 hours and normal- ized with starting time‐point. 2.3 \| RNA extraction and quantitative real‐time PCR (Q‐PCR) Total RNA from patient samples and cultured cells was extracted using TRIzol reagent. High‐capacity cDNA reverse transcription kit was used for cDNA synthesis. Q‐PCR was conducted on a CFX96™ with SYBR Green Q‐PCR reagents. The 2‐ΔΔCt method was used for relative quantification. The primer pairs used were as follows: GLI1, 5′‐GGGTGCCGGAAGTCATACTC‐3′ (forward) and 5′‐GC TAGGA‐TCTGTATAGCGTTTGG‐3′ (reverse); BCL2, 5′‐CTCAGCAGG TATCACATGG‐GG‐3′ (forward) and 5′‐CCAAGGTCTTGCGTACAAA TTCC‐3′ (reverse); ACTIN, 5′‐GATCATTGCTCCTCCTGAGC‐3′ (for- ward) and 5′‐ACTCCTGCTTGCTGAT‐CCAC‐3′ (reverse). 2.1 \| NSCLC cell lines and patient samples Human NSCLC cell lines (A549 and H1299) were purchased from the ATCC and cultured in Dulbecco's modified Eagle's medium (Gibco, Gaithersburg, MD, USA) containing 10% FBS and 1% peni- cillin/streptomycin (Sangon Biotech, Shanghai, China). MiR‐520b mimic (MC11115) and inhibitor (MH11115) were purchase from Thermo and added into the cells at final concentration of 20 nmol/L. Fresh‐frozen NSCLC samples and their paired normal samples were obtained from patients who were undergoing surgical resection at 2 \| MATERIALS AND METHODS For miRNA expression detection, Taqman miRNA assays were employed to quantify the expression of mature miR‐520b. The rela- tive expression level of miR‐520b was normalized to RNU6B. the First Affiliated Hospital of Nanchang University (Nanchang, China). None of the patients had received any radiochemotherapy. All the samples were divided into two parts for RNA extraction and western blot, respectively. The use of human samples was approved by Clinical Research Ethics Committee of Nanchang University, China. transcriptional factor GLI2/3 is detained in the cytoplasm to degrade by β‐TrCP E3 ligase. In the presence of Hh ligand, GLI2/3 translo- cates into the nucleus to turn on the expression of target genes, including PTCH1 and BCL2.13 On the other hand, the nuclear GLI2/3 is ubiquitinated and destabilized by another E3 ligase SPOP,14 ensur- ing that the proper Hh pathway output. For this respect, SPOP plays an anti‐tumour role in Hh‐related NSCLC. Micro RNAs (miRNAs) are small (about 19‐25 bp), noncoding RNAs that silence gene expression through repression of mRNA sta- bility or translation. Recent studies have demonstrated that many miRNAs show aberrant expression in tumour tissues, indicating that miRNAs are possibly involved in tumourigenesis. On the other hand, miRNAs also provide putative biomarkers for tumour diag- noses.15 Increasing findings show that several miRNAs plays impor- tant roles in NSCLC tumourigenesis. MiR‐21 promotes NSCLC cell proliferation and invasion through inhibiting the expression of PTEN, a well‐known tumour suppressor.16 In contrast, miR‐1253 suppresses NSCLC progression via blocking WNT5A expression.17 Therefore, miRNAs likely play dual roles in NSCLC tumourigenesis through silencing distinct targets. Although human genome encodes more than 400 kinds of miRNAs, the functions of many miRNAs are still unknown. It will be fruitful to investigate the roles of miRNAs in NSCLC progression. 1 \| INTRODUCTION most of NSCLS patients are diagnosed at the late stage and die with- out successful treatment. Thus, studies on the mechanisms of NSCLC tumourigenesis are critical for both early prognosis and the develop- ment of novel therapeutic approaches for NSCLC. Lung cancer is the most frequent cause of cancer‐related death in men and the second most frequent in women after breast cancer. Many risk factors are strong correlated with lung cancer tumourigen- esis, including smoking and air pollution.1,2 Despite significant advancements have been made in surgeries, radiotherapies and chemotherapies in the past decades, the survival rate of lung cancer is still low.3 Lung cancers comprise two types, small cell lung cancer (SCLC) and non‐small cell lung cancer (NSCLC).4 NSCLC contributes to over 80% of lung cancers. In past years, although many genes, such as P53 and EGFR, have been reported to regulate NSCLC development, In NSCLC tumourigenesis, multiple pathways are deregulated due to genetic or epigenetic mutations. Constitutive activation of Shh pathway is a common cause of NSCLC.5 Lots of mutations on Hh signalling components have identified in NSCLC samples.6,7 Besides, several Hh pathway inhibitors, including Cyclopamine and Forskolin, serve as attractive anti‐cancer agents for NSCLC treat- ment.8-10 The Hh pathway is first identified through a large gene screening that is required for embryonic patterning in Drosophila.11 Subsequent studies show that the Hh pathway is highly conserved from Drosophila to mammals.12 In the absence of Hh ligand, the d. and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. J Cell Mol Med. 2019;23:205–215. LIU ET AL. 206 LIU ET AL. 3.1 \| MiR‐520b is up‐regulated and plays a oncogenic role in NSCLC Although it is known miR‐520b plays roles in several types of cancer, its function in NSCLC is still unknown. We examined the expression of miR‐520b in NSCLC patients using Q‐PCR and found that miR‐520 was uniformly increased in 12 NSLCL samples compared with the paratumour samples (Figure 1A). To further investigate the function of miR‐520b, we treated the NSCLC cells with miR‐520b mimic or miR‐ 520b inhibitor. Compared with control, miR‐520b mimic promoted, while miR‐520b inhibitor suppressed cell proliferation of A549 and H1299 cells (Figure 1B, C). In addition, wound healing assay showed that miR‐520b mimic quickened the wound healing, but miR‐520b inhibitor exerted an opposite effect (Figure 1D). Consistently, tran- swell results revealed that miR‐520b mimic increased, whereas miR‐ 520b inhibitor decreased H1299 cell invasion (Figure 1E). Taken together, our results reveal that miR‐520b is up‐regulated in NSCLC samples and miR‐520b exerts oncogenic function in NSCLC cells. 2.5 \| Transfection and western blot Cells were transfected using lipofectamine 2000 according to the manufacturer's instructions. Forty‐eight hours after transfection, cells were harvested for immunoprecipitation and western blot analysis with standard protocols. To examine the ubiquitination levels of GLI2 and GLI3, A549 cells were transfected with Myc‐GLI2, HA‐GLI3 and different SPOP mutants. Before cell harvesting, the cells were treated by MG132 (50 mmol/L/mL) for 4 hours to prevent protein destabiliza- tion. Cells were first lysed by 100 mL denaturing buffer (1% SDS, 50 mM Tris, pH 7.5, 0.5 mmol/L EDTA and 1 mmol/L DTT) and incu- bated at 100°C for 5 minutes. The lysates were diluted with 900 mL lysis buffer and subjected to immunoprecipitation and western blot. The antibodies used for western blot analyses were as follows: mouse anti‐Fg (Sigma, Darmstadt, Germany); mouse anti‐ACTIN (Genscript, Corporation, Piscataway, NJ, USA); rabbit anti‐GLI1 (ABclonal, Woburn, MA, USA); rabbit anti‐PTCH1 (ABclonal); rabbit anti‐BCL2 (ABclonal); rabbit anti‐HHIP (ABclonal); rabbit anti‐AXIN2 (ABclonal); rabbit anti‐c‐Myc (ABclonal); rabbit anti‐CTGF (ABclonal); rabbit anti‐ AREG (ABclonal); rabbit anti‐SPOP (ABclonal); mouse anti‐Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti‐HA (Santa Cruz); mouse anti‐Ub (Santa Cruz); goat antimouse HRP (Abmax) and goat anti‐rabbit HRP (Abmax, Beijing, China). 3.2 During NSCLC tumourigenesis, multiple oncogenic pathways are involved in, including Hh, Wnt and Hippo.18-20 We next wanted to test whether miR‐520b turns on these pathways. We found that the expressions of Hh pathway targets (GLI1 and BCL2) were apparently increased in NSCLC specimens (Figure 2A, B). Furthermore, miR‐ 520b mimic treatment indeed increased Hh target gene expression (GLI1, PTCH1, BCL2 and HHIP), but with no any detectable effect on Wnt pathway (AXIN2 and c-Myc) and Hippo pathway (CTGF and AREG) (Figure 2C). In contrast, the inhibitor attenuated Hh pathway, not Wnt and Hippo pathways (Figure 2C). Furthermore, we found that the mimic elevated Gli‐Luciferase activity, while the inhibitor played an opposite role (Figure 2D). Q‐PCR in A549 cells also con- firmed these results (Figure 2E). These data suggest that miR‐520b possibly specifically activates Hh pathway in NSCLC cells. 2.7 All statistical analysis was performed by SPSS software. The reported data are representative of at least three independent experiments. A two‐tailed P value of less than 0.05 was considered statistically sig- nificant and the P < 0.001 was considered highly significant. In this study, exact P values were not shown, statistical significance was as follows: P > 0.05 (NS, no significance), P < 0.05 (), P < 0.01 () and P < 0.001 (). To silence SPOP, GLI2 or GLI3 in NSCLC cells, small interfering RNAs (siRNAs) were transfected at a final concentration of 200 nmol/ L via Lipofectamine™RNAiMAX transfection reagent according to the protocols (Invitrogen, Carlsbad, CA, USA). The siRNAs sequences were shown as follows: CTR‐siRNA, 5′‐CAAACACUUCCUUGGAAUGdTdT‐ 3′; SPOP‐siRNA‐1, 5′‐CUCACCGGGGCAUCGACUCdTdT‐3′; SPOP‐ siRNA‐2, 5′‐G‐GUUUCGAUACCUCUCAGUdTdT‐3′; GLI2‐siRNA, 5′‐ GUUCCUCACGGCGU‐ CGUAGdTdT‐3′ and GLI3‐siRNA, 5′‐UGGAA- GUUGUAGCUCACUGdTdT‐3′. SPOP mutant plasmids were generated using PCR‐based site‐direc- ted mutagenesis at the background of CMV‐Fg‐SPOP. was cotransfected with Renilla luciferase plasmid into A549 cells. Dual Luciferase Reporter Assay System was employed to check the luciferase activity after 24 hours according to the manufacturer's instruction. All luciferase activity data are presented as means ± SD from at least three independent experiments. To knock out the endogenous SPOP, we used CRISPR/Cas9 tool. The sgRNA targeting SPOP was CCTCCGGCAGAAATGTCGAGTGG. It was annealed to the complementary oligo and cloned into pGL3‐U6‐sgRNA‐ PGK‐puromycin vector (Addgene, Cambridge, MA, USA). A549 cells were cotransfected with this plasmid and pST‐NLS‐Cas9 plasmid (Addgene). 48 hours after transfection, the cells were treated by puro- mycin (0.02 mg/mL, Invivogen, San Diego, USA) and blasticidin (0.75 mg/ mL, Invivogen) for additional 48 hours. After cells form colonies, pick the small colonies into 96‐well plates. Genomic DNA from the cells is ampli- fied by PCR. Putative mutants were further validated by sequencing. 2.4 \| Constructs, Cas9 and RNAi To generate Fg‐SPOP, Myc‐GLI2, Myc‐GLI3 and HA‐GLI3 constructs, we amplified the corresponding cDNA using Primer STAR DNA poly- merase (TAKARA, Kusatsu,Shiga, Japan) and then cloned them into CMV‐Fg, pcDNA3.1‐Myc or pcDNA3.1‐HA vectors respectively. LIU ET AL. 207 2.6 \| Luciferase assays The putative miR‐520b binding site in 3′‐UTR of SPOP was sub- cloned into pGL3‐Basic‐Luc vector (Promega, Woods Hollow Road, USA). Meanwhile, the corresponding mutant construct was gener- ated by mutation of the complementary sequence of miR‐520b seed region (AGCACTTA to TCGTGAAT). The firefly luciferase construct 208 \| LIU ET AL. 208 LIU ET AL. 208 To test whether miR‐520b exerts the oncogenic effect through Hh (Figure 2F) ensuring the efficiency of this compound The up‐regu- FIGURE 2 miR‐520b activates Hh pathway in NSCLC cells. (A, B) Relative mRNA levels of GLI1 and BCL2 from NSCLC samples and matched normal samples were examined by Q‐PCR. (C) WB results of A549 cells with miR‐520b mimic or miR‐520b inhibitor treatment. (D) GliBS‐luciferase reporter assay in A549 cells under indicated treatment. GliBS luciferase activities were normalized to Renilla luciferase activities. (E) Q‐PCR assay to test GLI1 and BCL2 with miR‐520b mimic or miR‐520b inhibitor treatment in A549 cells. (F) WB results of A549 cells treated by Cyclopamine at indicated concentrations. (G) MTT and transwell assays of A549 cells under indicated treatment. All values are mean ± SD (n = 3, P < 0.05,P < 0.01 and P < 0.001) LIU ET AL. \| 209 LIU ET AL. 209 FIGURE 2 miR‐520b activates Hh pathway in NSCLC cells. (A, B) Relative mRNA levels of GLI1 and BCL2 from NSCLC samples and matched normal samples were examined by Q‐PCR. (C) WB results of A549 cells with miR‐520b mimic or miR‐520b inhibitor treatment. (D) GliBS‐luciferase reporter assay in A549 cells under indicated treatment. GliBS luciferase activities were normalized to Renilla luciferase activities. (E) Q‐PCR assay to test GLI1 and BCL2 with miR‐520b mimic or miR‐520b inhibitor treatment in A549 cells. (F) WB results of A549 cells treated by Cyclopamine at indicated concentrations. (G) MTT and transwell assays of A549 cells under indicated treatment. All values are mean ± SD (n = 3, P < 0.05,P < 0.01 and P < 0.001) (Figure 2F), ensuring the efficiency of this compound. The up‐regu- lated proliferation and invasion caused by miR‐520b mimic was neu- tralized by cyclopamine (Figure 2G), indicating that miR‐520b promotes NSCLC cell proliferation and invasion through Hh signalling. To test whether miR‐520b exerts the oncogenic effect through Hh signalling, a well‐known Hh antagonist cyclopamine was employed to block Hh pathway. 2.6 \| Luciferase assays Treated A549 cells with distinct concentrations of cyclopamine indeed decreased the expression of PTCH1 and GLI1 FIGURE 1 miR‐520b is up‐regulated in NSCLC samples and exerts an oncogenic role in NSCLC cells. (A) Q‐PCR analysis of miR‐520b expression in 12 pairs of NSCLC samples and paratumour normal tissues (N, paratumour normal samples; C, cancer samples). (B) 3‐day MTT proliferation results of A549 cells with miR‐520b mimic or miR‐520b inhibitor treatment. (C) MTT assays of H1299 cells treated with miR‐ 520b mimic or miR‐520b inhibitor. (D) Wound healing assays of A549 cells treated with miR‐520b mimic or miR‐520b inhibitor. Quantification of wound closure at indicated time‐points was shown on the right. (E) Transwell assay of A549 cells treated with miR‐520b mimic or miR‐520b inhibitor. Numbers of invasive cells were shown on the right. Scale bar, 20 μm. All values are mean ± SD (n = 3, P < 0.05,P < 0.01 and P < 0.001) 210 \| LIU ET AL. 210 \| LIU E FIGURE 3 miR‐520b directly targets SPOP in NSCLC cells. (A) A putative miR‐520b binding site in the 3′‐UTR of SPOP. (B) WB analyses of SPOP protein levels in 12 pairs of NSCLC samples and paratumour normal samples. (C) Low miR‐520b expression was associated with a poor disease free survival in TCGA cohort (P < 0.0001). (D) WB assay of A549 cells treated by miR‐520b mimic or miR‐520b inhibitor. (E) Neither miR‐520b mimic nor miR‐520b inhibitor affects exogenous SPOP expression in A549 cells. (F) WB assay showed SPOP protein level with SPOP‐siRNA treatment. (G) MTT assays of A549 cells transfected with indicated siRNA or plasmid. (H) Alignment of Sanger sequencing results of PCR amplicons encompassing the target site, SgRNAs targets are highlighted in red and the PAM sequence is underlined. (I) WB analysis of SPOP expression of WT or KO A549 cells. (J) MTT analyses of WT or SPOP KO A549 cells treated with miR‐520b mimic or miR‐520b inhibitor. (K) Transwell analyses of WT or SPOP KO A549 cells treated with miR‐520b mimic or miR‐520b inhibitor. (L) miR‐520b only suppressed the relative luciferase activity in the construct, which contains the wild‐type sequence of the binding site in the 3′‐UTR of SPOP. WT, luciferase construct containing wild‐type binding site in SPOP 3′‐UTR; Mu, mutated nucleotides were introduced to the complementary seed sequence. 2.6 \| Luciferase assays All values are mean ± SD (n = 3, P < 0.05, **P < 0.001 and NS, no significance) 210 \| LIU ET AL. FIGURE 3 miR‐520b directly targets SPOP in NSCLC cells. (A) A putative miR‐520b binding site in the 3′‐UTR of SPOP. (B) WB analyses of SPOP protein levels in 12 pairs of NSCLC samples and paratumour normal samples. (C) Low miR‐520b expression was associated with a poor disease free survival in TCGA cohort (P < 0.0001). (D) WB assay of A549 cells treated by miR‐520b mimic or miR‐520b inhibitor. (E) Neither miR‐520b mimic nor miR‐520b inhibitor affects exogenous SPOP expression in A549 cells. (F) WB assay showed SPOP protein level with SPOP‐siRNA treatment. (G) MTT assays of A549 cells transfected with indicated siRNA or plasmid. (H) Alignment of Sanger sequencing results of PCR amplicons encompassing the target site, SgRNAs targets are highlighted in red and the PAM sequence is underlined. (I) WB analysis of SPOP expression of WT or KO A549 cells. (J) MTT analyses of WT or SPOP KO A549 cells treated with miR‐520b mimic or miR‐520b inhibitor. (K) Transwell analyses of WT or SPOP KO A549 cells treated with miR‐520b mimic or miR‐520b inhibitor. (L) miR‐520b only suppressed the relative luciferase activity in the construct, which contains the wild‐type sequence of the binding site in the 3′‐UTR of SPOP. WT, luciferase construct containing wild‐type binding site in SPOP 3′‐UTR; Mu, mutated nucleotides were introduced to the complementary seed sequence. All values are mean ± SD (n = 3, P < 0.05, *P < 0.001 and NS, no significance) (Figure 3A). We demonstrated that the expression of SPOP was decreased in NSCLC samples (Figure 3B), showing negative correla- tion with miR‐520b. Consistently, data from TCGA (http://cancerge nome.nih.gov/) revealed that patients with low SPOP expression 3.3 \| SPOP is a target of miR‐520b To investigate the target of miR‐520b, we employed bioinformatic analysis (www.targesan.org) and found SPOP was a putative target \| 211 \| 211 211 LIU ET AL. FIGURE 4 SPOP promotes GLI2/3 ubiquitination and degradation. (A, B) Fg‐SPOP could pull down Myc‐GLI2 (A) and Myc‐GLI3 (B) in A549 cells. (C) Endogenous SPOP interacted with endogenous GLI2 and GLI3 in A549 cells. (D) SPOP promoted GLI2/3 degradation in A549 cells, which was abolished by MG132 treatment. (E) WB analysis of WT and SPOP KO A549 cells. (F) SPOP bound GLI2/3 through its N‐terminal MATH domain. (G) WB assay of A549 cells transfected with WT SPOP or different SPOP mutants. (H) Ubiquitination assay of A549 cells transfected with indicated plasmids FIGURE 4 SPOP promotes GLI2/3 ubiquitination and degradation. (A, B) Fg‐SPOP could pull down Myc‐GLI2 (A) and Myc‐GLI3 (B) in A549 cells. (C) Endogenous SPOP interacted with endogenous GLI2 and GLI3 in A549 cells. (D) SPOP promoted GLI2/3 degradation in A549 cells, which was abolished by MG132 treatment. (E) WB analysis of WT and SPOP KO A549 cells. (F) SPOP bound GLI2/3 through its N‐terminal MATH domain. (G) WB assay of A549 cells transfected with WT SPOP or different SPOP mutants. (H) Ubiquitination assay of A549 cells transfected with indicated plasmids (n = 965) had a poorer survival compared with those with high miR‐ 520b expression (n = 961, P < 0.0001) (Figure 3C). Furthermore, we found that miR‐520b mimic decreased, while miR‐520b inhibitor increased SPOP protein level in A549 cells (Figure 3D). On the other hand, neither mimic nor inhibitor showed any effect on exogenous SPOP level (Figure 3E), suggesting that miR‐520b decreases SPOP through targeting SPOP 3′‐UTR region. essential for miR‐520 regulating NSCLC cell proliferation and invasion. To test whether SPOP is the direct target of miR‐520b, luciferase reporter assays were performed. The fragment of the SPOP 3′‐UTR containing the predicted or mutant miR‐520b site was cloned into the pGL3‐Basic‐Luc vector. We found that miR‐ 520b exerted inhibitory effects on the luciferase activity in the construct which contains the wild‐type binding site, whereas no suppressive effects on the binding site mutant construct (Fig- ure 3L). To examine the anti‐tumour role of SPOP on NSCLC, we silenced endogenous SPOP via siRNA and found that knockdown of SPOP promoted A549 cell proliferation, whereas overexpression of SPOP inhibited cell proliferation (Figure 3G). 3.3 \| SPOP is a target of miR‐520b In addition, CRISPR/Cas9 technique was used to knock out SPOP in A549 cells (Figure 3H). We picked up 10 cell lines and found a stop codon was induced in a cell line (Figure 3H). Western blot analysis showed complete depletion of SPOP protein in the (stop) cell line. Therefore, we chose this cell line (stop) for subsequent experi- ments. Knockout of SPOP indeed promoted cell proliferation (Fig- ure 3J) and invasion (Figure 3K), and this oncogenic effect could not be relieved by miR‐520b mimic or miR‐520b inhibitor (Fig- ure 3J, K). Furthermore, A549 cells with SPOP knockout failed to be modulated by miR‐520b (Figure 3J, K), suggesting that SPOP is 3.4 \| SPOP suppresses Hh pathway through ubiquitinating GLI2 and GLI3 The previous studies have demonstrated that Rdx (SPOP homologue) binds and ubiquitinates Ci (GLI2/3 homologue) to inhibit Hh sig- nalling activity in Drosophila.14 We tried to test whether SPOP destabilizes GLI2/3 to negatively regulate Hh pathway in NSCLC cells. In A549 cells, SPOP could bind GLI2/3 (Figure 4A, B), and pro- moted proteasome‐mediated GLI2/3 degradation in a dose‐depen- dent manner (Figure 4C). Moreover, GLI2/3 showed increased expression in SPOP knockout cells compared with wild‐type cells 212 \| 212 212 LIU ET AL. IGURE 5 miR‐520b exerts oncogenic effect on NSCLC cells through GLI2/3. (A, B) MTT assays of A549 cells (A) and H1299 cells (B) ansfected with indicated constructs. (C) MTT assays of A549 cells (left) and H1299 cells (right) under indicated treatment. (D) Wound healing ssays of A549 cells under indicated treatment. Quantification of wound closure at indicated time‐points was shown on the right. All values e mean ± SD (n = 3, P < 0.001 and NS, no significance) FIGURE 5 miR‐520b exerts oncogenic effect on NSCLC cells through GLI2/3. (A, B) MTT assays of A549 cells (A) and H1299 cells (B) ransfected with indicated constructs. (C) MTT assays of A549 cells (left) and H1299 cells (right) under indicated treatment. (D) Wound heali assays of A549 cells under indicated treatment Quantification of wound closure at indicated time‐points was shown on the right All values FIGURE 5 miR‐520b exerts oncogenic effect on NSCLC cells through GLI2/3. (A, B) MTT assays of A549 cells (A) and H1299 cells (B) transfected with indicated constructs. (C) MTT assays of A549 cells (left) and H1299 cells (right) under indicated treatment. (D) Wound healing assays of A549 cells under indicated treatment. Quantification of wound closure at indicated time‐points was shown on the right. All values are mean ± SD (n = 3, **P < 0.001 and NS, no significance) Previous GWAS screening has shown that SPOP is mutated in several human cancers, including prostate cancer and gastric can- cer.21,22 Interestingly, most SPOP mutations localize in the MATH domain, which presumably impair its ability to bind substrates. To explore whether the cancer‐derived mutations affect SPOP (Figure 4D). SPOP protein comprises two functional domains: the N‐ terminal MATH domain and the C‐terminal BTB domain. Loss of MATH domain deprived SPOP association with GLI2/3 (Figure 4E), indicating that SPOP interacts with GLI2/3 via its N‐terminal MATH domain. (Figure 4D). 3.4 \| SPOP suppresses Hh pathway through ubiquitinating GLI2 and GLI3 SPOP protein comprises two functional domains: the N‐ terminal MATH domain and the C‐terminal BTB domain. Loss of MATH domain deprived SPOP association with GLI2/3 (Figure 4E), indicating that SPOP interacts with GLI2/3 via its N‐terminal MATH domain. LIU ET AL. LIU ET AL. FIGURE 6 The model of miR‐520b regulating NSCLC tumourigenesis. (A) In normal lung cells, SPOP ubiquitinates GLI2/ 3, resulting in GLI2/3 degradation and Hh pathway suppression. Under this condition, Hh pathway activity keeps at the basal level. (B) In NSCLC cells, miR‐520b is up‐ regulated and targets SPOP mRNA, removing SPOP‐mediated GLI2/3 destabilization. In turn, Hh pathway is hyperactivated LIU ET AL. \| 213 FIGURE 6 The model of miR‐520b regulating NSCLC tumourigenesis. (A) In normal lung cells, SPOP ubiquitinates GLI2/ 3, resulting in GLI2/3 degradation and Hh pathway suppression. Under this condition, Hh pathway activity keeps at the basal level. (B) In NSCLC cells, miR‐520b is up‐ regulated and targets SPOP mRNA, removing SPOP‐mediated GLI2/3 destabilization. In turn, Hh pathway is hyperactivated FIGURE 6 The model of miR‐520b regulating NSCLC tumourigenesis. (A) In normal lung cells, SPOP ubiquitinates GLI2/ 3, resulting in GLI2/3 degradation and Hh pathway suppression. Under this condition, Hh pathway activity keeps at the basal level. (B) In NSCLC cells, miR‐520b is up‐ regulated and targets SPOP mRNA, removing SPOP‐mediated GLI2/3 destabilization. In turn, Hh pathway is hyperactivated early stage owing to lack of apparent symptoms. Thus, it is urgent to identify the bio‐marker for NSCLC early diagnosis. In this study, we found that miR‐520b was up‐regulated in NSCLC specimens and positively correlated with NSCLC cell proliferation, migration and invasion. Furthermore, we demonstrated that miR‐520b could acti- vate Hh signalling in NSCLC cells. Through bioinformatic and bio- chemical analyses, we revealed that SPOP was a target of miR‐520b. SPOP bound GLI2/3 via its N‐terminal MATH domain, resulting in GLI2/3 proteasome‐mediated degradation and Hh pathway inactiva- tion. Finally, we found that knockdown of GLI2/3 effectively neutral- ized the effects of miR‐520b on NSCLC cell proliferation and migration. Taken together, our findings illustrated that miR‐520b exerted oncogenic effect in NSCLC through SPOP‐GLI2/3 signalling axis. This study provides miR‐520b as a putative bio‐marker for NSCLC diagnosis and therapeutic target for NSCLC clinic intervention. interaction with GLI2/3, we carried out co‐IP assays and found that only wild‐type SPOP interacted with GLI2/3 (Figure 4F). 3.4 \| SPOP suppresses Hh pathway through ubiquitinating GLI2 and GLI3 Given that SPOP acts as an E3 ligase, we next wanted to examine the ubiquiti- nation level of GLI2/3. Consistently, SPOP indeed elevated the ubiq- uitination of GLI2/3, whereas these SPOP mutants failed to promote GLI2/3 ubiquitination (Figure 4G). Collectively, these results show that SPOP inhibits Hh pathway through destabilizing the transcrip- tional factor GLI2/3, which is abolished in cancers due to SPOP mutations. 3.5 \| MiR‐520b regulates NSCLC through SPOP‐ GLI2/3 axis The above results have clearly demonstrated that mutations on MATH domain destroy SPOP binding to GLI2/3. Consistently, wild‐ type SPOP attenuated the proliferation of A549 and H1299 cells, whereas SPOP mutants did not show any affects (Figure 5A, B), indi- cating that SPOP regulates NSCLC cell proliferation possibly through binding and ubiquitinating GLI2/3. We next wanted to explore whether miR‐520b plays oncogenic roles via GLI2/3. The up‐regu- lated cell proliferation caused by miR‐520b mimic was effectively restored by silencing GLI2 or GLI3 (Figure 5C), suggesting that miR‐ 520b sits upstream of GLI2/3 to promote NSCLC cell proliferation. Furthermore, wound healing assays also showed that miR‐520b pro- moted NSCLC cell migration through GLI2/3. As a matter of fact, accumulating studies show that miR‐520b plays important roles in tumourigenesis. In oestrogen receptor neg- ative breast cancer, miR‐520b functions as a tumour suppressor by targeting NF‐κB and TGF‐β pathways.23 Besides, it is also reported that miR‐520b suppresses cell migration and invasion through inhibiting CD44 in head‐neck cancer.24 Based on previous data and our present study, miR‐520b likely plays oncogenic or anti‐ tumour roles in different cancers through targeting distinct genes. It will be fruitful to explore the roles of miR‐520b in other can- cers. The Hh pathway is a central regulator of development and tumourigenesis.25 During embryogenesis, Hh pathway is indispens- able for the development of multiple tissues, including brain, limbs and lung.26-28 In adult, Hh signalling keeps basal activity to regu- late injury‐induce regeneration and stem cell maintenance. Its ACKNOWLEDGEMENTS 11. Nusslein-Volhard C, Wieschaus E. Mutations affecting segment num- ber and polarity in Drosophila. Nature. 1980;287:795‐801. This study was supported by Jiangxi Provincial Natural Science Foundation of China (2012ZBBG70008), Jiangxi Provincial Education Department Support Program (GJJ60139), Major Project of Jiangxi Natural Science Foundation (20161ACB20012) and National Natural Science Foundation of China (81660009). 12. Wilson CW, Chuang PT. Mechanism and evolution of cytosolic Hedgehog signal transduction. Development. 2010;137:2079‐2094. https://doi.org/10.1242/dev.045021 13. Kasper M, Regl G, Frischauf AM, Aberger F. 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https://openalex.org/W2973579071	https://www.gt-crust.ru/jour/article/download/23/25	Russian	null	About the wave mechanism of activation of faults in seismic zones of the lithosphere in Mongolia	Geodinamika i tektonofizika	2,013	cc-by	8,051	Recommended by V.A. San’kov Citation: Mel’nikov M.G., 2013. About the wave mechanism of activation of faults in seismic zones of the litho sphere in Mongolia. Geodynamics & Tectonophysics 4 (1), 69–81. doi:10.5800/GT2013410092. Институт земной коры СО РАН, Иркутск, Россия Институт земной коры СО РАН, Иркутск, Россия Аннотация: Проведено исследование миграций землетрясений вдоль активных разломов в сейсмических зонах Мон голии, которые интерпретируются как результат воздействия деформационных волн, изучены их векторные скоро сти и другие параметры. Проведено сравнительное изучение локальных разломов по крупномасштабным картогра фическим материалам, охарактеризованы различия и общность параметров волн в разломах различных рангов. Ключевые слова: деформационные волны, землетрясение, сейсмическая зона, активные разломы, параметры, Монго лия. - P U B L I S H E D B Y T H E I N S T I T U T E O F T H E E A R T H ’ S C R U S T S I B E R I A N B R A N C H O F R U S S I A N A C A D E M Y O F S C I E N C E S http://dx.doi.org/10.5800/GT-2013-4-1-0092 Institute of the Earth’s Crust, SB RAS, Irkutsk, Russia Institute of the Earth’s Crust, SB RAS, Irkutsk, Russia Abstract: The study is focused on earthquake migrations along active faults in seismic zones of Mongolia. The earthquake migrations are interpreted as a result of the influence of deformational waves. Vector velocities and other parameters of the deformational waves are studied. Based on data from largescale maps, local faults are compared, and differences and simi larities of parameters of waves related to faults of different ranks are described. Key words: deformational waves, earthquake, seismic zone, active faults, Mongolia. О ВОЛНОВОМ МЕХАНИЗМЕ АКТИВИЗАЦИИ РАЗЛОМОВ В СЕЙСМИЧЕСКИХ ЗОНАХ ЛИТОСФЕРЫ МОНГОЛИИ М. Г. Мельников Институт земной коры СО РАН, Иркутск, Россия M. G. Mel’nikov T e c t o n o p h y s i c s ABOUT THE WAVE MECHANISM OF ACTIVATION OF FAULTS IN SEISMIC ZONES OF THE LITHOSPHERE IN MONGOLIA M. G. Mel’nikov GEODYNAMICS & TECTONOPHYSICS ISSN 2078-502X 3. МЕТОДИКА ОБРАБОТКИ ДАННЫХ ПО РАЗЛОМАМ ТЕРРИТОРИИ МОНГОЛИИ Для объяснения миграции очагов землетрясений и пространственновременной периодичности их рас пределения в литературу введено понятие деформаци онных волн. Фундаментом концепции деформацион ных волн являются результаты исследований про странственновременного распределения землетрясе ний в различных регионах Земли, процессов медлен ного деформирования земной коры и перемещения аномалий геофизических полей вблизи зон разломов [Bykov, 2005; Sherman, 2009]. Короткопериодная активизация разрывов в реаль ном времени и пространственновременная локализа ция очагов землетрясений [Sherman et al., 2005a] воз буждаются и контролируются деформационными вол нами, которые рассматриваются как триггерный меха низм нарушения метастабильного состояния разлом ноблоковой среды литосферы, находящейся под по стоянным воздействием регионального поля напряже ний. Принято, что активизация происходит при про хождении через разлом фронта волны. В работах [Nikolaevsky, Ramazanov, 1985; Nikolaev sky, 1991] объясняется происхождение деформацион ных волн. Обработка данных по сейсмичности и разломной тектонике невозможна без специальных современных ГИСтехнологий, так как каталоги землетрясений со держат огромное количество данных и обработка их без использования специальных программ была бы слишком трудоемкой. В связи с этим С.И. Шерманом и Е.А. Горбуновой был разработан специальный про граммный пакет Digital Faults [Gorbunova, Sherman, 2012], который позволяет импортировать необходи мую информацию из баз данных (БД) по разломной тектонике, определять длины разломов и области их активного динамического влияния (ОАДВ), импорти ровать данные из БД по сейсмичности, оценивать ме стоположения землетрясений в ОАДВ конкретных разрывов, а также экспортировать и сохранять мате риалы в виде таблиц и графиков в координатах «вре мяместо» для конкретных разломов, определять па раметры волн и давать оценку достоверности линий регрессий. Новый метод классификации сейсмоактивных раз ломов предложен в работе [Sherman et al., 2005b], в которой также введены параметры количественной и магнитудной характеристик разломов. Благодаря этим новым параметрам были выявлены дополнительные геодинамические свойства разломов, а также вероят ные волновые источники их активизаций в пределах коротких промежутков времени [Sherman, Gorbunova, 2008]. Активизация разломов вызывается медленными де формационными волнами, нарушающими неустойчи вое динамическое состояние разломной зоны. Для многих сейсмоактивных разломов такие нарушения динамического равновесия фиксируются несколько раз, то есть активизация разломов происходит перио дически с относительно высокой, в масштабах реаль ного времени, частотой [Sherman, 2009; Sherman et al., 2012b]. На графиках «времяместо» по оси абсцисс откла дываются длины разломов, так чтобы отметка «0» со ответствовала западной оконечности разлома, по оси ординат – время событий (рис. 1). Затем выявляются последовательности событий, проходящие по всей длине разлома, в пределах заданной области динами ческого влияния. Устанавливается теснота корреляци онной связи для выборки «времяместо локализации». 1. ВВЕДЕНИЕ дований [Vikulin, 2003; Bykov, 2005; Sherman, Gor bunova, 2008]. По его решению опубликовано множе ство материалов, но вопрос попрежнему остается от крытым изза многочисленных геологогеофизических факторов, вызывающих сейсмический процесс, и мно говариантности причин, контролирующих пространст История изучения направленной миграции очагов землетрясений вдоль разломов земной коры насчиты вает около пятидесяти лет, и сейчас этот вопрос вхо дит в число теоретически и социально важных иссле 69 M.G. Mel’nikov: About the wave mechanism of activation of faults… тоники МНР [Khil’ko et al., 1985], Карта современной геодинамики Азии [Levi et al., 2007] и карты, предос тавленные Исследовательским центром астрономии и геофизики Монгольской академии наук [Gangaadorzh, 2010]). венновременную локализацию ощутимых землетря сений в конкретных сейсмических зонах. Задачей статьи ставится детальная обработка дан ных по сейсмическому процессу на территории Мон голии, выявление тенденций векторной направленно сти миграции очагов землетрясений, установление общей тенденции для данного региона, анализ полу ченных параметров деформационных волн и изучение их взаимосвязей. Для выбранных разломов оценены области динами ческого влияния [Sherman et al., 1983] и по методике [Sherman, Gorbunova, 2008] проведено исследование направления миграций очагов землетрясений. 3. МЕТОДИКА ОБРАБОТКИ ДАННЫХ ПО РАЗЛОМАМ ТЕРРИТОРИИ МОНГОЛИИ Определяются параметры деформационных волн по методике, описанной в работе [Sherman, Gorbunova, 2008]. В случае нескольких возможных вариантов ин терпретации положения линии регрессии выбирается тот, у которого наиболее высокие коэффициенты кор реляции. Каждая такая регрессионная прямая соответ Хорошо известно, что преобладающее количество очагов землетрясений приурочено к зонам разломов и областям их динамического влияния [Sherman et al., 1983]. Разломы являются основными структурами, контролирующими сейсмический процесс и эпицен тральное поле землетрясений в сейсмических зонах континентальной литосферы [Sherman, 2009]. Нами проведена оценка сейсмической активности и неболь шое уточнение положения главных сейсмоактивных разломов территории Монголии. Использованы раз личные геологические, сейсмотектонические карты и карты сейсмотектоники МНР, составленные в разные годы различными исследователями (Карта сейсмотек 70 Geodynamics & Tectonophysics 2013 Volume 4 Issue 1 Pages 69–81 Geodynamics & Tectonophysics 2013 Volume 4 Issue 1 Pages 69–81 Рис. 1. Методика построения графика для определения вектора миграции очагов землетрясений по простиранию разлома и оценки средней векторной скорости деформационной волны их возбуждения. На оси абсцисс жирной линией показана длина ак тивизированной части разлома, пунктирной – его полная длина. Квадратиками показаны сейсмические события в соответствии с их локализацией на разломе и временем свершения события. Цифры у трендов – временная последовательность прохождения фронтов деформационных волн [Gorbunova, Sherman, 2012]. нитудой от 3 до 8, получены новые данные по активи зации разломов и векторной направленности деформа ционных волн и их параметрам. нитудой от 3 до 8, получены новые данные по активи зации разломов и векторной направленности деформа ционных волн и их параметрам. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Миграция очагов землетрясений в сейсмических зонах, деформационные процессы в разломных зонах обсуждаются многими исследователями [Kasahara, 1979; Bykov, 2005; Ulomov, 1993; Malamud, Nikolaev sky, 1989]. В соответствии с работой [Sherman, Zlogodukhova, 2011], сейсмическая зона – это территория в тектони ческиактивной области, ограниченная контуром, вну три которого за заданный интервал времени количест во сейсмических событий с М>3 (К>9) выше первого десятка (или не ниже определенной статистически значимой величины). Внешний контур сейсмической зоны должен проводиться по изолинии, соответству ющей плотности ощутимых землетрясений с М≥3 не менее трех событий на заданную площадь. Сейсмиче ская зона может рассматриваться как «самостоятель ная структурная область литосферы. Ее образуют ли нейно вытянутая зона современной деструкции лито сферы (концентратор наиболее сильных землетрясе ний) и окружающие зону разноранговые разломы, многие из которых одно или многократно в реальном времени (месяцы, годы, десятилетия) селективно во влекались в кратковременный процесс активизации» [Sherman, Zlogodukhova, 2011]. Рис. 1. Методика построения графика для определения вектора миграции очагов землетрясений по простиранию разлома и оценки средней векторной скорости деформационной волны их возбуждения. На оси абсцисс жирной линией показана длина ак тивизированной части разлома, пунктирной – его полная длина. Квадратиками показаны сейсмические события в соответствии с их локализацией на разломе и временем свершения события. Цифры у трендов – временная последовательность прохождения фронтов деформационных волн [Gorbunova, Sherman, 2012]. Fig. 1. The method of graphics to determine vectors of earthquake foci migration along faults and to estimate average velocities of de formation waves of excitation. On the Xaxis, the thick line shows the length of the activated fault, and the dashed line shows the full length of the fault. Squares show seismic events according to their locations at the fault and the time of their occurrence. Numbers on the trends show the temporal sequence of occurrence of deformation waves’ fronts [Gorbunova, Sherman, 2012]. На территории Монголии выделяется ряд сейсми ческих зон, которые были исследованы в работах предшественников [Khil’ko et al., 1985; Gangaadorzh, 2010; Levi et al., 1996] и часть из которых была ис пользована нами при анализе: МонголоАлтайская (зо на I), Болнайская (зона II), Хубсугульская (зона III), ГобиАлтайская (зона IV) (рис. 2). По основным сегментам этих зон и их активным разломам нами была изучена специфика пространст венновременных локализаций очагов землетрясений, соответствующая, как принято в концепции [Sherman, 2009], скорости продвижения деформационных волн в соответствующих сегментах и разломах. ствует прохождению через разлом фронта волны, вы звавшего его активизацию. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ 1 – границы деструктивных зон Монголии (а) и нумерация их сейсмических зон (б); 2 – сейсмические события, цветом и размером показан их энергетический класс (от больших красных до маленьких бирюзовых – соответственно от 18го до 10го класса); 3 – государственная граница Монголии. Fig. 2. The map of seismic zones of Mongolia (according to [Khil'ko et al., 1985; Gangaadorzh, 2010]). 1 – borders of destructive zones of Mon l ( ) d b f (б) l h b d l (f l d ll Рис. 2. Карта сейсмических зон Монголии в соответствии с [Khil’ko et al., 1985; Gangaadorzh, 2010]. 1 – границы деструктивных зон Монголии (а) и нумерация их сейсмических зон (б); 2 – сейсмические события, цветом и размером показан их энергетический класс (от больших красных до маленьких бирюзовых – соответственно от 18го до 10го класса); 3 – государственная граница Монголии. Fig. 2. The map of seismic zones of Mongolia (according to [Khil'ko et al., 1985; Gangaadorzh, 2010]). 1 – borders of destructive zones of Mon golia (a) and numbers of seismic zones (б); 2 – seismic events; energy classes are shown by size and colours (from large red to small turquoise, re spectively, from the 18th to 10th classes); 3 – the state border of Mongolia. Fig. 2. The map of seismic zones of Mongolia (according to [Khil'ko et al., 1985; Gangaadorzh, 2010]). 1 – borders of destructive zones of Mon golia (a) and numbers of seismic zones (б); 2 – seismic events; energy classes are shown by size and colours (from large red to small turquoise, re spectively, from the 18th to 10th classes); 3 – the state border of Mongolia. личие волны, которая просто не отразилась на разломе в виде миграционной последовательности, скорее все го, изза неполноты данных по фиксации слабых по энергии очагов землетрясений. ривались различные возможные варианты их построе ния, но для оценки параметров волн были выбраны последовательности с наибольшими коэффициентами детерминации. Серыми точками на графиках обозна чены события, которые были исключены из анализа. Так, к примеру, в левой части графика 2b (рис. 4) име ется группа событий, которую не удалось интерпре тировать с точки зрения миграции, так как миграции в этом случае нет. События здесь происходят в одном и том же участке разлома, но равномерно распределены во времени. Эти события инициируются узлом пересе чения разломов на местности и «выпадают» из стан дартного анализа соотношения «времяместо». 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Чаще всего по одному раз лому фиксируется несколько линий активизаций, суб параллельных друг другу, что говорит о периодично сти процесса прохождения волн через конкретные раз ломы и, следовательно, о нескольких периодах их ак тивизации. у щ р С использованием карты (рис. 3) были изучены три крупных разлома из разных сейсмических зон Монго лии (обозначены синим цветом на рис. 3). Для опреде ления принадлежности сейсмических событий к кон кретным разломам принимались области их динамиче ского влияния шириной 20 км в обе стороны от обо значенных на карте осевых линий разломов. Выборка для составления графиков и оценки параметров де формационных волн и периодичности активизаций разломов содержит события с K≥10 (M≥3.1). Графики «времяместо» для данных разломов приведены на рис. 4. При выявлении пространственных закономер ностей локализации событий на графиках рассмат Как показано в работах [Sherman, Gorbunova, 2008; Gorbunova, Sherman, 2012], наклон линий тренда гра фика отражает направление распространения очагов землетрясений вдоль простирания разлома: с левого фланга к правому или наоборот; тангенс угла наклона линий трендов (α) показывает среднюю скорость ми грации очагов или скорость деформационной волны в рассматриваемом разломе. В результате обработки базы данных по 21 разлому, с которыми связано около 2300 землетрясений с маг 71 M.G. Mel’nikov: About the wave mechanism of activation of faults… Рис. 2. Карта сейсмических зон Монголии в соответствии с [Khil’ko et al., 1985; Gangaadorzh, 2010]. 1 – границы деструктивных зон Монголии (а) и нумерация их сейсмических зон (б); 2 – сейсмические события, цветом и размером показан их энергетический класс (от больших красных до маленьких бирюзовых – соответственно от 18го до 10го класса); 3 – государственная граница Монголии. Fig. 2. The map of seismic zones of Mongolia (according to [Khil'ko et al., 1985; Gangaadorzh, 2010]). 1 – borders of destructive zones of Mon golia (a) and numbers of seismic zones (б); 2 – seismic events; energy classes are shown by size and colours (from large red to small turquoise, re spectively, from the 18th to 10th classes); 3 – the state border of Mongolia. M.G. Mel’nikov: About the wave mechanism of activation of faults… G. Mel nikov: About the wave mechanism of activation of faults… Рис. 2. Карта сейсмических зон Монголии в соответствии с [Khil’ko et al., 1985; Gangaadorzh, 2010]. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Также из анализа исключена группа событий в правой части того же графика, которая могла бы интерпретировать ся как волна (как показано пунктиром), но в этом слу чае на промежутке 450–600 км должна быть еще одна последовательность (также показана пунктиром), то гда бы сохранилось значение длины волны и периода для всего графика. Однако на этом промежутке собы тий слишком мало, и можно только предполагать на Пунктирными линиями на графиках показаны ин терполяции и экстраполяции линий активизаций, для которых недостаточно данных, чтобы построить ли нию тренда. Для разломов с высокой корреляцией эксперимен тальных данных «времяместо» были составлены таб лицы параметров деформационных волн (табл. 1, 2). Средняя скорость деформационных волн для раз ломов из табл. 1 – 6.5 км/год, средняя длина волны – 150 км; в целом направление волн для данных разло мов – с запада на восток, что согласуется с соседними регионами, для которых были проведены подобные исследования [Sherman et al., 2012a]. Существуют мнения, что миграция очагов земле трясений связана с продвижением фронта деформации вдоль активных разломов [Novopashina, San’kov, 2010]. Скорости миграций здесь варьируются от первых ки 72 Geodynamics & Tectonophysics 2013 Volume 4 Issue 1 Pages 69–81 Geodynamics & Tectonophysics 2013 Volume 4 Issue 1 Pages 69–81 Рис. 3. Активные разломы Монголии. (Выкопировка из Карты современной геодинамики Азии [Levi et al., 2007] с использованием мате риалов [Khil’ko et al., 1985] и авторскими добавлениями). 1 – активные разломы Монголии; 2 – активные разломы, выбранные для ана лиза и их номера; 3 – государственная граница Монголии. Рис. 3. Активные разломы Монголии. (Выкопировка из Карты современной геодинамики Азии [Levi et al., 2007] с использованием мате риалов [Khil’ko et al., 1985] и авторскими добавлениями). 1 – активные разломы Монголии; 2 – активные разломы, выбранные для ана лиза и их номера; 3 – государственная граница Монголии. Fig. 3. Active faults of Mongolia (this section is copied from the map of modern geodynamics of Asia published in [Levi et al., 2007]; data from [Khil'ko et al., 1985] and the author’s data are included). 1 – active faults in Mongolia; 2 – active faults selected for analysis and their numbers; 3 – the state border of Mongolia. Fig. 3. Active faults of Mongolia (this section is copied from the map of modern geodynamics of Asia published in [Levi et al., 2007]; data from [Khil'ko et al., 1985] and the author’s data are included). 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ 1 – active faults in Mongolia; 2 – active faults selected for analysis and their numbers; 3 – the state border of Mongolia. лометров до первых десятков километров в год, что в целом согласуется с результатами, полученными в на стоящей работе и в работах других авторов. Зависимость между скоростями волн и направлениями разломов не прослеживается, так же как и между пе риодами волн и длинами разломов. р д д р Линейную зависимость «длина волны/длина разло ма» (рис. 6) можно объяснить тем, что при детальном анализе были использованы разломы с длинами разно го порядка. На графиках более протяженных разломов (более 200 км) (рис. 7, а) фиксируются «регрессион ные цепочки» с достаточно высокими скоростями и длинами волн (серые линии). В выделенной части это го графика (рис. 7, а) в более крупном масштабе видна измененная картина: тренды с достаточно высокими коэффициентами детерминации, но более низкими скоростями (рис. 7, б, черные линии). В данном случае скорость равна 1.4 км/год, в отличие от мелкомас штабной ситуации первоначального оригинального графика для данного разлома (см. рис. 4, разлом 2b), где скорость была 6 км/год (см. табл. 1). В случае, если рассматривается изначально короткий разлом (менее 200 км), зафиксировать более быстрые волны и уви деть полную картину невозможно, так как нельзя про анализировать ситуацию, сложившуюся за пределами его окончаний. В связи с этим, переходя к более Для более детального анализа параметров дефор мационных волн в зонах сопутствующих разломов на ми проведено изучение их параметров по более де тальной и обновленной более поздними исследова ниями карте (рис. 5). На ней видно, что изученные выше разломы принадлежат к различным сейсмиче ским зонам (I, II, IV). Проведено изучение параметров деформационных волн для сопутствующих и оперяю щих разломов (рис. 5). Полученные параметры интег рированы в таблицах 3 и 4 [Melnikov, Gangaadorzh, 2012]. На рис. 5 стрелками показаны направления дефор мационных волн для каждого разлома. Преобладаю щее направление во всех сейсмических зонах, как вид но из рисунка, с запада на восток. Для рассматриваемых разломов были построены графики взаимосвязей их параметров и параметров волн (рис. 6). Из графиков видно, что существует линейная зави симость между длинами разломов и длинами волн. 73 M.G. Mel’nikov: About the wave mechanism of activation of faults… с. 4. Графики в координатах «времяместо» для выбранных активных разломов Монголии (см. рис. 3). Зеленым и синим цветом чена принадлежность событий к определенной линии тренда. Серые точки – события, исключенные из анализа. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Coefficients of determination and timelocation regression equation for faults of Mongolia Номер разлома (протяженность, км) Колво сейсмических событий, используемых в анализе, и диапазон магнитуд (классов) Количество временных совокупностей (периодов) Коэффициент детерминации Уравнение регрессии 1 (552) 24, M=3.0–4.4 (K=10–12) 2 R1 2=0.93 R2 2=0.8 t = (0.1549∙ℓ + 1917) t = (0.1001∙ℓ + 1961.1) 2 (713) 100, M=3.0–6.1 (K=10–15) 2 R1 2=0.72 R2 2=0.72 t = (0.1759∙ℓ + 1917) t = (0.157∙ℓ + 1943.8) 3 (384) 74, M=3.5–5.7 (K=11–14) 3 R1 2=0.97 R2 2=0.84 R3 2=0.74 t = (0.1901∙ℓ + 1913.4) t = (0.177∙ℓ + 1933.9) t = (0.164∙ℓ + 1955.3) Рис. 5. Карта деструктивных зон Монголии. 1 – границы деструктивных зон Монголии (а) и нумерация их сейсмических зон (б); 2 – ак тивные разломы в сейсмических зонах (а), номера разломов и обозначение их сегментов (б), цветом показана принадлежность к опреде ленной сейсмической зоне; 3 – вектор направленности деформационных волн; 4 – начальная точка расчетных данных по разлому; 5 – го сударственная граница Монголии. Fig. 5. The map of destructive zones of Mongolia. 1 – borders of destructive zones of Mongolia (a) and the numbers of seismic zones (б); 2 – ac tive faults in seismic zones (a), number of faults and their segments (б); colours correspond to seismic zones; 3 – vector showing direction of de formation waves; 4 – starting point of estimations for the fault; 5 – the state border of Mongolia. Т а б л и ц а 2. Коэффициенты детерминации и уравнения регрессии графиков «времяместо» разломов Монголии T a b l e 2. Coefficients of determination and timelocation regression equation for faults of Mongolia Номер разлома (протяженность, км) Колво сейсмических событий, используемых в анализе, и диапазон магнитуд (классов) Количество временных совокупностей (периодов) Коэффициент детерминации Уравнение регрессии 1 (552) 24, M=3.0–4.4 (K=10–12) 2 R1 2=0.93 R2 2=0.8 t = (0.1549∙ℓ + 1917) t = (0.1001∙ℓ + 1961.1) 2 (713) 100, M=3.0–6.1 (K=10–15) 2 R1 2=0.72 R2 2=0.72 t = (0.1759∙ℓ + 1917) t = (0.157∙ℓ + 1943.8) 3 (384) 74, M=3.5–5.7 (K=11–14) 3 R1 2=0.97 R2 2=0.84 R3 2=0.74 t = (0.1901∙ℓ + 1913.4) t = (0.177∙ℓ + 1933.9) t = (0.164∙ℓ + 1955.3) ц а 2. Коэффициенты детерминации и уравнения регрессии графиков «времяместо» разломов Монголии 2. Coefficients of determination and timelocation regression equation for faults of Mongolia Рис. 5. Карта деструктивных зон Монголии. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ 4 Timelocation curves for selected active faults of Mongolia (see Fig 3) Events marked in green and blue belong to corresponding t Рис. 4. Графики в координатах «времяместо» для выбранных активных разломов Монголии (см. рис. 3). Зеленым и синим цветом обо значена принадлежность событий к определенной линии тренда. Серые точки – события, исключенные из анализа. Fig. 4. Timelocation curves for selected active faults of Mongolia (see Fig. 3). Events marked in green and blue belong to corresponding trends. Grey dots show events excluded from the analysis. Geodynamics & Tectonophysics 2013 Volume 4 Issue 1 Pages 69–81 Т а б л и ц а 1. Параметры деформационных волн – триггерного механизма возбуждения сейсмических событий Монголии T a b l e 1. Parameters of deformation waves acting as the trigger mechanism of seismic events in Mongolia Номер разлома (протяженность, км) Простирание Колво сейсмических событий, используемых в анализе, и диапазон магнитуд (классов) Длина волны (км) Скорость волны (км/год) Период волны (год) 1 (552) 340º 24, M=3.0–4.4 (K=10–12) 200 8 ССЗ – ЮЮВ 25 2 (713) 90º 100, M=3.0–6.1 (K=10–15) 126 6 З – В 21 3 (384) 280º 74, M=3.5–5.7 (K=11–14) 108 6 З – В 18 ы деформационных волн – триггерного механизма возбуждения сейсмических событий Монголии 1. Параметры деформационных волн – триггерного механизма возбуждения сейсмических событий Монгол Т а б л и ц а 1. Параметры деформационных волн – триггерного механизма возбуждения сейсмических со b l f d f h h f l л и ц а 1. Параметры деформационных волн – триггерного механизма возбуждения сейсмических событи l e 1. Parameters of deformation waves acting as the trigger mechanism of seismic events in Mongolia meters of deformation waves acting as the trigger mechanism of seismic events in Mongolia Т а б л и ц а 2. Коэффициенты детерминации и уравнения регрессии графиков «времяместо» разломов Монголии T a b l e 2. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ 1 – границы деструктивных зон Монголии (а) и нумерация их сейсмических зон (б); 2 – ак тивные разломы в сейсмических зонах (а), номера разломов и обозначение их сегментов (б), цветом показана принадлежность к опреде ленной сейсмической зоне; 3 – вектор направленности деформационных волн; 4 – начальная точка расчетных данных по разлому; 5 – го сударственная граница Монголии. Fig. 5. The map of destructive zones of Mongolia. 1 – borders of destructive zones of Mongolia (a) and the numbers of seismic zones (б); 2 – ac tive faults in seismic zones (a), number of faults and their segments (б); colours correspond to seismic zones; 3 – vector showing direction of de formation waves; 4 – starting point of estimations for the fault; 5 – the state border of Mongolia. Рис. 5. Карта деструктивных зон Монголии. 1 – границы деструктивных зон Монголии (а) и нумерация их сейсмических зон (б); 2 – ак тивные разломы в сейсмических зонах (а), номера разломов и обозначение их сегментов (б), цветом показана принадлежность к опреде ленной сейсмической зоне; 3 – вектор направленности деформационных волн; 4 – начальная точка расчетных данных по разлому; 5 – го сударственная граница Монголии. Fig. 5. The map of destructive zones of Mongolia. 1 – borders of destructive zones of Mongolia (a) and the numbers of seismic zones (б); 2 – ac tive faults in seismic zones (a), number of faults and their segments (б); colours correspond to seismic zones; 3 – vector showing direction of de formation waves; 4 – starting point of estimations for the fault; 5 – the state border of Mongolia. 75 M.G. Mel’nikov: About the wave mechanism of activation of faults… M.G. Mel nikov: About the wave mechanism of activation of faults… Т а б л и ц а 3. Параметры деформационных волн – триггерного механизма возбуждения сейсмических событий Монголии T a b l e 3. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Coefficients of determination and timelocation regression equations for faults of Mongolia Номер разлома (протяженность, км) Колво сейсмических событий, используемых в анализе, и диапазон магнитуд (классов) Количество временных совокупностей (периодов) Коэффициент детерминации Уравнени I2(1) (86) 133, M=3.0–5.5 (K=10–14) 5 R1 2=0.74 R2 2=0.77 R3 2=0.65 R4 2=0.7 R5 2=0.73 t = (0.477 t = (0.717 t = (0.500 t = (0.806 t = (0.373 I2(2) (86) 133, M=3.0–5.5 (K=10–14) 4 R1 2=0.4 R2 2=0.33 R3 2=0.61 R4 2=0.54 t = (–1.80 t = (–1.31 t = (–2.09 t = (–0.60 I4(1) (271) 18, M=3–5 (K=10–13) 3 R1 2=0.65 R2 2=0.9 R3 2=0.99 t = (0.457 t = (0.460 t = (0.354 I4(2) (271) 18, M=3–5 (K=10–13) 2 R1 2=0.77 R2 2=0.93 t = (–0.19 t = (–0.24 I5 (312) 46, M=3.0–5.5 (K=10–14) 4 R1 2=0.53 R2 2=0.34 R3 2=0.89 R4 2=0.83 t = (0.167 t = (0.252 t = (0.412 t = (0.400 I7 (517) 52, M=3.0–4.6 (K=10–12) 3 R1 2=0.81 R2 2=0.74 R3 2=0.33 t = (0.18∙ℓ t = (0.309 t = (0.241 I8б (143) 14, M=3.1–4.9 (K=10–13) 2 R1 2=0.62 R2 2=0.33 t = (0.557 t = (0.121 I8в (235) 13, M=3.0–4.4 (K=10–12) 1 R1 2=0.75 t = (0.364 I8г (115) 17, M=3.0–4.4 (K=10–12) 1 R1 2=0.6 t = (–0.48 I10(1) (309) 60, M=3–6 (K=10–15) 3 R1 2=0.63 R2 2=0.82 R3 2=0.84 t = (0.271 t = (0.299 t = (0.392 I10(2) (309) 60, M=3–6 (K=10–15) 3 R1 2=0.44 R2 2=0.75 R3 2=0.74 t = (–0.29 t = (–0.21 t = (–0.33 II1 (247) 134, M=3.0–4.9 (K=10–13) 3 R1 2=0.65 R2 2=0.84 R3 2=0.55 t = (0.297 t = (0.336 t = (0.266 II2 (434) 74, M=3–6 (K=10–15) 2 R1 2=0.62 R2 2=0.73 t = (0.548 t = (0.186 II3(1) (74) 13, M=3.0–4.4 (K=10–12) 1 R1 2=0.65 t = (1.261 II3(2) (74) 13, M=3.0–4.4 (K=10–12) 1 R1 2=0.8 t = (–0.67 III1а (27) 14, M=3.2–4.2 (K=10–12) 1 R1 2=0.55 t = (1.723 III1б (33) 54, M=3.0–5.5 (K=10–14) 4 R1 2=0.64 R2 2=0.64 R3 2=0.25 R4 2=0.45 t = (–2.74 t = (–2.74 t = (–2.17 t = (–2.74 III1в (24) 62, M=3.0–5.5 (K=10–14) 2 R1 2=0.65 R2 2=0.3 t = (1.542 t = (1.267 III2а (102) 19, M=3.6–4.4 (K=11–12) 3 R1 2=0.46 R2 2=0.93 R3 2=0.77 t = (1.929 t = (2.078 t = (2.352 III2б (121) 41, M=3.6–5.3 (K=11–14) 2 R1 2=0.91 R2 2=0.4 t = (1.140 t = (0.707 III3а (76) 50, M=3.0–4.4 (K=10–12) 3 R1 2=0.87 R2 2=0.43 R3 2=0.73 t = (0.686 t = (0.724 t = (0.508 III3б (111) 31, M=3.0–4.5 (K=10–12) 1 R1 2=0.75 t = (0.463 Т а б л и ц а 4. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Коэффициенты детерминации и уравнения регрессии графиков «времяместо» разломов Монголии T a b l e 4. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Coefficients of determination and timelocation regression equations for faults of Mongolia Номер разлома (протяженность, км) Колво сейсмических событий, используемых в анализе, и диапазон магнитуд (классов) Количество временных совокупностей (периодов) Коэффициент детерминации Уравнение регрессии I2(1) (86) 133, M=3.0–5.5 (K=10–14) 5 R1 2=0.74 R2 2=0.77 R3 2=0.65 R4 2=0.7 R5 2=0.73 t = (0.4777∙ℓ + 1934.6) t = (0.7177∙ℓ + 1927) t = (0.5007∙ℓ + 1952.5) t = (0.8064∙ℓ + 1959.4) t = (0.3739∙ℓ + 1994.7) I2(2) (86) 133, M=3.0–5.5 (K=10–14) 4 R1 2=0.4 R2 2=0.33 R3 2=0.61 R4 2=0.54 t = (–1.8049∙ℓ + 2039) t = (–1.3109∙ℓ + 2039.2) t = (–2.0988∙ℓ + 2099.4) t = (–0.6069∙ℓ + 2043.5) I4(1) (271) 18, M=3–5 (K=10–13) 3 R1 2=0.65 R2 2=0.9 R3 2=0.99 t = (0.4577∙ℓ + 1898.2) t = (0.4603∙ℓ + 1930.1) t = (0.3544∙ℓ + 1969) I4(2) (271) 18, M=3–5 (K=10–13) 2 R1 2=0.77 R2 2=0.93 t = (–0.1944∙ℓ + 2000.8) t = (–0.247∙ℓ + 2049.9) I5 (312) 46, M=3.0–5.5 (K=10–14) 4 R1 2=0.53 R2 2=0.34 R3 2=0.89 R4 2=0.83 t = (0.1676∙ℓ + 1921.7) t = (0.2526∙ℓ + 1921.3) t = (0.4122∙ℓ + 1927.7) t = (0.4004∙ℓ + 1959.5) I7 (517) 52, M=3.0–4.6 (K=10–12) 3 R1 2=0.81 R2 2=0.74 R3 2=0.33 t = (0.18∙ℓ + 1931.5) t = (0.3096∙ℓ + 1946.2) t = (0.2418∙ℓ + 1980.9) I8б (143) 14, M=3.1–4.9 (K=10–13) 2 R1 2=0.62 R2 2=0.33 t = (0.5575∙ℓ + 1945.3) t = (0.1218∙ℓ + 1994.5) I8в (235) 13, M=3.0–4.4 (K=10–12) 1 R1 2=0.75 t = (0.3641∙ℓ + 1958.8) I8г (115) 17, M=3.0–4.4 (K=10–12) 1 R1 2=0.6 t = (–0.4811∙ℓ + 2037.3) I10(1) (309) 60, M=3–6 (K=10–15) 3 R1 2=0.63 R2 2=0.82 R3 2=0.84 t = (0.2712∙ℓ + 1931.2) t = (0.2998∙ℓ + 1942) t = (0.3929∙ℓ + 1962.2) I10(2) (309) 60, M=3–6 (K=10–15) 3 R1 2=0.44 R2 2=0.75 R3 2=0.74 t = (–0.2999∙ℓ + 1997.2) t = (–0.2178∙ℓ + 2018.4) t = (–0.3335∙ℓ + 2057.2) II1 (247) 134, M=3.0–4.9 (K=10–13) 3 R1 2=0.65 R2 2=0.84 R3 2=0.55 t = (0.297∙ℓ + 1940.7) t = (0.3368∙ℓ + 1951.3) t = (0.2668∙ℓ + 1973.5) II2 (434) 74, M=3–6 (K=10–15) 2 R1 2=0.62 R2 2=0.73 t = (0.5484∙ℓ + 1935.9) t = (0.1868∙ℓ + 1953.8) II3(1) (74) 13, M=3.0–4.4 (K=10–12) 1 R1 2=0.65 t = (1.261∙ℓ + 1970.3) II3(2) (74) 13, M=3.0–4.4 (K=10–12) 1 R1 2=0.8 t = (–0.6786∙ℓ + 2007.9) III1а (27) 14, M=3.2–4.2 (K=10–12) 1 R1 2=0.55 t = (1.7238∙ℓ + 1971.7) III1б (33) 54, M=3.0–5.5 (K=10–14) 4 R1 2=0.64 R2 2=0.64 R3 2=0.25 R4 2=0.45 t = (–2.74∙ℓ + 1993.3) t = (–2.7482∙ℓ + 2011) t = (–2.1771∙ℓ + 2033.4) t = (–2.7458∙ℓ + 2073.3) III1в (24) 62, M=3.0–5.5 (K=10–14) 2 R1 2=0.65 R2 2=0.3 t = (1.5423∙ℓ + 1956.5) t = (1.2676∙ℓ + 1985.7) III2а (102) 19, M=3.6–4.4 (K=11–12) 3 R1 2=0.46 R2 2=0.93 R3 2=0.77 t = (1.9294∙ℓ + 1852.7) t = (2.0781∙ℓ + 1901.2) t = (2.3525∙ℓ + 1946.8) III2б (121) 41, M=3.6–5.3 (K=11–14) 2 R1 2=0.91 R2 2=0.4 t = (1.1403∙ℓ + 1898.8) t = (0.7079∙ℓ + 1958.6) III3а (76) 50, M=3.0–4.4 (K=10–12) 3 R1 2=0.87 R 2=0 43 t = (0.6869∙ℓ + 1937.5) t = (0 7243 ℓ+ 1957) эффициенты детерминации и уравнения регрессии графиков «времяместо» разломов Монголии f d d l f f l f l ц а 4. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Parameters of deformation waves acting as the trigger mechanism of seismic events in Mongolia Номер разлома (протяженность, км) Простирание Колво сейсмических событий, используемых в анализе, и диапазон магнитуд (классов) Длина волны (км) Скорость волны (км/год) Период вол (год) I2(1) (86) 300º 133, M=3.0–5.5 (K=10–14) 31 2 ЗСЗ–ВЮВ 17 I2(2) (86) 300º 133, M=3.0–5.5 (K=10–14) 22 1 ВЮВ–ЗСЗ 25 I4(1) (271) 340º 18, M=3–5 (K=10–13) 79 2 СЗ–ЮВ 33 I4(2) (271) 340º 18, M=3–5 (K=10–13) 184 5 ЮВ–СЗ 40 I5 (312) 335º 46, M=3.0–5.5 (K=10–14) 114 4 СЗ–ЮВ 31 I7 (517) 320º 52, M=3.0–4.6 (K=10–12) 142 5 ССЗ–ЮЮВ 33 I8б (143) 320º 14, M=3.1–4.9 (K=10–13) 135 5 СЗ–ЮВ 27 I8в (235) 325º 13, M=3.0–4.4 (K=10–12) 3 СЗ–ЮВ I8г (115) 300º 17, M=3.0–4.4 (K=10–12) 2 ЮВ–СЗ I10(1) (309) 350º 60, M=3–6 (K=10–15) 73 3 ССЗ–ЮЮВ 23 I10(2) (309) 350º 60, M=3–6 (K=10–15) 86 4 ЮЮВ–ССЗ 24 II1 (247) 75º 134, M=3.0–4.9 (K=10–13) 54 4 ЗЮЗ–ВСВ 16 II2 (434) 270º 74, M=3–6 (K=10–15) 157 6 З–В 26 II3(1) (74) 340º 13, M=3.0–4.4 (K=10–12) 1 СЗ–ЮВ II3(2) (74) 340º 13, M=3.0–4.4 (K=10–12) 1 ЮВ–СЗ III1а (27) 40º 14, M=3.2–4.2 (K=10–12) 1 ЮЗ–СВ III1б (33) 55º 54, M=3.0–5.5 (K=10–14) 10 0.5 СВ–ЮЗ 25 III1в (24) 30º 62, M=3.0–5.5 (K=10–14) 19 1 ЮЗ–СВ 26 III2а (102) 280º 19, M=3.6–4.4 (K=11–12) 24 0.5 З–В 52 III2б (121) 75º 41, M=3.6–5.3 (K=11–14) 40 1 З–В 35 III3а (76) 340º 50, M=3.0–4.4 (K=10–12) 33 2 ССЗ–ЮЮВ 21 III3б (111) 45º 31, M=3.0–4.5 (K=10–12) 2 ЮВ–СВ III4 (150) 0º 22, M=3.0–4.9 (K=10–13) 33 1 С–Ю 23 IV1а (127) 290º 147, M=3.0–7.9 (K=10–18) 34 1 ЗСЗ–ВЮВ 26 IV1б(1) (88) 280º 67, M=3–6 (K=10–15) 23 1 ЗСЗ–ВЮВ 22 IV1б(2) (88) 280º 67, M=3–6 (K=10–15) 38 1 ВЮВ–ЗСЗ 30 IV1в (100) 300º 57, M=3–6 (K=10–15) 29 1 ЗСЗ–ВЮВ 24 Т а б л и ц а 3. Параметры деформационных волн – триггерного механизма возбуждения сейсмических событий Монголии T a b l e 3. Parameters of deformation waves acting as the trigger mechanism of seismic events in Mongolia л и ц а 3. Параметры деформационных волн – триггерного механизма возбуждения сейсмических событий l e 3. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Parameters of deformation waves acting as the trigger mechanism of seismic events in Mongolia 76 Geodynamics & Tectonophysics 2013 Volume 4 Issue 1 Pages 69–81 mics & Tectonophysics 2013 Volume 4 Issue 1 Pages 69 Т а б л и ц а 4. Коэффициенты детерминации и уравнения регрессии графиков «времяместо» разломов Монголии T a b l e 4. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Коэффициенты детерминации и уравнения регрессии графиков «времяместо» разломов Монголии 4. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Coefficients of determination and timelocation regression equations for faults of Mongolia яместо» разломов Монголии f Mongolia Коэффициент детерминации Уравнение регрессии R1 2=0.74 R2 2=0.77 R3 2=0.65 R4 2=0.7 R5 2=0.73 t = (0.4777∙ℓ + 1934.6) t = (0.7177∙ℓ + 1927) t = (0.5007∙ℓ + 1952.5) t = (0.8064∙ℓ + 1959.4) t = (0.3739∙ℓ + 1994.7) R1 2=0.4 R2 2=0.33 R3 2=0.61 R4 2=0.54 t = (–1.8049∙ℓ + 2039) t = (–1.3109∙ℓ + 2039.2) t = (–2.0988∙ℓ + 2099.4) t = (–0.6069∙ℓ + 2043.5) R1 2=0.65 R2 2=0.9 R3 2=0.99 t = (0.4577∙ℓ + 1898.2) t = (0.4603∙ℓ + 1930.1) t = (0.3544∙ℓ + 1969) R1 2=0.77 R2 2=0.93 t = (–0.1944∙ℓ + 2000.8) t = (–0.247∙ℓ + 2049.9) R1 2=0.53 R2 2=0.34 R3 2=0.89 R4 2=0.83 t = (0.1676∙ℓ + 1921.7) t = (0.2526∙ℓ + 1921.3) t = (0.4122∙ℓ + 1927.7) t = (0.4004∙ℓ + 1959.5) R1 2=0.81 R2 2=0.74 R3 2=0.33 t = (0.18∙ℓ + 1931.5) t = (0.3096∙ℓ + 1946.2) t = (0.2418∙ℓ + 1980.9) R1 2=0.62 R2 2=0.33 t = (0.5575∙ℓ + 1945.3) t = (0.1218∙ℓ + 1994.5) R1 2=0.75 t = (0.3641∙ℓ + 1958.8) R1 2=0.6 t = (–0.4811∙ℓ + 2037.3) R1 2=0.63 R2 2=0.82 R3 2=0.84 t = (0.2712∙ℓ + 1931.2) t = (0.2998∙ℓ + 1942) t = (0.3929∙ℓ + 1962.2) R1 2=0.44 R2 2=0.75 R3 2=0.74 t = (–0.2999∙ℓ + 1997.2) t = (–0.2178∙ℓ + 2018.4) t = (–0.3335∙ℓ + 2057.2) R1 2=0.65 R2 2=0.84 R3 2=0.55 t = (0.297∙ℓ + 1940.7) t = (0.3368∙ℓ + 1951.3) t = (0.2668∙ℓ + 1973.5) R1 2=0.62 R2 2=0.73 t = (0.5484∙ℓ + 1935.9) t = (0.1868∙ℓ + 1953.8) R1 2=0.65 t = (1.261∙ℓ + 1970.3) R1 2=0.8 t = (–0.6786∙ℓ + 2007.9) R1 2=0.55 t = (1.7238∙ℓ + 1971.7) R1 2=0.64 R2 2=0.64 R3 2=0.25 R4 2=0.45 t = (–2.74∙ℓ + 1993.3) t = (–2.7482∙ℓ + 2011) t = (–2.1771∙ℓ + 2033.4) t = (–2.7458∙ℓ + 2073.3) R1 2=0.65 R2 2=0.3 t = (1.5423∙ℓ + 1956.5) t = (1.2676∙ℓ + 1985.7) R1 2=0.46 R2 2=0.93 R3 2=0.77 t = (1.9294∙ℓ + 1852.7) t = (2.0781∙ℓ + 1901.2) t = (2.3525∙ℓ + 1946.8) R1 2=0.91 R2 2=0.4 t = (1.1403∙ℓ + 1898.8) t = (0.7079∙ℓ + 1958.6) R1 2=0.87 R2 2=0.43 R3 2=0.73 t = (0.6869∙ℓ + 1937.5) t = (0.7243∙ℓ + 1957) t = (0.5082∙ℓ + 1981.3) R1 2=0.75 t = (0.463∙ℓ + 1962) R1 2=0.6 R2 2=0.95 R3 2=0.87 t = (–0.9035∙ℓ + 1995.5) t = (–0.6503∙ℓ + 2012.7) t = (–0.5739∙ℓ + 2030.4) 77 M.G. 4. АНАЛИЗ РЕЗУЛЬТАТОВ ИССЛЕДОВАНИЙ Mel’nikov: About the wave mechanism of activation of faults… О к о н ч а н и е т а б л и ц ы 4. T a b l e 4 ( c o n t i n u e d ). IV1а (127) 147, M=3.0–7.9 (K=10–18) 4 R1 2=0.66 R2 2=0.77 R3 2=0.79 R4 2=0.78 t = (0.5419∙ℓ + 1912) t = (1.0154∙ℓ + 1881.9) t = (0.7657∙ℓ + 1929) t = (0.8609∙ℓ + 1954.9) IV1б(1) (88) 67, M=3–6 (K=10–15) 5 R1 2=0.65 R2 2=0.88 R3 2=0.67 R4 2=0.66 R5 2=0.32 t = (0.8913∙ℓ + 1893.9) t = (1.3682∙ℓ + 1885) t = (0.6845∙ℓ + 1946) t = (0.8794∙ℓ + 1956.9) t = (1.3244∙ℓ + 1970.6) IV1б(2) (88) 67, M=3–6 (K=10–15) 3 R1 2=0.61 R2 2=0.35 R3 2=0.33 t = (–0.9556∙ℓ + 1997.5) t = (–0.942∙ℓ + 2030.8) t = (–0.607∙ℓ + 2031.5) IV1в (100) 57, M=3–6 (K=10–15) 3 R1 2=0.83 R2 2=0.88 R3 2=0.88 t = (0.7144∙ℓ + 1936) t = (0.7788∙ℓ + 1957.2) t = (1.0059∙ℓ + 1975.6) M.G. Mel’nikov: About the wave mechanism of activation of faults… Такие же связи обнаружены для районов Китая и При байкалья [Sherman et al., 2012b; Sherman, Gorbunova, 2008]. Особенно хорошо эта зависимость просматри вается на графиках, построенных для отдельных тер детальному анализу, следует рассматривать разломы с длинами одного порядка. д д р д Отношения скоростей волн и длин разломов опи сываются экспоненциальной зависимостью (рис. 8). Рис. 6. Графики взаимосвязей параметров волн и разломов. Fig. 6. Relationships between parameters of waves and faults. Рис. 6. Графики взаимосвязей параметров волн и разломов. Fig. 6. Relationships between parameters of waves and faults. Geodynamics & Tectonophysics 2013 Volume 4 Issue 1 Pages 69–81 Рис. 7. Различие фиксируемых параметров деформационных волн в зависимости от длины разломов. a – график разлома 2b; b – график участка разлома 2b длиной 140 км в укрупненном масштабе, выделенный на рис. 7, а, вертикальными линиями. Серые линии – интер претация данных в длинном разломе; черные линии – новый вариант интерпретации с низкими скоростями в коротком разломе. Fig. 7. Variations of recorded parameters of deformation waves with regard to fault length. a – curve for Fault 2b; b – curve for the section of Fault 2b (140 km long) in the larger scale, which is marked in Fig. 7, a by vertical lines. Gray lines show data interpretation for the long fault; black lines show a new version of interpretation with regard to low velocities for the short fault. Рис. 7. Различие фиксируемых параметров деформационных волн в зависимости от длины разломов. a – график разлома 2b; b – график участка разлома 2b длиной 140 км в укрупненном масштабе, выделенный на рис. 7, а, вертикальными линиями. Серые линии – интер претация данных в длинном разломе; черные линии – новый вариант интерпретации с низкими скоростями в коротком разломе. Fig. 7. Variations of recorded parameters of deformation waves with regard to fault length. a – curve for Fault 2b; b – curve for the section of Fault 2b (140 km long) in the larger scale, which is marked in Fig. 7, a by vertical lines. Gray lines show data interpretation for the long fault; black lines show a new version of interpretation with regard to low velocities for the short fault. Fig. 7. Variations of recorded parameters of deformation waves with regard to fault length. a – curve for Fault 2b; b – curve for the section of Fault 2b (140 km long) in the larger scale, which is marked in Fig. 7, a by vertical lines. Gray lines show data interpretation for the long fault; black lines show a new version of interpretation with regard to low velocities for the short fault. Рис. 8. График зависимости скорости волны от длины разлома. Рис. 8. График зависимости скорости волны от длины разлома. Рис. 8. График зависимости скорости волны от длины разлома. Fig. 8. Wave velocity versus fault length. Fig. 8. Wave velocity versus fault length. Fig. 8. Fig. 8. Wave velocity versus fault length. Wave velocity versus fault length. 79 M.G. Mel’nikov: About the wave mechanism of activation of faults… тенденций векторной направленности миграции оча гов землетрясений. риторий с относительно однородной геологогеофизи ческой обстановкой, таких как Байкальская рифтовая зона и некоторые районы Китая [Sherman et al., 2012a]. Физический смысл зависимости между скоростями волн и длинами разломов объясняется тем, что более длинные разломы относительно более чувствительны к нагрузке [Kocharyan, Spivak, 2003], так как менее ус тойчивы. Их реакция на деформационные волны вы ражается более интенсивными активизациями, что су щественно отражается на скоростях миграций очагов землетрясений. В работе систематизированы параметры, определе на общая векторная направленность деформационных волн у разломов разных рангов. Установлено, что фик сируемые длины и скорости волн во многом зависят от длин рассматриваемых разломов, и для наиболее точ ного анализа параметров собственно деформационных волн следует использовать наиболее протяженные раз ломы с соизмеримыми длинами. 7. ЛИТЕРАТУРА Bykov V.G., 2005. Strain waves in the Earth: theory, field data, and models. Russian Geology and Geophysics 46 (11), 1176– 1190. Bykov V.G., 2005. Strain waves in the Earth: theory, field data, and models. Russian Geology and Geophysics 46 (11), 1176– 1190. Gangaadorzh B., 2010. Seismicity of Mongolia and Adjacent Territories. Abstract ... Thesis for the degree of Candidate of Geology and Mineralogy. Irkutsk, 16 p. (in Russian) [Гангаадорж Б. Сейсмичность Монголии и сопредельных тер риторий: Автореф. дис. … канд. геол.мин. наук. Иркутск, 2010. 16 с.]. Gorbunova E.A., Sherman S.I., 2012. Slow Deformation Waves in the Lithosphere: Registration, Parameters, and Geody namic Analysis (Central Asia). Russian Journal of Pacific Geology 6 (1), 1–13. http://dx.doi.org/10.1134/S181971401 201006X. Kasahara K., 1979. Migration of crustal deformation. Tectonophysics 52 (1–4,) 329–341. http://dx.doi.org/10.1016/0040 1951(79)902403. Khil’ko S.D., Kurushin R.A., Kochetkov V.M., Misharina L.A., Mel’nikova V.I., Gileva N.A., Lastochkin S.V., Balzhinnyam I., Monhoo D., 1985. Earthquakes and fundamentals of seismic zoning of Mongolia. Nauka, Moscow, 224 p. (in Russian) [Хилько С.Д., Курушин Р.А., Кочетков В.М., Мишарина Л.А., Мельникова В.И., Гилева Н.А., Ласточкин С.В., Балжинням И., Монхоо Д. Землетрясения и основы сейсмического районирования Монголии. М.: Наука, 1985. 224 с.]. Kocharyan G.G., Spivak A.A., 2003. The Dynamics of Deformation of Rock Blocks. Akademkniga, Moscow, 423 p. (in Rus sian) [Кочарян Г.Г., Спивак А.А. Динамика деформирования блочных массивов горных пород. М.: ИКЦ «Академ книга», 2003. 423 с.]. Levi K.G., Khromovskikh V.S., Kochetkov V.M., Nikolaev V.V., Semenov R.M., Serebrennikov S.P., Chipizubov A.V., Dem’ yanovich M.G., Arzhannikov S.G., Del’yansky E.A., Smekalin O.P., Ruzhich V.V., Buddo V.Yu., Masal’sky O.K., Potapov V.A., Berzhinsky Yu.A., Radziminovich Ya.B., 1996. Modern geodynamics: seismotectonics, earthquake prediction, earth quake risk (fundamental and applied aspects) (Article II). In: Logachev N.A. (Ed.), The Lithosphere of Central Asia. Main Findings of the Institute of the Earth's Crust in 1992–1996. Nauka, Novosibirsk, p. 150–182 (in Russian) [Леви К.Г., Хромовских В.С., Кочетков В.М., Николаев В.В., Семенов Р.М., Серебренников С.П., Чипизубов А.В., Демья нович М.Г., Аржанников С.Г., Дельянский Е.А., Смекалин О.П., Ружич В.В., Буддо В.Ю., Масальский О.К., Пота пов В.А., Бержинский Ю.А., Радзиминович Я.Б. Современная геодинамика: сейсмотектоника, прогноз землетря сений, сейсмический риск (фундаментальные и прикладные аспекты) (Статья II) // Литосфера Центральной Азии. Основные результаты исследований Института земной коры СО РАН в 1992–1996 гг. / Под ред. Н.А. Логачева. Новосибирск: Наука, 1996. С. 150–182]. Levi K.G., Sherman S.I., San’kov V.A., Lunina O.V., Lukhnev A.V., 2007. The Map of Modern Geodynamics of Asia. 5. ЗАКЛЮЧЕНИЕ Автор выражает благодарность профессору С.И. Шерману за постановку исследований и обсужде ние результатов. Задачей работы ставился анализ основных парамет ров деформационных волн, изучение их взаимосвязей посредством детальной обработки данных по сейсми ческим событиям Монголии, а также выявление общих Работа поддержана грантом РФФИ (120591161 ГФЕН_а), проектами СО РАН VII.64.1.4. и ОНЗ 7.7. Работа поддержана грантом РФФИ (120591161 ГФЕН_а), проектами СО РАН VII.64.1.4. и ОНЗ 7.7. Melnikov M.G., Gangaadorzh B., 2012. Seismically active areas of destruction in Mongolia and wavetriggering mechanisms of their activation. In: Modern geodynamics of Central Asia and hazardous natural processes: quantitative research re sults. Proceedings of the AllRussia conference and youth school on modern geodynamics (Irkutsk, September 23–29, 7. ЛИТЕРАТУРА GP 475 VKF, Irkutsk (in Russian) [Леви К.Г., Шерман С.И., Саньков В.А., Лунина О.В., Лухнев А.В. Карта современной геодинамики Азии. Иркутск: ГП 475 ВКФ, 2007]. Malamud A.S., Nikolaevsky V.N., 1989. Earthquake Cycles and Tectonic Waves. Donish, Dushanbe, 132 p. (in Russian) [Маламуд А.С., Николаевский В.Н. Циклы землетрясений и тектонические волны. Душанбе: Дониш, 1989. 132 c.]. Melnikov M.G., Gangaadorzh B., 2012. Seismically active areas of destruction in Mongolia and wavetriggering mechanisms of their activation. In: Modern geodynamics of Central Asia and hazardous natural processes: quantitative research re sults. Proceedings of the AllRussia conference and youth school on modern geodynamics (Irkutsk, September 23–29, 80 Geodynamics & Tectonophysics 2013 Volume 4 Issue 1 Pages 69–81 2012). IEC SB RAS, Irkutsk, V. 1. P. 161–164 (in Russian) [Мельников М.Г., Гангаадорж Б. Сейсмоактивные дест руктивные зоны Монголии и триггерные волновые механизмы их активизации // Современная геодинамика Цен тральной Азии и опасные природные процессы: результаты исследований на количественной основе: Материалы Всероссийского совещания и молодежной школы по современной геодинамике (г. Иркутск, 23–29 сентября 2012 г.). Иркутск: ИЗК СО РАН, 2012. Т. 1. С. 161–164]. Nikolaevsky V.N., 1991. Tectonic waves of the Earth. Priroda 8, 17–23 (in Russian) [Николаевский В.Н. Тектонические волны Земли // Природа. 1991. № 8. С. 17–23]. Nikolaevsky V.N., Ramazanov T.K., 1985. The theory of fast tectonic waves. Prikladnaya Matematika i Mechanika 49 (3), 426–469 (in Russian) [Николаевский В.Н., Рамазанов Т.К. Теория быстрых тектонических волн // Прикладная ма тематика и механика. 1985. Т. 49. № 3. С. 426–469]. Novopashina А.V., San’kov V.А., 2010. Velocities of slow migration of seismic activity in CisBaikal region. Geodynamics & Tectonophysics 1 (2), 197–203. http://dx.doi.org/10.5800/GT2010120015. Sherman S.I., 2009. A Tectonophysical Model of a Seismic Zone: Experience of Development Based on the Example of the Baikal Rift System. Izvestiya, Physics of the Solid Earth 45 (11), 938–951. Sherman S.I., Bornyakov S.A., Buddo V.Yu., 1983. Areas of Dynamic Influence of Faults (Modeling Results). Nauka, No vosibirsk, 112 p. (in Russian) [Шерман С.И., Борняков С.А., Буддо В.Ю. Области динамического влияния разломов (результаты моделирования). Новосибирск: Наука, 1983. 112 с.]. Sherman S.I., Gorbunova E.A., 2008. Wave origin of fault activation in the Central Asia on the basis of seismic monitoring. Physical Mesomechanics 11 (1), 115–122. Sherman S.I., Gorbunova E.A., Mel’nikov M.G., 2012a. Deformation waves as trigger mechanisms of excitation of earth quakes in areas of dynamic influence of faults. 7. ЛИТЕРАТУРА In: Book of abstracts 33rd General Assembly of the European Seismol ogical Commission (GA ESC 2012), 19–24 August 2012, Moscow and Young Seismologist Training Course (YSTC 2012), August 25–30, 2012, Obninsk – Moscow, PH “Poligrafik”, 2012, p. 243–244. Sherman S.I., Lunina O.V., Savitskii V.A., 2005a. The state of stresses and recent lithospheric destruction zones of Asia. In: Problems and Prospects of Mining Sciences. Geomechanics. Mining Institute, SB RAS, Novosibirsk. V. 1. P. 34–39 (in Russian) [Шерман С.И., Лунина О.В., Савитский В.А. Напряженное состояние и зоны современной деструкции литосферы Азии // Проблемы и перспективы развития горных наук. Геомеханика. Новосибирск: Институт горно го дела СО РАН, 2005. Т. 1. С. 34–39]. Sherman S.I., Lysak S.V., Gorbunova E.A., 2012b. A tectonophysical model of the Baikal seismic zone: testing and implica tions for mediumterm earthquake prediction. Russian Geology and Geophysics 53 (4), 392–405. http://dx.doi.org/10. 1016/j.rgg.2012.03.003. Sherman S.I, Sorokin A.P., Savitskii V.A., 2005b. New methods for the classification of seismoactive lithosph based on the index of seismicity. Doklady Earth Sciences 401 (3), 413–416. Sherman S.I., Zlogodukhova О.G., 2011. Seismic belts and zones of the Earth: formalization of notions, positions in the litho sphere, and structural control. Geodynamics & Tectonophysics 2 (1), 1–34. http://dx.doi.org/10.5800/GT2011210031. Ulomov V.I., 1993. Waves of seismogeodynamic activation and longterm earthquake prediction. Fizika Zemli 4, 43–53 (in Russian) [Уломов В.И. Волны сейсмогеодинамической активизации и долгосрочный прогноз землетрясений // Физика Земли. 1993. № 4. С. 43–53]. Vikulin A.V., 2003. The Physics of Wave Seismic Process. KGPI, PetropavlovskKamchatsky, 150 p. (in Russian) [Викулин А.В. Физика волнового сейсмического процесса. ПетропавловскКамчатский: КГПИ, 2003. 150 с.]. Geodynamics & Tectonophysics 2013 Volume 4 Issue 1 Pages 69–81 Мельников Михаил Геннадьевич, аспирант, лаборатория тектонофизики Институт земной коры СО РАН 664033, Иркутск, ул. Лермонтова, 128, Россия email: mg_melnikov@crust.irk.ru Mel’nikov, Mikhail G., Postgraduate Student, Laboratory of Tectonophysics Institute of the Earth’s Crust, Siberian Branch of RAS 128 Lermontov street, Irkutsk 664033, Russia email: mg_melnikov@crust.irk.ru Мельников Михаил Геннадьевич, аспирант, лаборатория тектонофизики Институт земной коры СО РАН 664033, Иркутск, ул. Лермонтова, 128, Россия email: mg_melnikov@crust.irk.ru Mel’nikov, Mikhail G., Postgraduate Student, Laboratory of Tectonophysics Institute of the Earth’s Crust, Siberian Branch of RAS 128 Lermontov street, Irkutsk 664033, Russia email: mg_melnikov@crust.irk.ru 81
https://openalex.org/W4298035420	https://zenodo.org/records/6364482/files/478-480.pdf	English	null	Cu +2, Ni+2 and Co+2 Chelates of N-N'-Hexamethylene-bis (2:5:di-OH, Acetophenone/ Propiophenone/Benzophenone)	Zenodo (CERN European Organization for Nuclear Research)	1,980	cc-by	1,552	(i) N-N'-hexametbylene-bis ( 2 : 5 : di-OH-aceto- pbenone) (i) N-N'-hexametbylene-bis ( 2 : 5 : di-OH-aceto- pbenone) (ii) N-N' -hexamethylene-bis (2 : 5 : di-OH-propio· phenone) (c) Analytical : All the nine chelates were decomposed by perchlorfc acid and the estimation of metal ions were carried out by standard methodsM, The molecular weights determined cryscopically using Camphor as a solvent showed monomeric in nature•. The conductivity of all the chelates was determined in D.M.F. on 'Toshniwal Conductivity Bridge' proving their non-electrolytic uature 11 • The percentage of nitrogen in each case was estimated by Kjeldabls method. The results of several such determinations are present in Table 1 (iii) N-N'-hexamethylene-bis (2: 5: di-OH-benzo- phenone) J. Indian Chem. Soc., Vol. L VJI, May 1980, PPo 478-480 J. Indian Chem. Soc., Vol. L VJI, May 1980, PPo 478-480 Cu +2, Ni+ 2 and Co+ 2 Chelates of N-N~-Hexamethylene-bis (2:5:di-OH, Acetophenone/ Propiophenone/Benzophenone) R. B. PATEL and J. K. VERMA Govt. College, Daman and R. K. SEVAK R. B. PATEL and J. K. VERMA Govt. College, Daman and R. K. SEVAK Govt. College, Daman and R. K. SEVAK Arts, Science and Commerce College, Kholwad and R. K. SEVAK Arts, Science and Commerce College, Kholwad Manuscript received 21 August 1979, accepted 22 December 1979 The Schiff bases N-N'-hexamethylene bis·( 2:S:di-OH, acetophenone, propiophe· none and benzophenone) are used to study their com.Piex forming behaviour with the copper(II), nickel(II) and cobalt(IJ) ions. Elemental, molecular weight determination, conductivity, magnetic moments and electronic spectra were carried out to assign a square plannar structure for copper( II), nickel(II) and cobalt(II) chelates. The I.R. spectra shows the characteristic stretching vibrations of v (M-N) and v (M-0) below 500 cm·t. M U M UCH research work is being done now a days on metal chelates of Schiff bases1 ta), tb>, The present paper reports the study of the complex forming behaviour of the following Schiff bases with the ions copper(II), nickel(II) and cobalt(ll) hours. The cobalt(II) chelates of all the three Schiff bases were prepared by passing hydrogen gas during the preparation of these cbelates. The reddish brown, yellowish brown and reddish black precipitates of copper(II), nlckel(ll) and cobalt(II) respectively formed were filtered and washed with water to remove ex~:ess of metal ions and then with 50% absolute alcohol to remove excess of Schiff bases. They were dried at room temperature. Because of less solubility in chloroform, benzene etc, they were not recrystallised. They are soluble only in D.M.F. hours. The cobalt(II) chelates of all the three Schiff bases were prepared by passing hydrogen gas during the preparation of these cbelates. The reddish brown, yellowish brown and reddish black precipitates of copper(II), nlckel(ll) and cobalt(II) respectively formed were filtered and washed with water to remove ex~:ess of metal ions and then with 50% absolute alcohol to remove excess of Schiff bases. They were dried at room temperature. Because of less solubility in chloroform, benzene etc, they were not recrystallised. They are soluble only in D.M.F. (i) N-N'-hexametbylene-bis ( 2 : 5 : di-OH-aceto- pbenone) Experimental (a) Preparation of Schiff bases : The two moles of 2 : 5 : di-OH-acetophenone, 2 : 5 : di-OH·propio- pbenone and 2: 5: di-OH-benzophenone and one mole of hexamethylene diamine were refluxed in presence of absolute alcohol on water for thirty minutes. The yellow-brown precipitates of all the three Schiff bases formed were filtered and washed with 50% alcohol to remove unreacted ketones and finally they were dried and recrystallised from chloroform giving m.p. 189°, 130" and 169° respectively. The structure of all the three divalent metal chclates can be depicted as shown below : (b) Preparallon of Chelates : The solid Schiff bases and buffered metal ions solutions of copper, nickel and cobalt were refiuxed on water bath for two Where M=Cu+ 2 , Ni+• and Co' 2 and R- -CH,-+H1SB1 -C.H~-+H1SB, -C0H 1r·+H1SB, Where M=Cu+ 2 , Ni+• and Co' 2 and R- -CH,-+H1SB1 -C.H~-+H1SB, -C0H 1r·+H1SB, Where M=Cu+ 2 , Ni+• and Co' 2 and R- -CH,-+H1SB1 -C.H~-+H1SB, -C0H 1r·+H1SB, PATEL, VERMA & SBVAKJ: N•N1·HEXAMBTHYLENB-bi.s' (2:5: DI·OH, ACETOPHENONE ETC. TABLE 1 No. Name of Molecular formula Mol. Weight %of Metal % of Nitrogen Conducti- Magnetic chela!es Exp. Found Bxp. Found Exp. Found vity mhos moment ]. CuSB1 C. 1H 260,N2' - Cu 445.54 440 14.27 2. CuSB;' C 04Ha 00 4N. - Cu 473.54 490 13.43 3. CuSBa c .• HaoO•N• - Cu 569.54 580 11.16 4. NiSB 1 ChH86 0,N:~ - Ni 440.71 425 13.32 s. NiSB;' C04H 100 4 N. - Ni 468.71 480 12.52 6. NiSB, Ca.H800 4 N 8 - Ni 564.71 540 10.40 7. CoSBl c .. H •• o.N. -Co 440.94 435 13.38 8. CoSB. C 04H 800 4N• -Co 468.94 490 12.57 9. CoSB1 CuHa 00,N, -Co 564.94 550 10.45 14.90 6.28 12.89 5.91 11.86 4.91 12.68 6.35 13.10 5.97 10.89 4.96 13.98 6.35 11.82 5.91 10.90 4.96 cm 2/mole 6 45 10 5.78 10 4.70 12 6.55 5 5.80 10 4.72' 5 ·6.18 10 5.76 15 4.82 10 in B.M. (e) Electronic Spectra: The electronic spectra of all the chelates were recorded on 'Spectronic-20'. Th~ solvent used was chloroform. The copper chelates show the following bands 8. KAzuo NAKAMOTO "Infrared Spectra of Inorganic and Co-ordination Compounds'\ John Wiley and Sons, New York, 1963, p. 146, 162, 422. 1. (a) R.. H. HoLM, G. W. EVERETT, JR. and A. CHAKRA• VARTY cited in "Progress in Inorganic Chemistry" Edited by F. A. Cotton Vol. (VII) p. 83 : Inter Science Publication 1966 and references therein. Experimental SHARPE, p. 303, Cambridge University Press, 1968 and references therein. The band at ...... 405 nm could be an intraligand band. The coblat chelates show the following bands 660 nm (E= 300) 510 nm (E=2500) 400 nm (E = 7100) 660 nm (E= 300) 510 nm (E=2500) 400 nm (E = 7100) 2. CUMMING and KEY "Quantitative Analysis", Ed. Robert Alexander Chalmers (Oliver and Boyd Publishers, London) 1956, p. 257. The first two bands at,....,660 nm and,.....,510 nm could be due to the transitions 3. A. I. VoGEL, "Quantitative Inorganic Analysis'' {Longman), 1969, p. ~26. liB10+-9 A111 and sEu+-sA1u liB10+-9 A111 and sEu+-sA1u 4. A. I. VOGEL, A Text Book of Practical Organic Chemistry (including qualitative organic analysis) Third Ed. Longman, 1961 p. 1037. The band at--400 nm could be a characteristic of charge transfer band. 5. A. P. AMES and P. G. SBARS, J Phys. Chern. Ithaca, 1955, 59, 16. (f) I. R. Spectra: The I. R. Spectra reveal the M-N and M-0 stretching vibrations 8 at,....,480 and,...., 420 cm- 1 respectively. The rest of the frequencies fit very well with the depicted structure. 6. W. A. EARNSHAW, Introduction to Magnetio Chemistry (Academic Press, New York), 1968, p. 35. 7. B. N. Fmms, "Introduction to Ligand Fields" (Wiley) 1966,318. Experimental ~eft .\ E 1.80 640 ( 108) 565 ( 230) 390 (5800) 1.84 630 ( 105) 570 ( 172) 390 (5600) 1.78 630 ( 102) 570 ( 170) 385 (5500) dia 590 ( 410) 515 (1050) 405 (4700) dia 590 ( 420) 510 (1040) 410 (4700) dia 585 ( 410) 510 (1060) 410 (4800) 1.89 660 ( 300) 510 (2500) 400 (7100) 1.92 670 ( 290) 510 (2500) 400 (6700) 1,93 670 ( 264) 500 (2880) 390 (7800) TABLE 1 (d) Magnetism : The magnetic susceptibilities of all the chelates were determined on a Gouy balance at room temperature. The copper chelates show one unpaired electron as the magnetic moment f-Ie 11 ='"" 1:8 B.M. on the basis of a square planar structure 6 • 7 • The nickel chelates are found to be diamagnetic having zero unpaired electron on the basis of square planar structure 6 •7 • The cobalt chelates show one unpaired electron as the magnetic moment p 611 =/"'0Jl.9 B.M. on the basis of square planar 6 • 7 structure. The square planar cobalt(II) chelates show higher magnetic moment than the value----1.7 B.M. expected on the basis of a spin only formula p 6 ff = yn(n + 2) B.M. for one unpaired electron due to orbital contribution which is due to mixing 2 A 217 into 2A 117 • The value.....,J.9 B.M. is consistant with a square planar 6 ' 7 structure. 640 nm (e= 108) 565 nm (E = 230) 390 nm ( E = 5800) These may be assigned on an idealised D , 1, symmetry to the following transitions. 640 nm 2 A10-+-2 B 10 565 nm 2Eg-+-2 B10 640 nm 2 A10-+-2 B 10 565 nm 2Eg-+-2 B10 The band at ...... 390 nm is quite intense and seems to be a charge transfer band. The nickel chelates show the following bands 590 nm (E= 410) 515 nm (E=l050) 405 nm (E=4700) (e) Electronic Spectra: The electronic spectra of all the chelates were recorded on 'Spectronic-20'. Th~ solvent used was chloroform. The copper chelates show the following bands The first two bands.-590 nm and--515 nm could be due to the electronic transitic;ms ~B 10 -+- 1 A 10 and ~B,+-tA,a. 479 J, INDIAN CHBM. SOC., VOL, LVII, MAY 1980 (b) B. 0. WEsT, cited in "New Pathways in Inorganic Chemistry'' edited by EAV. Eds. worth, A. G. MADDORK and A. G. References 8. KAzuo NAKAMOTO "Infrared Spectra of Inorganic and Co-ordination Compounds'\ John Wiley and Sons, New York, 1963, p. 146, 162, 422.
https://openalex.org/W3177188254	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/A5E061E95632BE8E66486904CE769192/S2071832221000250a.pdf/div-class-title-the-application-of-international-law-in-the-court-of-justice-of-the-european-union-proportionality-rising-div.pdf	English	null	The Application of International Law in the Court of Justice of the European Union: Proportionality Rising	German law journal	2,021	cc-by	32,724	Abstract Application of international treaty and customary international law at the Court of Justice of the European Union (CJEU) is increasingly recognized by scholars as problematic regarding legal certainty. This Article seeks to illustrate why this is and to propose reform. Through comparing judicial approaches in the appli- cation of international law at the CJEU to its approach in internal case law, it is argued that in the frequent absence of proportionality in external case law the Court has utilized, redeployed, or varied other judicial devices in an effort to retain the discretion which proportionality affords. These are argued to effect legal certainty and established concepts of justice within the EU legal system. Accordingly, it is submitted that proportionality should be transplanted fully and openly to external relations case law and that support for this can be extrapolated from existing literature. Keywords: External relations case law; proportionality; direct effect; legal certainty; international law © The Author(s), 2021. Published by Cambridge University Press on behalf of the German Law Journal. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Dr. Dunbar is currently a senior lecturer at Kingston University’s School of Law, Social and Behavioral Sciences. His research currently concerns the relationship between international and domestic law, debating and testing notions of justice, and legal certainty in domestic courts’ case law. His PhD criticized the current approaches adopted by the Court of Justice of the European Union for applying international law within the EU legal system. The Application of International Law in the Court of Justice of the European Union: Proportionality Rising Rupert Dunbar* Department of Law, Kingston University, Surrey, England *Corresponding author: Rupert.Dunbar@kingston.ac.uk (Received 16 July 2020; accepted 16 November 2020) (Received 16 July 2020; accepted 16 November 2020) German Law Journal (2021), 22, pp. 557–592 doi:10.1017/glj.2021.25 German Law Journal (2021), 22, pp. 557–592 doi:10.1017/glj.2021.25 2TAKIS TRIDIMAS, GENERAL PRINCIPLES OF EU LAW 136–38 (2d ed. 2006). See also Opinion of Advocate General Jacobs at 1533, ECJ, Case C-120/94, Comm’n v. Greece, ECLI:EU:C:1995:109 (Apr. 6, 1995), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-120/94. p pp y g g y 1GUNNAR BECK, THE LEGAL REASONING OF THE COURT OF JUSTICE OF THE EU 347 (2012). https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 3For example, free movement, fundamental rights, environmental protection, et cetera. Dr. Dunbar is currently a senior lecturer at Kingston University’s School of Law, Social and Behavioral Sciences. Hi research currently concerns the relationship between international and domestic law, debating and testing notions of justice and legal certainty in domestic courts’ case law. His PhD criticized the current approaches adopted by the Court of Justice o the European Union for applying international law within the EU legal system. 1GUNNAR BECK, THE LEGAL REASONING OF THE COURT OF JUSTICE OF THE EU 347 (2012). 2TAKIS TRIDIMAS, GENERAL PRINCIPLES OF EU LAW 136–38 (2d ed. 2006). See also Opinion of Advocate General Jacobs a 1533, ECJ, Case C-120/94, Comm’n v. Greece, ECLI:EU:C:1995:109 (Apr. 6, 1995), http://curia.europa.eu/juris/liste.jsf language=en&num=C-120/94. 3For example, free movement, fundamental rights, environmental protection, et cetera. g g For example, free movement, fundamental rights, environmental protection, et cetera. and legal certainty in domestic courts case law. His PhD criticized the current approaches adopted by the Court of Justice of the European Union for applying international law within the EU legal system. 1GUNNAR BECK, THE LEGAL REASONING OF THE COURT OF JUSTICE OF THE EU 347 (2012). 2TAKIS TRIDIMAS, GENERAL PRINCIPLES OF EU LAW 136–38 (2d ed. 2006). See also Opinion of Advocate General Jacobs at 1533, ECJ, Case C-120/94, Comm’n v. Greece, ECLI:EU:C:1995:109 (Apr. 6, 1995), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-120/94. 3F l f f d l i h i l i A. Introduction Proportionality is the predominant tool for resolving cases in EU law.1 Areas in which its role is excluded or limited are few,2 however, case law applying international law—external relations case law—has emerged as one. Proportionality ensures that a given action does not go beyond what is necessary and does not infringe unacceptably on other rights or legitimate aims. Its impact on the EU legal system is notable; norms of EU law emerge as non-hierarchical, with the potential for each to prove most important in the unique facts of a given case.3 This enables the Court to man- age and develop norms within EU law and provides it significant discretion too. The large absence of proportionality in external relations case law means that a significant mechanism for resolving disputes is missing. The result has been a strict hierarchy in which sup- posedly mechanical rules govern when international law can be relied upon. The reality is very different though; scholars have become increasingly vocal in exposing the Court’s discretion and https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar Rupert Dunbar 558 activism in external relations case law.4 But discretion here, unlike proportionality in internal case law, is fragmented and manifests itself across inter alia rules of reliance, especially the direct effect test, opportunistic approaches to interpretation, and recourse to the concept of autonomy. This significantly influences legal certainty, which the Court has called a “fundamental prin- ciple of Community law.”5 It recognizes that legal certainty, “requires, in particular, that rules should be clear and precise, so that individuals may ascertain unequivocally what their rights and obligations are and may take steps accordingly.”6 We will see that external case law falls far short of this aim, affecting participants. This is particularly problematic given that legal certainty and justice are all that law ought to achieve.7 A significant shortcoming in one is concerning, as they are, “a couple that cannot be ignored in a functioning and legitimate system of Law.”8 However, our analysis will also reveal issues in exter- nal case law concerning justice, by which is meant, achieving the right outcome in a given case. Substantive, or “thick,” justice is contestable, but “thin” justice is widely accepted. 4See MARIO MENDEZ, THE LEGAL EFFECTS OF EU AGREEMENTS: MAXIMALIST TREATY ENFORCEMENT AND JUDICIAL AVOIDANCE TECHNIQUES (2013); Mario Mendez, The Legal Effect of Community Agreements: Maximalist Treaty Enforcement and Judicial Avoidance Techniques, 21 EUR. J. INT’L L. 83 (2010). 4See MARIO MENDEZ, THE LEGAL EFFECTS OF EU AGREEMENTS: MAXIMALIST TREATY ENFORCEMENT AND JUDICIAL AVOIDANCE TECHNIQUES (2013); Mario Mendez, The Legal Effect of Community Agreements: Maximalist Treaty Enforcement and Judicial Avoidance Techniques, 21 EUR. J. INT’L L. 83 (2010). 5See ECJ, Case C-308/06, Intertanko and Others, ECLI:EU:C:2008:312 (June 3, 2008), para. 69, http://curia.europa.eu/juris/ liste.jsf?language=en&num=C-308/06. See also ECJ, Case C-344/04, IATA and ELFAA, ECLI:EU:C:2006:10 (Jan. 10, 2006), para 68, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-344/04. 6ECJ, Case C-366/10, Air Transport Association of America and Others, ECLI:EU:C:2011:864 (Dec. 21, 2011), para. 68, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-366/10. 7Joseph Raz, Legal Principles and the Limits of Law, 81 YALE L.J. 823, 841 (1972); LORD DENNING, THE DISCIPLINE OF LAW 293 (1979); JÜRGEN HABERMAS, BETWEEN FACTS AND NORMS 199 (William Rehg trans., 1997); Stefano Bertea, Certainty, Reasonableness and Argumentation in Law, 18 ARGUMENTATION 465, 475 (2004); Robert Alexy, The Dual Nature of Law, 23 RATIO JURIS 167 (2010); WILLIAM TWINING, KARL LLEWELLYN AND THE REALIST MOVEMENT 157 (2d ed. 2012); and BECK, supra note 1, at 274. 8Bertea, supra note 7, at 475. 9ARISTOTLE, THE NICOMACHEAN ETHICS 80–88 (David Ross trans., Oxford University Press 2009). 10See, e.g., C.H. PERELMAN, JUSTICE 21–24 (1967); Kenneth I. Winston, On Treating Like Case Alike, 62 CALIF. L. REV. 1, 22 (1974); R.M. HARE, MORAL THINKING: ITS LEVELS, METHOD AND POINT 157 (1981); H.L.A. HART, THE CONCEPT OF LAW 159 (3d ed. 2012); GERARD CONWAY, THE LIMITS OF LEGAL REASONING AND THE EUROPEAN COURT OF JUSTICE 244 (2012); and SIONAIDH DOUGLAS-SCOTT, LAW AFTER MODERNITY 185 (2013). 11See, e.g., ECJ, Case C-459/03, Commission v Ireland, ECLI:EU:C:2006:345 (May 30, 2006), para 123, http://curia.europa. eu/juris/liste.jsf?language=en&num=C-459/03. See also Joined Cases C–402/05 P and C–415/05 P, Kadi & Al Barakaat Int’l Found. v. Council and Comm’n, ECLI:EU:C:2008:461 (Sep. 3, 2008), para. 282, https://curia.europa.eu/juris/liste.jsf?oqp= &for=&mat=or&jge=&td=%3BALL&jur=C%2CT%2CF&num=402%252F05&page=1&dates=&pcs=Oor&lg=&pro=&nat= or&cit=none%252CC%252CCJ%252CR%252C2008E%252C%252C%252C%252C%252C%252C%252C%252C%252C%252Ctrue %252Cfalse%252Cfalse&language=en&avg=&cid=12821209. 12ECJ, Case C-181/73, Haegeman v. Belgium, ECLI:EU:C:1974:41 (Apr. 30, 1974), para. 5, http://curia.europa.eu/juris/liste. jsf?language=en&num=C-181/73. 13Tor-Inge Harbo, The Function of the Proportionality Principle in EU Law, 16 EUR. L.J. 158, 159 (2010) (citing NICHOLAS A. Introduction Thin justice can be derived from Aristotle9 and amounts to the assertion that “like cases should be treated alike and unalike cases unalike to the extent of their unalikeness.”10 Through comparing internal and external case law we will be well-placed to see that fundamental aspects of the EU legal system are modified in external case law. This is so despite the Court stating that “an international agreement cannot affect the allocation of responsibilities defined in the Treaties”11 and asserting that international law is “an integral part of [EU] law.”12 It will be argued that fundamental changes largely flow as a result of the large absence of proportionality in external case law. The extent of “unalikeness”—which would justify divergence from internal case law—and the consequent exclusion of proportionality will be challenged, particularly given that general principles of EU law are anticipated to have relevance “in a series of infinite applications of the law,”13 and that proportionality and other general principles have on occasion been applied in external case law. 7Joseph Raz, Legal Principles and the Limits of Law, 81 YALE L.J. 823, 841 (1972); LORD DENNING, THE DISCIPLINE OF LAW 293 (1979); JÜRGEN HABERMAS, BETWEEN FACTS AND NORMS 199 (William Rehg trans., 1997); Stefano Bertea, Certainty, Reasonableness and Argumentation in Law, 18 ARGUMENTATION 465, 475 (2004); Robert Alexy, The Dual Nature of Law, 23 RATIO JURIS 167 (2010); WILLIAM TWINING, KARL LLEWELLYN AND THE REALIST MOVEMENT 157 (2d ed. 2012); and BECK, supra note 1, at 274. TLE, THE NICOMACHEAN ETHICS 80–88 (David Ross trans., Oxford University Press 2009). y 10See, e.g., C.H. PERELMAN, JUSTICE 21–24 (1967); Kenneth I. Winston, On Treating Like Case Alike, 62 CALIF. L. REV. 1, 22 (1974); R.M. HARE, MORAL THINKING: ITS LEVELS, METHOD AND POINT 157 (1981); H.L.A. HART, THE CONCEPT OF LAW 159 (3d ed. 2012); GERARD CONWAY, THE LIMITS OF LEGAL REASONING AND THE EUROPEAN COURT OF JUSTICE 244 (2012); and SIONAIDH DOUGLAS-SCOTT, LAW AFTER MODERNITY 185 (2013). 11See, e.g., ECJ, Case C-459/03, Commission v Ireland, ECLI:EU:C:2006:345 (May 30, 2006), para 123, http://curia.europa. eu/juris/liste.jsf?language=en&num=C-459/03. See also Joined Cases C–402/05 P and C–415/05 P, Kadi & Al Barakaat Int’l Found. v. Council and Comm’n, ECLI:EU:C:2008:461 (Sep. 3, 2008), para. 282, https://curia.europa.eu/juris/liste.jsf?oqp= &for=&mat=or&jge=&td=%3BALL&jur=C%2CT%2CF&num=402%252F05&page=1&dates=&pcs=Oor&lg=&pro=&nat= or&cit=none%252CC%252CCJ%252CR%252C2008E%252C%252C%252C%252C%252C%252C%252C%252C%252C%252Ctrue %252Cfalse%252Cfalse&language=en&avg=&cid=12821209. g g g C-181/73, Haegeman v. Belgium, ECLI:EU:C:1974:41 (Apr. 30, 1974), para. 5, http://curia.europa.eu/juris/liste. en&num=C-181/73. 13Tor-Inge Harbo, The Function of the Proportionality Principle in EU Law, 16 EUR. L.J. 7Joseph Raz, Legal Principles and the Limits of Law, 81 YALE L.J. 823, 841 (1972); LORD DENNING, THE DISCIPLINE OF LAW 293 (1979); JÜRGEN HABERMAS, BETWEEN FACTS AND NORMS 199 (William Rehg trans., 1997); Stefano Bertea, Certainty, Reasonableness and Argumentation in Law, 18 ARGUMENTATION 465, 475 (2004); Robert Alexy, The Dual Nature of Law, 23 RATIO JURIS 167 (2010); WILLIAM TWINING, KARL LLEWELLYN AND THE REALIST MOVEMENT 157 (2d ed. 2012); and BECK, supra note 1, at 274. 5See ECJ, Case C-308/06, Intertanko and Others, ECLI:EU:C:2008:312 (June 3, 2008), para. 69, http://curia.europa.eu/juris/ liste.jsf?language=en&num=C-308/06. See also ECJ, Case C-344/04, IATA and ELFAA, ECLI:EU:C:2006:10 (Jan. 10, 2006), para 68, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-344/04. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 14Robert Alexy, The Dual Nature of Law, 23 RATIO JURIS 167 (2010). See also Joseph Raz, Legal Principles and the Limits of Law, 81 YALE L.J. 823, 841 (1972); DENNING, supra note 7, at 293; HABERMAS, supra note 7, at 199; Bertea, supra note 7, at 475; TWINING, supra note 7, at 157; and BECK, supra note 1, at 274. 15RAAS HOLDGAARD, EXTERNAL RELATIONS LAW OF THE EUROPEAN COMMUNITY: LEGAL REASONING AND LEGAL DISCOURSES 235 (2008). 16Achilles Skordas, Völkerrechtsfreundlichkeit as Comity and the Disquiet of Neoformalism: A Response to Jan Klabbers, in EUROPEAN FOREIGN POLICY: LEGAL AND POLITICAL PERSPECTIVES 116 (Panos Koutrakos ed., 2011). 17Jed Odermatt, The Court of Justice of the European Union: International or Domestic Court?, 3 Cambridge J. Int’l & Comp. L. 696, 702 (2014). 18Judicaël Etienne, Loyalty Towards International Law as a Constitutional Principle of EU Law? 14 (Jean Monnet Working Paper Series, 03/2011, 2011), http://jeanmonnetprogram.org/wp-content/uploads/2014/12/110301.pdf (last accessed Apr. 30, 2021). 19Id. at 39. B. The Rise of Proportionality in External Relations Case Law Scholarship? I. The Acknowledged Need for Reform of External Relations Case Law Scholarship can be drawn upon to confirm two primary observations regarding external relations case law: First, legal certainty is problematic, and second, the case law evidences some balancing of EU law/interests against international law. The former is important, as the “dual nature” of law suggests that legal certainty, which is defined as being able to anticipate an outcome, and justice, which is defined as achieving the right outcome in a given case, are all law ought to achieve.14 In lieu of an authoritative balance between these aims one would need to be cautious in proposing reforms. However, due to shortcomings in legal certainty, scholars have been liberated in propos- ing modifications to the Court’s approach, with most favoring a continuance of the balance between EU and international law already present, but in modified form. Surprisingly though, only a few scholars mention proportionality expressly, and those do so only briefly. A. Introduction 158, 159 (2010) (cit EMILIOU, THE PRINCIPLE OF PROPORTIONALITY IN EU LAW: A COMPARATIVE STUDY 115 (1996)). https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 559 German Law Journal The need for the Court to address shortcomings in this area is reflected in the mass of proposals from scholars for reform. However, proportionality has received little sustained attention in literature concerning external relations case law. It is submitted, though, that scholars’ proposals for reform—somewhat like the Court’s approaches in external relations case law—often converge towards proportionality without fully arriving there. Accordingly, this Article offers an initial exploration of the possibility for extension of proportionality, in its full form, to external relations case law. It seems this could both improve legal certainty and form a more just basis for the appli- cation of international law in the CJEU moving forward. This Article is structured as follows: First, a lack of legal certainty and the necessity of balancing EU law and interests against international law in external relations case law will be highlighted in prominent scholarship, with proportionality identified as warranting closer attention; second, the context and prominence of the application of proportionality in internal case law will be analyzed; third, the absence of proportionality in external relations case law will be analyzed and reflected upon through consideration of the judicial tools which have been deployed to facilitate discretion in its stead. This Article concludes with the pragmatic call for proportionality to be deployed more openly, frequently and fully in external relations case law. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press LDGAARD, EXTERNAL RELATIONS LAW OF THE EUROPEAN COMMUNITY: LEGAL REASONING AND LEGAL 35 (2008). II. Concerns Regarding Legal Certainty in External Relations Case Law Concerning legal certainty in the case law, Holdgaard speaks of a “manifest lack of clarity and coherence,”15 Skordas of ‘‘counter-systemic commotion,”16 and Odermatt encourages “a more consistent and principled approach when dealing with international law questions.”17 Etienne notes “[u]ncertainty” concerning the application of customary international law and direct effect of international agreements,18 and then calls on the Court generally to say “what it is actually doing and how it is doing [it].”19 Mendez criticizes the “curt and formalistic” reasoning in Intrertanko and draws attention to the fact that, concerning the direct effect test, “we are entitled Concerning legal certainty in the case law, Holdgaard speaks of a “manifest lack of clarity and coherence,”15 Skordas of ‘‘counter-systemic commotion,”16 and Odermatt encourages “a more https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 560 to expect a credible attempt at reasoned justification for why a particular Agreement will be deprived of its most potent internal legal effects.”20 Intertanko is also highlighted by Koutrakos as evidence that the process of direct effect “may be unpredictable,”21 whilst Air Transport is said to be “staggering in its brevity and lack of clarity.”22 p gg g y y Of course, criticisms of legal certainty will always be present, but they are particularly acute concerning the application of international law in the CJEU. Our analysis of external relations case law will confirm concerns of scholars regarding legal certainty; however, the absence of pro- portionality will be explained as the reason for this to a greater extent than has previously been undertaken—albeit most scholars favor some balance. III. Disposing of Two Possible Arguments in Favor of the Status Quo With legal certainty problematic, two possible avenues remain by which reform in external rela- tions case law may not be desirable: First, were this construct to be required by international law itself, and second, were the case law as it currently stands to benefit the EU—for instance, through a continuing projection of the “good citizen Europe” brand.23 First, it is submitted that international law does not generally dictate the effects which it shall have in domestic legal systems.24 The Danzig 25 case of the Permanent Court of International Justice provides a notable exception, but one which was ultimately based on “the very object of [that] international agreement, according to the intention of the contracting Parties,” and which the Court recognized went against “a well-established principle of international law . II. Concerns Regarding Legal Certainty in External Relations Case Law . . [that] an international agreement, cannot, as such, create direct rights and obligations for private individ- uals.”26 The narrow exception has not been returned to, indicating its exceptional nature.27 The more recent LaGrand28 and Avena29 cases arguably indicate the limited relevance of Danzig mov- ing forward. Both concerned Article 36 of the Vienna Convention on Consular Relations, which allows consulates access to detainees and which the US was accused of breaching. But even in the face of such specific links to individuals it has been recognized the International Court of Justice did “not stipulate or imply, that the US courts are required to give direct effect to the obligation.”30 It is thus, widely acknowledged by scholars that there is not an obligation to apply international law domestically.31 This has been suggested to apply equally to UN Security Council Resolutions, arguably the most binding of international legal instruments and so, one might think, more prone to direct effect.32 Of course, the scope for maneuver provided by international law has manifested 24On the interesting and seemingly expanding practice of EU agreements expressing when they will not have direct effect, see Narine Ghazaryan, Who are the ‘Gatekeepers’?: In Continuation of the Debate on the Direct Applicability and Direct Effect of EU International Agreements, 37 Y.B. EUR. L. 27 (2018). 25Jurisdiction of the Courts of Danzig (Pecuniary Claims of Danzig Railway Officials who have Passed into the Polish Service, against the Polish Railways Administration), Advisory Opinion, 1928 P.C.I.J. (ser. B) No. 15. 26 É NOLLKAEMPER, NATIONAL COURTS AND THE INTERNATIONAL RULE OF LAW 125 (2011). 8LaGrand Case (Ger. v. U.S.), Judgment, 2001 I.C.J. 466 (June 27). ena and Other Mexican Nationals (Mex. v. U.S.), Judgment, 2004 I.C.J. 12 (March 31). 31Yuval Shany, Toward a General Margin of Appreciation Doctrine in International Law?, 16 EUR. J. INT’L L. 907 (2006); Jean d'Aspremont & Frédéric Dopagne, Kadi: The ECJ’s Reminder of the Elementary Divide Between Legal Orders, 5 INT’L ORGS. L. REV. 371 (2008); NOLLKAEMPER, supra note 27, at 11, 120, 299–304; André Nollkaemper, The Duality of Direct Effect, 25 EUR. J. INT’L L. 105, 122 (2014). 20MENDEZ, supra note 4, at 319. 21PANOS KOUTRAKOS, EU INTERNATIONAL RELATIONS LAW 266 (2d ed. 2015). 22Id. at 317. 23For use of this term see Tim Dunne, Good Citizen Europe, 84 International Affairs 13 (2008). 24On the interesting and seemingly expanding practice of EU agreements expressing when they will not have direct effect, see Narine Ghazaryan, Who are the ‘Gatekeepers’?: In Continuation of the Debate on the Direct Applicability and Direct Effect of EU International Agreements, 37 Y.B. EUR. L. 27 (2018). 25Jurisdiction of the Courts of Danzig (Pecuniary Claims of Danzig Railway Officials who have Passed into the Polish Service, against the Polish Railways Administration), Advisory Opinion, 1928 P.C.I.J. (ser. B) No. 15. 26Id. at 17–18. 27ANDRÉ NOLLKAEMPER, NATIONAL COURTS AND THE INTERNATIONAL RULE OF LAW 125 (2011). 28LaGrand Case (Ger. v. U.S.), Judgment, 2001 I.C.J. 466 (June 27). 29Avena and Other Mexican Nationals (Mex. v. U.S.), Judgment, 2004 I.C.J. 12 (March 31). 30NOLLKAEMPER, supra note 27, at 11. 31Yuval Shany, Toward a General Margin of Appreciation Doctrine in International Law?, 16 EUR. J. INT’L L. 907 (2006); Jean d'Aspremont & Frédéric Dopagne, Kadi: The ECJ’s Reminder of the Elementary Divide Between Legal Orders, 5 INT’L ORGS. L. REV. 371 (2008); NOLLKAEMPER, supra note 27, at 11, 120, 299–304; André Nollkaemper, The Duality of Direct Effect, 25 EUR. J. INT’L L. 105, 122 (2014). 32CHRISTINA ECKES, EU-COUNTER TERRORIST POLICIES AND FUNDAMENTAL RIGHTS 233 (2009); Matthias Forteau, The Role of the International Rules of Interpretation for the Determination of Direct Effect of International Agreements, in THE INTERPRETATION OF INTERNATIONAL LAW BY DOMESTIC COURTS: UNITY, DIVERSITY AND CONVERGENCE 99 (Helmut 23For use of this term see Tim Dunne, Good Citizen Europe, 84 International Affairs 13 (2008). 27ANDRÉ NOLLKAEMPER, NATIONAL COURTS AND THE INTERNATIONAL RULE OF LAW 125 (2011). 29Avena and Other Mexican Nationals (Mex. v. U.S.), Judgment, 2004 I.C.J. 12 (March 31). 30 28LaGrand Case (Ger. v. U.S.), Judgment, 2001 I.C.J. 466 (June 27). https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 21PANOS KOUTRAKOS, EU INTERNATIONAL RELATIONS LAW 266 (2d ed. 2015). 22Id. at 317. 20MENDEZ, supra note 4, at 319. 24On the interesting and seemingly expanding practice of EU agreements expressing when they will not have direct effect, see Narine Ghazaryan, Who are the ‘Gatekeepers’?: In Continuation of the Debate on the Direct Applicability and Direct Effect of EU International Agreements, 37 Y.B. EUR. L. 27 (2018). 25Jurisdiction of the Courts of Danzig (Pecuniary Claims of Danzig Railway Officials who have Passed into the Polish Service, against the Polish Railways Administration), Advisory Opinion, 1928 P.C.I.J. (ser. B) No. 15. 26Id. at 17–18. II. Concerns Regarding Legal Certainty in External Relations Case Law ff 32CHRISTINA ECKES, EU-COUNTER TERRORIST POLICIES AND FUNDAMENTAL RIGHTS 233 (2009); Matthias Forteau, The Role of the International Rules of Interpretation for the Determination of Direct Effect of International Agreements, in THE INTERPRETATION OF INTERNATIONAL LAW BY DOMESTIC COURTS: UNITY, DIVERSITY AND CONVERGENCE 99 (Helmut 32CHRISTINA ECKES, EU-COUNTER TERRORIST POLICIES AND FUNDAMENTAL RIGHTS 233 (2009); Matthias Forteau, The Role of the International Rules of Interpretation for the Determination of Direct Effect of International Agreements, in THE INTERPRETATION OF INTERNATIONAL LAW BY DOMESTIC COURTS: UNITY, DIVERSITY AND CONVERGENCE 99 (Helmut 561 German Law Journal itself in the EU through its own tests of direct effect and its utilization of the concept of an autono- mous legal system which, “cannot be prejudiced by an international agreement.”33Accordingly, discretion is both afforded and already taken, as we will see immediately below, and when con- sidering external relations case law more fully, but direct effect and autonomy have not proven satisfactory utilizations of it. Second, accepting that the perception other nations and parties may have is important to international standing, including the capacity to function effectively on the international plane, the Court’s case law still falls short.34 Ultimately, remarkably few scholars are supportive of the current external relations case law of the CJEU without equivocation.35 For instance, the 2008 cases, Intertanko and Kadi, have been described as an annus horribilis for the Court, showcasing the declining perception of its open approach towards international law.36 Such views have even become mainstream; Goldsmith and Posner’s brief—and very negative—appraisal of the Court’s case law appeared in the Wall Street Journal.37 pp With external relations case law at a remarkably low ebb concerning its utility in raising the esteem in which the EU is held, problems regarding legal certainty, and international law provid- ing discretion for domestic courts in applying international law, it is not surprising that many suggestions for reform have been forthcoming.38 These can now be considered. IV. Acknowledgement of the Need for Balance Between Protection of EU Law and Respect for International Law in the Interests of Justice 34For discussion see LOUIS HENKIN, HOW NATIONS BEHAVE (2d ed. 1968); Harold Hongju Koh, Why Do Nations Obey International Law?, 106 YALE L.J. 2599 (1997); Andrew T. Guzman, A Compliance-Based Theory of International Law, 90 CALIF. L. REV. 1823 (2002); and JACK L. GOLDSMITH & ERIK A. POSNER, THE LIMITS OF INTERNATIONAL LAW (2007). 35But see Eileen Denza, Placing the European Union in International Context: Legitimacy of the Case Law, in JUDGING EUROPE’S JUDGES (Maurice Adams et al. eds., 2013). Pieter Jan Kuijper, “It Shall Contribute to : : : the Strict Observance and Development of International Law : : : ”, in THE COURT OF JUSTICE AND THE CONSTRUCTION OF EUROPE: ANALYSES AND PERSPECTIVES ON SIXTY YEARS OF CASE-LAW 611 (2013) (concluding that “[a] lot of critique has been lavished on the Court . . . much of this critique seems exaggerated”). However, Jan Kuijper’s contribution was to the Court’s sixth anni- versary publication of essays and one ought not to be too rude to one’s host. And he did point out that “the Court perhaps needs to improve its treatment of binding decisions of (other) international courts and tribunals.” Id. at 611. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 33Kadi and Al Barakaat International Foundation v. Council and Commission, Joined Cases C–402/05 P P 316. Phillip Aust & Georg Nolte eds., 2016). Contra Erika de Wet & Jure Vidmar, Conclusions, in HIERARCHY IN INTERNATIONAL LAW: THE PLACE OF HUMAN RIGHTS 304 (Erika de Wet & Jure Vidmar eds., 2012). Phillip Aust & Georg Nolte eds., 2016). Contra Erika de Wet & Jure Vidmar, Conclusions, in HIERARCHY IN INTERNATIONAL LAW: THE PLACE OF HUMAN RIGHTS 304 (Erika de Wet & Jure Vidmar eds., 2012). 33Kadi and Al Barakaat International Foundation v. Council and Commission, Joined Cases C–402/05 P and C–415/05 P 316. 34For discussion see LOUIS HENKIN, HOW NATIONS BEHAVE (2d ed. 1968); Harold Hongju Koh, Why Do Nations Obey International Law?, 106 YALE L.J. 2599 (1997); Andrew T. Guzman, A Compliance-Based Theory of International Law, 90 CALIF. L. REV. 1823 (2002); and JACK L. GOLDSMITH & ERIK A. POSNER, THE LIMITS OF INTERNATIONAL LAW (2007). 35But see Eileen Denza, Placing the European Union in International Context: Legitimacy of the Case Law, in JUDGING EUROPE’S JUDGES (Maurice Adams et al. eds., 2013). Pieter Jan Kuijper, “It Shall Contribute to : : : the Strict Observance and Development of International Law : : : ”, in THE COURT OF JUSTICE AND THE CONSTRUCTION OF EUROPE: ANALYSES AND PERSPECTIVES ON SIXTY YEARS OF CASE-LAW 611 (2013) (concluding that “[a] lot of critique has been lavished on the Court . . . much of this critique seems exaggerated”). However, Jan Kuijper’s contribution was to the Court’s sixth anni- versary publication of essays and one ought not to be too rude to one’s host. And he did point out that “the Court perhaps needs to improve its treatment of binding decisions of (other) international courts and tribunals.” Id. at 611. 36MATTHIAS KOTTMANN, INTROVERTIERTE RECHTSGEMEINSCHAFT 233 (2014). 37Jack Goldsmith & Eric Posner, Does Europe Believe in International Law? Based on the record it has no grounds to criticize the U.S., WALL STREET JOURNAL (Nov. 25, 2008), http://www.wsj.com/articles/SB122757164701554711 (last accessed Apr. 30, p g y g p Phillip Aust & Georg Nolte eds., 2016). Contra Erika de Wet & Jure Vidmar, Conclusions, in HIERARCHY IN INTERNATIONAL LAW: THE PLACE OF HUMAN RIGHTS 304 (Erika de Wet & Jure Vidmar eds., 2012). 33Kadi and Al Barakaat International Foundation v. Council and Commission, Joined Cases C–402/05 P and C–415/05 P 316. 34For discussion see LOUIS HENKIN, HOW NATIONS BEHAVE (2d ed. 1968); Harold Hongju Koh, Why Do Nations Obey International Law?, 106 YALE L.J. 2599 (1997); Andrew T. Guzman, A Compliance-Based Theory of International Law, 90 CALIF. L. REV. 1823 (2002); and JACK L. GOLDSMITH & ERIK A. POSNER, THE LIMITS OF INTERNATIONAL LAW (2007). 35But see Eileen Denza, Placing the European Union in International Context: Legitimacy of the Case Law, in JUDGING EUROPE’S JUDGES (Maurice Adams et al. eds., 2013). Pieter Jan Kuijper, “It Shall Contribute to : : : the Strict Observance and Development of International Law : : : ”, in THE COURT OF JUSTICE AND THE CONSTRUCTION OF EUROPE: ANALYSES AND PERSPECTIVES ON SIXTY YEARS OF CASE-LAW 611 (2013) (concluding that “[a] lot of critique has been lavished on the Court . . . much of this critique seems exaggerated”). However, Jan Kuijper’s contribution was to the Court’s sixth anni- versary publication of essays and one ought not to be too rude to one’s host. And he did point out that “the Court perhaps needs to improve its treatment of binding decisions of (other) international courts and tribunals.” Id. at 611. 36MATTHIAS KOTTMANN, INTROVERTIERTE RECHTSGEMEINSCHAFT 233 (2014). 37Jack Goldsmith & Eric Posner, Does Europe Believe in International Law? Based on the record it has no grounds to criticize the U.S., WALL STREET JOURNAL (Nov. 25, 2008), http://www.wsj.com/articles/SB122757164701554711 (last accessed Apr. 30, 2021). 38On treatment of the CJEU as a domestic court, see Piet Eeckhout, Community Terrorism Listings, Fundamental Rights, and UN Security Council Resolutions: In Search of the Right Fit, 3 EUR. CONST. L. REV. 183, 196 (2007); Daniel Halberstam, Local, Global and Plural Constitutionalism: Europe Meets the World, in THE WORLDS OF EUROPEAN CONSTITUTIONALISM 198 (Gráinne de Búrca & J.H.H. Weiler eds., 2012); Allen Rosas, International Responsibility of the EU and the European Court of Justice, in INTERNATIONAL RESPONSIBILITY OF THE EUROPEAN UNION: EUROPEAN AND INTERNATIONAL PERSPECTIVES 159 (Malcolm Evans & Panos Koutrakos eds., 2013); Helmut Philipp Aust, Alejandro Rodiles & Peter Staubach, Unity or Uniformity? Domestic Courts and Treaty Interpretation, 27 Leiden J. Int’l L. 75, 100 (2014). Phillip Aust & Georg Nolte eds., 2016). Contra Erika de Wet & Jure Vidmar, Conclusions, in HIERARCHY IN INTERNATIONAL LAW: THE PLACE OF HUMAN RIGHTS 304 (Erika de Wet & Jure Vidmar eds., 2012). 33Kadi and Al Barakaat International Foundation v. Council and Commission, Joined Cases C–402/05 P and C–415/05 P 316. and Al Barakaat International Foundation v. Council and Commission, Joined Cases C–402/05 P and C–415 Phillip Aust & Georg Nolte eds., 2016). Contra Erika de Wet & Jure Vidmar, Conclusions, in HIERARCHY IN INTERNATIONAL LAW: THE PLACE OF HUMAN RIGHTS 304 (Erika de Wet & Jure Vidmar eds., 2012). 33Kadi and Al Barakaat International Foundation v. Council and Commission, Joined Cases C–402/05 P and C–415/05 p g 36MATTHIAS KOTTMANN, INTROVERTIERTE RECHTSGEMEINSCHAFT 233 (2014). LAW: THE PLACE OF HUMAN RIGHTS 304 (Erika de Wet & Jure Vidmar eds., 2012). 33Kadi and Al Barakaat International Foundation v. Council and Commission, Joined Cases C–402/05 P and C–415/05 P 316. IV. Acknowledgement of the Need for Balance Between Protection of EU Law and Respect for International Law in the Interests of Justice In their analyses of external relations case law, scholars tend to arrive at an abrogated position between respecting international law and protecting/promoting EU law/interests. This is achieved either through recognizing and endorsing elements of balance within current case law or propos- ing such a balance more generally. Accordingly, it has been noted that “the Court case-law reflects the balanced relationship between international law and EU law,”39 “[t]he Court is still trying to find a balance between protecting the autonomy of its own legal order and the openness towards international law that 37Jack Goldsmith & Eric Posner, Does Europe Believe in International Law? Based on the record it has no grounds to criticize the U.S., WALL STREET JOURNAL (Nov. 25, 2008), http://www.wsj.com/articles/SB122757164701554711 (last accessed Apr. 30, 2021). 38On treatment of the CJEU as a domestic court, see Piet Eeckhout, Community Terrorism Listings, Fundamental Rights, and UN Security Council Resolutions: In Search of the Right Fit, 3 EUR. CONST. L. REV. 183, 196 (2007); Daniel Halberstam, Local, Global and Plural Constitutionalism: Europe Meets the World, in THE WORLDS OF EUROPEAN CONSTITUTIONALISM 198 (Gráinne de Búrca & J.H.H. Weiler eds., 2012); Allen Rosas, International Responsibility of the EU and the European Court of Justice, in INTERNATIONAL RESPONSIBILITY OF THE EUROPEAN UNION: EUROPEAN AND INTERNATIONAL PERSPECTIVES 159 (Malcolm Evans & Panos Koutrakos eds., 2013); Helmut Philipp Aust, Alejandro Rodiles & Peter Staubach, Unity or Uniformity? Domestic Courts and Treaty Interpretation, 27 Leiden J. Int’l L. 75, 100 (2014). 39Etienne, supra note 18, at 2. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 562 is enshrined in the EU Treaties,”40 and that within the case law there is “commitment to comply with international law and the defence of EU autonomy.”41 In accommodating this balance moving forward, Cannizzarro suggests neo-monism as a con- cept which can “mitigate some of the consequences deriving from the supremacy of international law.”42 de Búrca suggests a “soft constitutionalist approach . . . which does not insist on a clear hierarchy of rules but rather on commonly negotiated and shared principles for addressing conflict.”43 Klabbers adopts the term Völkerrechtsfreundlichkeit to mean the EU respecting international law on the international plane and to “apply it in the internal legal order if and when appropriate.”44 In the same collection of essays Skordas responds to Klabbers. 40Odermatt, supra note 17, at 718. 41Francesca Martines, Direct Effect of International Agreements of the European Union, 25 EUR. J. INT’L L. 129, 144 (2014). 42Id. at 52. 43Gráinne de Búrca, The European Court of Justice and the International Legal Order After Kadi, 51 HARV. L. REV. 1, 39 (2010). 44Jan Klabbers, Völkerrechtsfreundlichkeit? International law and the Union Legal Order, in EUROPEAN FOREIGN POLICY: LEGAL AND POLITICAL PERSPECTIVES 98 (Panos Koutrakos ed., 2011). 45Contrasted with hierarchy and presented implicitly as the opposite. Id. at 116. 46Skordas, supra note 16, at 142. 47Id. at 126. 48Etienne, supra note 18, 34. 49See id. at 34 (“Meta-constitutional rules would relate to these general principles on principles of primary law, simulta- neously overarching and providing the European Union with the essence of its constitutional structure.”). See also id. at 33 (“It is obvious and common sense that Articles on the basic functioning of the Union do not have the same scope that material provisions on the Union policies have.”). 50Martines, supra note 41, at 145. 51Id. 52Id. at 146. 52Id. at 146. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press IV. Acknowledgement of the Need for Balance Between Protection of EU Law and Respect for International Law in the Interests of Justice For Skordas, Völkerrechtsfreundlichkeit as comity45 functions as an ersatz meta-principle that enables the ECJ to recognize, interpret, and implement international law and, at the same time, develop and preserve the Union’s own separate identity. 46 Comity, Skordas states, “has a hybrid status, oscillating between social [local] practice and a ‘sense of’ international legal obligation.”47 All of these proposals have at their heart a balance between international law and EU law. p p More precise proposals for the mechanics of this balance are also forthcoming in scholarship. Etienne considers that the Court “has opened the doors when international law commanded so and in turn has closed them when the integrity of essential EU acquis was at stake.” However, the extent to which only the essential EU acquis has been protected from the application of international law in the case law will appear below as debatable. Etienne proposes, moving forward at least, that meta-constitutional principles of EU law should be shielded from international law. These are, “ . . . the rules inherent to the fundaments of the European Union, providing for the essential values on which it is founded, [and] the general principles on its institutional framework and on its relationships with the member states and the international legal system.”48 The argument put forward implies that material, or policy, provisions of primary law are sub- ordinate and would not form a part of this cluster.49 Martines notes that “[t]here is of course a risk in approaching international law binding the EU selectively which can result in the very denial of international law, if pushed too far,”50 yet, “[o]n the other hand it should also be considered that the EU provides a way of protecting its legal order. . . .”51 One aspect of Martines’ suggestion arguably comes very close to proportionality, “[w]hat could be called the sub-constitutional supremacy of international law means that the incompatibility of an agreement's provision with primary law might be a limit to the direct application of its provisions.”52 p y g pp p Accordingly, this proposal is more liberal in balancing EU interests than that of Etienne and has a strong flavor of proportionality, without deploying that term. 50Martines, supra note 41, at 145. 48Etienne, supra note 18, 34. p p 50Martines, supra note 41, at 145. 51Id. 53PIET EECKHOUT, EU EXTERNAL RELATIONS LAW 383 (2d ed. 2011). 54MENDEZ, supra note 4, at 320. 55See generally TRIDIMAS, supra note 2. 56ECJ, Case C-294/83, Parti Ecologiste Les Verts v. European Parliament, ECLI:EU:C:1986:166 (Apr. 23, 1986), para. 23, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-294/83. 57Piet Eeckhout, Does Europe’s Constitution Stop at the Water’s Edge?: Law and Policy in the EU’s External Relations, 5 WALTER VAN GERVEN LECTURES 1, 27 (2005). See also Opinion of Advocate General Maduro at paras. 34–40, ECJ, Joined Cases C–402/05 P and C–415/05 P Kadi & Al Barakaat Int’l Found. v. Council & Comm’n, ECLI:EU:C:2008:11 (Jan. 16, 2008), https://curia.europa.eu/juris/liste.jsf?oqp=&for=&mat=or&jge=&td=%3BALL&jur=C%2CT%2CF& num=402%252F05&page=1&dates=&pcs=Oor&lg=&pro=&nat=or&cit=none%252CC%252CCJ%252CR%252C2008E %252C%252C%252C%252C%252C%252C%252C%252C%252C%252Ctrue%252Cfalse%252Cfalse&language=en&avg=& cid=12821209. V. Proportionality’s Place in the Literature and Potential Moving Forward Like a missing jigsaw piece, it is submitted that proportionality fits neatly into the literature. First, it can accommodate the aim sought by most scholars of achieving a balance between respect for international law and protection of EU law. This balance is now more clearly encap- sulated in the Treaties too, with Article 3(5) of the Treaty of the European Union (TEU) requiring strict observance of international law, but also that the EU uphold and promote its values and interests. The second way in which proportionality meets the aims of scholars is that its deployment would place less pressure on tests for direct effect of international law, which have given rise to the legal uncertainty to which scholars have objected. The process by which direct effect has become an awkward locus for discretion in the absence of proportionality in external relations case law will be further detailed below. The third benefit of proportionality is its familiarity and durability. With the proliferation of nomenclatures in external relations case law analyses there is the risk that scholars may speak past one another, whereas coalescence around proportionality can provide a more solid basis for devel- opment. Relevant elements of proportionality will be considered more fully below, but it is notable that proportionality is durable in its application to a range of contexts—governing competence and subsidiarity, reviewing EU and Member State action for legitimacy of aims and appropriate- ness of measures, and even weighing interests against one another stricto sensu.55 Its application in external relations case law could concern derogation from international law where an international treaty confers rights on individuals, but equally, due to proportionality’s reach it need not be limited to this. For example, in case law not applying international law the question of direct effect does not arise concerning review of EU legislative action under Article 263 or 267 of the Treaty on the Functioning of the European Union (TFEU), permissibility of review is pre- sumed, and the intensity of judicial review varies based on the complexity of the issue. A fourth merit of proportionality is that of facilitating coherence with fundamental tenets of the EU legal system. The rule of law principle from Les Verts provides that, “in a Community based on the rule of law . . . IV. Acknowledgement of the Need for Balance Between Protection of EU Law and Respect for International Law in the Interests of Justice Overall, prevarication is pal- pable in putting into print the “p word.” Eeckhout though does suggest that “general principles of EU law, such as the principle of proportionality” are an alternative to “putting up formal obstacles, https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 563 German Law Journal such as the lack of direct effect of an international agreement.”53 “[T]he interesting suggestion [of Eeckhout] that . . . the Court should more readily accept direct effect but show reluctance to find a breach [of international law] where EU legislation is concerned”54 was also noted by Mendez. These passages remain—to the author’s knowledge—the strongest endorsement of proportion- ality proper in the application of international law in the CJEU to date. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 58See MENDEZ, supra note 4 (concerning the Court’s tendency to limit Member State action more greatly than EU legislative action). 59Notwithstanding the weaker consequence of interpretation of EU legislation in light of non-directly effective international agreements and continued liability at international level. 60See Ghazaryan, supra note 24, at 64–74. 61BECK, supra note 1, at 438. 62See ECJ, Case C-26/62, Van Gend en Loos v. Administratie der Belastingen, ECLI:EU:C:1963:1 (Feb. 5, 1963), http://curia. europa.eu/juris/liste.jsf?language=en&num=C-26/62. 63ECJ, Case C-106/77, Amministrazione delle Finanze dello Stato v. Simmenthal, ECLI:EU:C:1978:49 (Mar. 9, 1978), para. 24, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-106/77. 64van Gend en Loos, Case C-26/62 at 13. 65The Court expanded the provision to allow jobseekers access to other Member States as “a strict interpretation of [Article 45(3) TFEU] would . . . make that provision ineffective.” ECJ, Case C-292/89, Antonissen, ECLI:EU:C:1991:80 (Feb. 26, 1991), para 12, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-292/89. 66Consolidated Version of the Treaty on the Functioning of the European Union art. 45(3), May 9, 2008, 2008 O.J. (C 115) [hereinafter TFEU] V. Proportionality’s Place in the Literature and Potential Moving Forward neither [the EU’s] Member States nor its institutions can avoid a review of the question whether the measures adopted by them are in conformity with the basic constitutional charter, the Treaty.”56 For EU external relations law the concept of a political question doctrine is not accepted, mean- ing that, in principle, the legislature’s action should be reviewable.57 Practically, though, without https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar Rupert Dunbar 564 direct effect of international law the EU legislature—or less frequently Member State58— essentially has freedom to ignore it.59 This seems in tension with the concept of international law as an integral part of EU law and Article 3(5) TEU. Moreover, we will see that at present, direct effect of international law precludes, in the absence of proportionality, the defense of the legislature or Member State that pursues an objective in the Treaty which was, in the circum- stances, more important than that of international law. Thus, international law can also, arguably, have too great an effect and it is notable that the Council in particular has sought to exclude direct effect from certain EU agreements.60 Proportionality can achieve review commensurate with com- plexity of circumstance, without excluding international law nor allowing it to overpower other EU norms. This would overcome the somewhat schizophrenic options currently available, whilst maintaining a closer correlation with recognized precepts of EU law. Having made the case that proportionality warrants more sustained consideration in the liter- ature, the following sections will consider how proportionality manages the relationship between other EU norms in internal case law. Reasons for it to be more fully and openly applied to external relations case law will then be developed in context. C. Judicial Discretion Manifests Itself Largely in Proportionality in Internal Case Law Claims to direct effect are arguably the distinctive feature of the EU legal order, and as a result, cover- age is prominent. However, direct effect sits within further rules of reliance. It is notable, though, that all of these rules are remarkably lenient in allowing EU law to be invoked easily. Invocation of EU law is simply the beginning in most cases and—as “EU primary law [itself] is characterized by a high degree of vagueness and pervasive norm collision across most of its substantive areas”61—resolution ultimately lies most frequently in the application of proportionality. I. Decline of Direct Effect and other Rules of Reliance as a Locus of Discretion in Interna The capability for individuals to rely on provisions of the EU Treaty against their Member State in domestic courts, so called vertical direct effect, first emerged in the van Gend en Loos case.62 Provided the relevant provision is clear, precise, and unconditional the Member State court must apply EU law and disapply conflicting national law.63 In Van Gend en Loos, the Court stated the relevant provision was “ideally adapted to produce direct effects,”64 and the criteria has been lib- erally applied to less ideal provisions since. For instance, free movement of workers “entail[s] the right [to accept offers of employment actually made65 in another Member State], subject to lim- itations justified on grounds of public policy, public security, or public health.”66 However, such 58See MENDEZ, supra note 4 (concerning the Court’s tendency to limit Member State action more greatly than EU legislative action). 59Notwithstanding the weaker consequence of interpretation of EU legislation in light of non-directly effective international agreements and continued liability at international level. lidated Version of the Treaty on the Functioning of the European Union art. 45(3), May 9, 2008, 2008 O.J. (C 1 r TFEU]. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 565 German Law Journal limitations are “subject to judicial control [at EU level]”67 and therefore provisions with such con- ditionality can have direct effect.68 Craig and de Búrca note that “[t]he idea that direct effect could apply even where the Member States possessed discretion . . . represented a significant juridical shift in thinking about direct effect.”69 g Similarly, the Court’s focus on negative obligation in Van Gend en Loos emerged as being non- dispositive in Lütticke.70 Here a positive obligation under The Treaty of Rome (EEC) Article 95 to repeal or amend discriminatory rules on taxation was considered “complete [and] legally perfect”71 and therefore capable of having direct effect. The case Defrenne v. Sabena (No. 2)72 marked a relaxation of the “clear, precise and unconditional” criteria.73 The case concerned Article 119 of the EEC Treaty which provided for the “principle that men and women should receive equal pay for equal work.” Advocate General Trabucchi conceded that the “form of words used . . . may seem too vague and the meaning of the word ‘principle’ itself not to be very specific” but asserted that “the purpose of the rule is nevertheless clear.”74 The Court conceded that where discretion was “indirect and disguised, . . I. Decline of Direct Effect and other Rules of Reliance as a Locus of Discretion in Interna . entire branches of industry and even of the economic system as a whole” would need to be addressed and therefore required legislative measures.75 However, in this case discrimination was direct and overt, meaning direct effect could be found. Scholars have noted the novelty in the fact that the provision “simultaneously [had] and did not have direct effect.”76 The concept of direct effect has also expanded to include directives,77 and even unwritten general principles of EU law may be directly effective.78 g p p y y Ultimately, there is the sense that “[d]irect effect is nothing but the ordinary state of [EU] law,”79 and it will be found wherever possible. Moreover, other rules governing reliance on EU law are equally liberal: First, Article 258 TFEU essentially presumes the Commission’s capacity to rely on the Treaty and any rules relating to it in order to challenge Member State action. Member States are empowered equally, as against one another, through Article 259 TFEU. Second, Article 263 TFEU provides a mechanism of direct judicial review before the CJEU. In analyzing “the validity and interpretation of acts of the institutions, bodies, offices, or agencies of the Union,”80 the Court applies the Treaty and rules emanating from it, including general e C-43/75, Defrenne v. Sabena, ECLI:EU:C:1976:56 (Apr. 8, 1976), http://curia.europa.eu/juris/liste.jsf? &num=C-43/75. 72ECJ, Case C-43/75, Defrenne v. Sabena, ECLI:EU:C:1976:56 (Apr. 8, 1976), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-43/75. g g 73JUHA RAITIO, THE PRINCIPLE OF LEGAL CERTAINTY IN EC LAW 353 (2003); ROBERT SCHÜTZE, EUROPEAN UNION LAW 85 (2015); CRAIG & DE BÚRCA, supra note 69, at 192. g g 73JUHA RAITIO, THE PRINCIPLE OF LEGAL CERTAINTY IN EC LAW 353 (2003); ROBERT SCHÜTZE, EUROPEAN UNION LAW 85 (2015); CRAIG & DE BÚRCA, supra note 69, at 192. p 74Opinion of Advocate General Trabucchi at 486, ECJ, Case C-43/75, Defrenne v. Sabena, ECLI:EU:C:1976:39 (Mar. 10, 1976), http://curia.europa.eu/juris/liste.jsf?language=en&num=C-43/75. 74Opinion of Advocate General Trabucchi at 486, ECJ, Case C-43/75, Defrenne v. Sabena, ECLI:EU:C:1976:39 (Mar. 10, 1976), http://curia.europa.eu/juris/liste.jsf?language=en&num=C-43/75. 75Defrenne v. Sabena, Case C-43/75 at paras. 18–19. f , p 76SIONAIDH DOUGLAS-SCOTT, CONSTITUTIONAL LAW OF THE EUROPEAN UNION 287 (2002). See also SCHÜTZE, supra note 73, at 85. 76SIONAIDH DOUGLAS-SCOTT, CONSTITUTIONAL LAW OF THE EUROPEAN UNION 287 (2002). See also SCHÜTZE, supra note 73, at 85. Van Duyn v. Home Office, Case C-41/74 at 1337. 78ECJ, Case C-260/89, Elliniki Radiophonia Tileorassi AE v. Dimotiki Etairia Pliroforissis, ECLI:EU:C:1991:254 (June 18, 1991), para 24, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-260/89. 67ECJ, Case C-41/74, Van Duyn v. Home Office, ECLI:EU:C:1974:133 (Dec. 4, 1974), para 7, http://curia.europa.eu/juris/ liste.jsf?language=en&num=C-41/74. 68See ECJ, Case C-2/74, Reyners v. Belgian State, ECLI:EU:C:1974:68 (June 21, 1974), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-2/74. 69PAUL CRAIG & GRÁINNE DE BÚRCA, EU LAW: TEXT, CASES AND MATERIALS 190 (6th ed. 2015). 70ECJ, Case C-48/65, Lütticke v. Comm’n of the EEC, ECLI:EU:C:1966:8 (Mar. 1, 1966), http://curia.europa.eu/juris/liste. jsf?language=en&num=C-48/65. 71Id. at 210. 72ECJ, Case C-43/75, Defrenne v. Sabena, ECLI:EU:C:1976:56 (Apr. 8, 1976), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-43/75. 73JUHA RAITIO, THE PRINCIPLE OF LEGAL CERTAINTY IN EC LAW 353 (2003); ROBERT SCHÜTZE, EUROPEAN UNION LAW 85 (2015); CRAIG & DE BÚRCA, supra note 69, at 192. 74Opinion of Advocate General Trabucchi at 486, ECJ, Case C-43/75, Defrenne v. Sabena, ECLI:EU:C:1976:39 (Mar. 10, 1976), http://curia.europa.eu/juris/liste.jsf?language=en&num=C-43/75. 75Defrenne v. Sabena, Case C-43/75 at paras. 18–19. 76SIONAIDH DOUGLAS-SCOTT, CONSTITUTIONAL LAW OF THE EUROPEAN UNION 287 (2002). See also SCHÜTZE, supra note 73, at 85. 77Van Duyn v. Home Office, Case C-41/74 at 1337. 78ECJ, Case C-260/89, Elliniki Radiophonia Tileorassi AE v. Dimotiki Etairia Pliroforissis, ECLI:EU:C:1991:254 (June 18, 1991), para 24, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-260/89. 79Pierre Pescatore, The Doctrine of Direct Effect: An Infant Disease of Community Law, 8 EUR. L. REV. 155, 177 (1983). See also DOUGLAS-SCOTT, supra note 76, at 287; ATHONY ARNULL, THE EUROPEAN UNION AND ITS COURT OF JUSTICE 157–79 (2d ed. 2006); Bruno de Witte, Direct Effect, Primacy and the Nature of the Legal Order, in THE EVOLUTION OF EU LAW 334 (Paul Craig & Gráinne de Búrca eds., 2d ed. 2011); and SCHÜTZE, supra note 73, at 87. 80TFEU art. 267(1)(b). I. Decline of Direct Effect and other Rules of Reliance as a Locus of Discretion in Interna 78ECJ, Case C-260/89, Elliniki Radiophonia Tileorassi AE v. Dimotiki Etairia Pliroforissis, ECLI:EU:C:1991:254 (June 18, 1991), para 24, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-260/89. 79Pierre Pescatore, The Doctrine of Direct Effect: An Infant Disease of Community Law, 8 EUR. L. REV. 155, 177 (1983). See also DOUGLAS-SCOTT, supra note 76, at 287; ATHONY ARNULL, THE EUROPEAN UNION AND ITS COURT OF JUSTICE 157–79 (2d ed. 2006); Bruno de Witte, Direct Effect, Primacy and the Nature of the Legal Order, in THE EVOLUTION OF EU LAW 334 (Paul Craig & Gráinne de Búrca eds., 2d ed. 2011); and SCHÜTZE, supra note 73, at 87. 80TFEU art 267(1)(b) 79Pierre Pescatore, The Doctrine of Direct Effect: An Infant Disease of Community Law, 8 EUR. L. REV. 155, 177 (1983). See also DOUGLAS-SCOTT, supra note 76, at 287; ATHONY ARNULL, THE EUROPEAN UNION AND ITS COURT OF JUSTICE 157–79 (2d ed. 2006); Bruno de Witte, Direct Effect, Primacy and the Nature of the Legal Order, in THE EVOLUTION OF EU LAW 334 (Paul Craig & Gráinne de Búrca eds., 2d ed. 2011); and SCHÜTZE, supra note 73, at 87. 80TFEU art. 267(1)(b). https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 566 principles,81 regardless of whether they have direct effect.82 Controversy regarding limited stand- ing for EU institutions under Article 263 TFEU has now been addressed, meaning that they, along with Member States, may challenge EU legislation without questions of direct effect arising. Private individuals must demonstrate that the act is of direct and individual concern to them, with the interpretation of this criteria being narrow.83 Minor changes in the language of the pro- vision in the Lisbon Treaty do not appear to have significantly affected this.84 However, if an indi- vidual does not fulfil the restrictive criteria of Article 263 TFEU they may proceed through the preliminary rulings procedure. Third, under Article 267 TFEU, preliminary rulings procedure, if the matter concerns the val- idity of EU legislation the national court’s question, it will prompt the CJEU to answer and to invalidate the legislation should this be required, regardless of direct effect.85 In all the instances above, the question as to whether a specific provision has direct effect is not enquired into: For Articles 258, 259, 263, and this aspect of Article 267 TFEU. 81See TRIDIMAS, supra note 2, at 36. 82Morten Broberg & Niels Fenger, PRELIMINARY REFERENCES TO THE EUROPEAN COURT OF JUSTICE 213–22 (2010). 83For the general approach, see ECJ, Case C-25/62, Plaumann & Co. v. Comm’n, ECLI:EU:C:1963:17 (July 15, 1963), http:// curia.europa.eu/juris/liste.jsf?language=en&num=C-25/62. But see F. Jacobs, Access by Individuals to Judicial Review in EU Law – Still an Issue of Concern?, in EUROPE: THE NEW LEGAL REALISM - ESSAYS IN HONOUR OF HJALTE RASMUSSEN 263–76 (H. Koch et al. eds., 2010), who discusses having previously advocated a broader interpretation in his role as Advocate General in ECJ, Case C-50/00 P, Uni´on de Peque˜nos Agricultores v. Council, ECLI:EU:C:2002:462 (July 25, 2002), http://curia.europa. eu/juris/liste.jsf?language=en&num=C-50/00, which was followed by a similar approach in the General Court but rejected on appeal by the Court of Justice in ECJ, Case C-263/02 P, Comm’n v. Jégo-Quéré, ECLI:EU:C:2004:210 (Apr. 1, 2004), http:// curia.europa.eu/juris/liste.jsf?language=en&num=C-263/02. 84ECJ, Case C-583/11 P, Inuit Tapiriit Kanatami and Others v. Parliament and Council, ECLI:EU:C:2013:625 (Oct. 3, 2013), paras. 55, 70, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-583/11. See also Carl Fredrik Bergström, Defending Restricted Standing for Individuals to Bring Direct Actions Against “Legislative” Measures, 10 EUR. CONST. L. REV. 481 (2014). 85For this reason, it is said not to be between parties stricto sensu. See BROBERG & FENGER, supra note 82, at 351. A limitation is that the question must be relevant for the national court to decide the case before it. See ECJ, Case C-104/79, Foglia v. Novello, ECLI:EU:C:1980:73 (Mar. 11, 1980), paras. 9–13, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-104/79. 86So-called indirect effect. 87See, e.g., ALLEN ROSAS & LORNA ARMATI, EU CONSTITUTIONAL LAW: AN INTRODUCTION 42 (2010). 81See TRIDIMAS, supra note 2, at 36. I. Decline of Direct Effect and other Rules of Reliance as a Locus of Discretion in Interna However, under Article 267 TFEU, cases brought by individuals against Member States and private individuals will require direct effect of the relevant provision; otherwise the provision may only be useful in the more limited capacity of interpreting another EU and/or national law provision.86 Although, as noted above, the test for direct effect is itself generous. The presumption, where relevant, of direct effect of EU legal provisions, combined with other rules which permit reliance creates a large opening, catches and then funnels cases through to the proportionality stage. It is submitted that proportionality—not direct effect—is the primary locus of the Court’s discretion in internal case law. The impact and practical application of this can now be considered. 87See, e.g., ALLEN ROSAS & LORNA ARMATI, EU CONSTITUTIONAL LAW: AN INTRODUCTION 42 (2010) https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 82Morten Broberg & Niels Fenger, PRELIMINARY REFERENCES TO THE EUROPEAN COURT OF JUSTICE 213–22 (2010). 83For the general approach, see ECJ, Case C-25/62, Plaumann & Co. v. Comm’n, ECLI:EU:C:1963:17 (July 15, 1963), http:// curia.europa.eu/juris/liste.jsf?language=en&num=C-25/62. But see F. Jacobs, Access by Individuals to Judicial Review in EU Law – Still an Issue of Concern?, in EUROPE: THE NEW LEGAL REALISM - ESSAYS IN HONOUR OF HJALTE RASMUSSEN 263–76 (H. Koch et al. eds., 2010), who discusses having previously advocated a broader interpretation in his role as Advocate General in ECJ, Case C-50/00 P, Uni´on de Peque˜nos Agricultores v. Council, ECLI:EU:C:2002:462 (July 25, 2002), http://curia.europa. eu/juris/liste.jsf?language=en&num=C-50/00, which was followed by a similar approach in the General Court but rejected on appeal by the Court of Justice in ECJ, Case C-263/02 P, Comm’n v. Jégo-Quéré, ECLI:EU:C:2004:210 (Apr. 1, 2004), http:// curia.europa.eu/juris/liste.jsf?language=en&num=C-263/02. 86So-called indirect effect. 84ECJ, Case C-583/11 P, Inuit Tapiriit Kanatami and Others v. Parliament and Council, ECLI:EU:C:2013:625 (Oct. 3, 2013), paras. 55, 70, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-583/11. See also Carl Fredrik Bergström, Defending Restricted Standing for Individuals to Bring Direct Actions Against “Legislative” Measures, 10 EUR. CONST. L. REV. 481 (2014). 85For this reason, it is said not to be between parties stricto sensu. See BROBERG & FENGER, supra note 82, at 351. A limitation is that the question must be relevant for the national court to decide the case before it. See ECJ, Case C-104/79, Foglia v. Novello, ECLI:EU:C:1980:73 (Mar. 11, 1980), paras. 9–13, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-104/79. 86 ll d d ff Corresponding Rise of Proportionality as a Locus of Discretion in Internal Case Law With direct effect and other related rules invariably allowing reliance on EU law provisions, cases must then progress to consider their application. While EU law features a hierarchy in which we identify the EU Treaty as sitting above secondary legislation and secondary legislation above national law,87 it is submitted that this hierarchy is not especially informative regarding the res- olution of most cases. Instead, the normative interest—pursued at any level of the hierarchy—is postulated at the EU treaty level, where multiple norms conflict and must be resolved through proportionality. p 82Morten Broberg & Niels Fenger, PRELIMINARY REFERENCES TO THE EUROPEAN COURT OF JUSTICE 213–22 (2010). 83For the general approach, see ECJ, Case C-25/62, Plaumann & Co. v. Comm’n, ECLI:EU:C:1963:17 (July 15, 1963), http:// curia.europa.eu/juris/liste.jsf?language=en&num=C-25/62. But see F. Jacobs, Access by Individuals to Judicial Review in EU Law – Still an Issue of Concern?, in EUROPE: THE NEW LEGAL REALISM - ESSAYS IN HONOUR OF HJALTE RASMUSSEN 263–76 (H. Koch et al. eds., 2010), who discusses having previously advocated a broader interpretation in his role as Advocate General in ECJ, Case C-50/00 P, Uni´on de Peque˜nos Agricultores v. Council, ECLI:EU:C:2002:462 (July 25, 2002), http://curia.europa. eu/juris/liste.jsf?language=en&num=C-50/00, which was followed by a similar approach in the General Court but rejected on appeal by the Court of Justice in ECJ, Case C-263/02 P, Comm’n v. Jégo-Quéré, ECLI:EU:C:2004:210 (Apr. 1, 2004), http:// curia.europa.eu/juris/liste.jsf?language=en&num=C-263/02. 84ECJ, Case C-583/11 P, Inuit Tapiriit Kanatami and Others v. Parliament and Council, ECLI:EU:C:2013:625 (Oct. 3, 2013), paras. 55, 70, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-583/11. See also Carl Fredrik Bergström, Defending Restricted Standing for Individuals to Bring Direct Actions Against “Legislative” Measures, 10 EUR. CONST. L. REV. 481 (2014). 85For this reason, it is said not to be between parties stricto sensu. See BROBERG & FENGER, supra note 82, at 351. A limitation is that the question must be relevant for the national court to decide the case before it. See ECJ, Case C-104/79, Foglia v. Novello, ECLI:EU:C:1980:73 (Mar. 11, 1980), paras. 9–13, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-104/79. 86So-called indirect effect. 86So-called indirect effect. e, e.g., ALLEN ROSAS & LORNA ARMATI, EU CONSTITUTIONAL LAW: AN INTRODUCTION 42 (2010). , p y g , , ( ) 89See in combination, ECJ, Case C-55/94, Gebhard v. Consiglio dell'Ordine degli Avvocati e Procuratori di Milano, ECLI: EU:C:1995:411 (Nov. 30, 1995), para 37, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-55/94, and ECJ, Case C-110/05, Comm’n v. Italy, ECLI:EU:C:2009:66 (Feb. 10, 2009), para 59, http://curia.europa.eu/juris/liste.jsf?language= en&num=C-110/05, and ECJ, Case C-112/00, Schmidberger, ECLI:EU:C:2003:333 (June 12, 2003), para 79, http://curia. europa.eu/juris/liste.jsf?language=en&num=C-112/00. Corresponding Rise of Proportionality as a Locus of Discretion in Internal Case Law https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 567 German Law Journal Proportionality arises in challenges to EU legislation and Member State action, essentially allowing defense of such actions through, in the case of the former, pursuit of another justified goal and, in the latter, recourse to derogations within the Treaty and those developed within the case law. The application of the proportionality test is noted to differ significantly depending upon whether EU or Member State action is challenged. The Court’s approach and presentation of rel- evant steps within proportionality analyses are also not consistently presented.88 p p p y y y p Nonetheless, the common steps for the Court in proportionality analyses are inqui p p p y y y p Nonetheless, the common steps for the Court in proportionality analyses are inquiries as to89: i. Whether the aim is recognized as legitimate by EU law. ii. Whether the measure was suitable to achieve the desired end. iii. Whether it was necessary to achieve the desired end. it was necessary to achieve the desired en iv. Whether the burden was excessive in relation to the objective sought to be achieved, proportionality stricto sensu. iv. Whether the burden was excessive in relation to the objective sought to be achieved, proportionality stricto sensu. It can be seen through this that proportionality requires, or at least facilitates, engagement with the facts and circumstances of a given case in a concrete way. 90A recent exception to this came in a line of case law showing tolerance of Member States adopting “easily managed and supervised” measures even if they were not the least restrictive means available. See, e.g., Commission v. Italy, Case C-110/05; ECJ, Case C-142/05, Åklagaren v. Mickelsson and Roos, ECLI:EU:C:2009:336 (June 4, 2009), para. 36, http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-142/05; ECJ, Case C-137/09, Josemans, ECLI:EU:C:2010:774 (Dec. 16, 2010), para. 82, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-142/05; and ECJ, Case C-512/13, Sopora, ECLI:EU:C:2015:108 (Feb. 24, 2015), para 33, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-512/13. 91See, e.g., BECK, supra note 1, at 422; SYBE A. DE VRIES, TENSIONS WITHIN THE INTERNAL MARKET: THE FUNCTIONING OF THE INTERNAL MARKET AND THE DEVELOPMENT OF HORIZONTAL FLANKING POLICIES 16 (2006); CRAIG & DE BÚRCA, supra note 69, at 532. 88Wolf Sauter, Proportionality in EU Law: A Balancing Act?, 15 CAMBRIDGE Y.B. EUR. STUD. 439, 448 (2013). p 93ECJ, Case C-127/07, Société Arcelor Atlantique et Lorraine and Others v. Premier Minister, ECLI:EU:C:2008:728 (Dec. 16, 2008), para. 58, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-127/07. 94Id. at para. 59. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 92Sauter, supra note 88, at 464–65. 88Wolf Sauter, Proportionality in EU Law: A Balancing Act?, 15 CAMBRIDGE Y.B. EUR. STUD. 439, 448 (2013). 89See in combination, ECJ, Case C-55/94, Gebhard v. Consiglio dell'Ordine degli Avvocati e Procuratori di Milano, ECLI: EU:C:1995:411 (Nov. 30, 1995), para 37, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-55/94, and ECJ, Case C-110/05, Comm’n v. Italy, ECLI:EU:C:2009:66 (Feb. 10, 2009), para 59, http://curia.europa.eu/juris/liste.jsf?language= en&num=C-110/05, and ECJ, Case C-112/00, Schmidberger, ECLI:EU:C:2003:333 (June 12, 2003), para 79, http://curia. europa.eu/juris/liste.jsf?language=en&num=C-112/00. 90A recent exception to this came in a line of case law showing tolerance of Member States adopting “easily managed and supervised” measures even if they were not the least restrictive means available. See, e.g., Commission v. Italy, Case C-110/05; ECJ, Case C-142/05, Åklagaren v. Mickelsson and Roos, ECLI:EU:C:2009:336 (June 4, 2009), para. 36, http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-142/05; ECJ, Case C-137/09, Josemans, ECLI:EU:C:2010:774 (Dec. 16, 2010), para. 82, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-142/05; and ECJ, Case C-512/13, Sopora, ECLI:EU:C:2015:108 (Feb. 24, 2015), para 33, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-512/13. 91See, e.g., BECK, supra note 1, at 422; SYBE A. DE VRIES, TENSIONS WITHIN THE INTERNAL MARKET: THE FUNCTIONING OF THE INTERNAL MARKET AND THE DEVELOPMENT OF HORIZONTAL FLANKING POLICIES 16 (2006); CRAIG & DE BÚRCA, supra note 69, at 532. 92Sauter, supra note 88, at 464–65. 93ECJ, Case C-127/07, Société Arcelor Atlantique et Lorraine and Others v. Premier Minister, ECLI:EU:C:2008:728 (Dec. 16, 2008), para. 58, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-127/07. 94Id. at para. 59. 95PAUL CRAIG, EU ADMINISTRATIVE LAW 656–63 (3d ed. 2018). 96Loïc Azoulai, The European Court of Justice and the Duty to Respect Sensitive National Interests, in JUDICIAL ACTIVISM AT THE EUROPEAN COURT OF JUSTICE 168 (Mark Dawson, Bruno de Witte & Elise Muir eds., 2013). 97See Harbo, supra note 13, at 172; CRAIG, supra note 95, at 653–54. 98ECJ, Case C-24/00, Commission v. France, ECLI:EU:C:2004:70 (Feb. 5, 2004), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-24/00. 99ECJ, Case C-34/79, Regina v. Henn and Darby, ECLI:EU:C:1979:295 (Dec. 14, 1979), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-34/79. 100ECJ, Case C-379/98, PreussenElektra v. Schleswag AG, ECLI:EU:C:2001:160 (Mar. 13, 2001), http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-379/98. 101See, e.g., Schmidberger, Case C-112/00. 102See, e.g., ECJ, Case C-313/94, Graffione v. Ditta Fransa, ECLI:EU:C:1996:450 (Nov. 26, 1996), http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-313/94. III. Proportionality Applied to EU Action As hinted at above, a key modification to the proportionality test comes where EU secondary legislation is reviewed, as it often need not solely be manifestly disproportionate, or inappropriate, whereas Member State action should typically adopt the least restrictive means available.90 The distinction results in deference to the EU legislature where it is exercising discretion, though Member State action is subject to more intense review.91 The consequence of a lighter touch review of EU secondary legislation, as compared to intru- sive analysis of Member State measures, is that case law reviewing EU action will also be less help- ful in informing participants of the current normative state of EU law for this or future cases.92 Nonetheless, “the Community legislature is obliged to base its choice on objective criteria appro- priate to the aim pursued by the legislation in question,” including consideration of all facts and technical and scientific data available.93 All interests involved must be considered, and “the Community legislature’s exercise of its discretion must not produce results that are manifestly less appropriate than those that would be produced by other measures that were also suitable for those objectives.”94 This means that both the outcome and the procedure are subject to review. p 93ECJ, Case C-127/07, Société Arcelor Atlantique et Lorraine and Others v. Premier Minister, ECLI:EU:C:2008:728 (Dec. 16, 2008), para. 58, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-127/07. 94Id. at para. 59. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 568 Rupert Dunbar Rupert Dunbar A further variation arises within proportionality review of EU action based on the nature of the conflicting rights. It is recognized that where a fundamental right is at stake, rather than a discre- tionary policy choice, the review of EU legislative action will be based on protecting the substance of the relevant right, and for this reason can be more intrusive.95 Proportionality and the nature of fundamental rights is developed further below. However, it should be noted that fundamental rights are readily balanced against other interests in review of both EU and Member State action. IV. Proportionality Applied to Member State Action As stated at the introduction, proportionality is relevant in most areas of EU law. However, consideration of internal market case law is particularly helpful in identifying the normatively relative—less hierarchical—nature of EU law, as other norms are often measured against the four fundamental freedoms.96 Another notable point is that, in this case law, these “other” norms manifest themselves in national rules creating barriers to free movement, and the hierarchical place of national law—at the very bottom—does not preclude their normative weight as interests recognized as being of importance in EU law. Once recognized as such, provided the measure was appropriate and necessary, the Court can consider whether the restriction on trade, or another norm, was excessive in relation to the objective sought to be achieved—proportionality stricto sensu. Even where the Court does not balance norms against one another stricto sensu, which it is sometimes reluctant to do unless prompted, it is recognized that the relevant norms also affect the intrusiveness of tests of appropriateness and necessity.97 For this reason, the term “balance” is used liberally by scholars, and this is also the case here. A snapshot of case law, therefore, reveals that, inter alia, protection of health can be more important than free movement of goods,98 as can public morality.99 Of course, these feature as express derogations in Article 36 TFEU, but environmental protection,100 fundamental rights,101 and consumer protection102 have the capacity to prevail similarly. The protection of norms beyond the closed-list of Article 36 TFEU illustrates the Court’s inclination to entertain clashes between norms, even when previous case law would have implied that this was not possible.103 The Court’s inclination to balance is strong, and it is not only the free movement norms—or “trade” norms—against which others are balanced, this is just the most prominent area of practice. By way of brief example, fundamental rights are frequently balanced against the public interest104—or 103For instance, in PreussenElektra, Case C-379/98, the national measure appeared discriminatory but was still justified as a mandatory requirement (environmental protection), which in the Court’s case law had hitherto only been available only to non-discriminatory measures. 103For instance, in PreussenElektra, Case C-379/98, the national measure appeared discriminatory but was still justified as a mandatory requirement (environmental protection), which in the Court’s case law had hitherto only been available only to non-discriminatory measures. y 104See, e.g., ECJ, Case C-4/73, Nold KG v. Comm’n, ECLI:EU:C:1975:114 (Sep. 24, 1975), para. 95PAUL CRAIG, EU ADMINISTRATIVE LAW 656–63 (3d ed. 2018). 96Loïc Azoulai, The European Court of Justice and the Duty to Respect Sensitive National Interests, in JUDICIAL ACTIVISM AT THE EUROPEAN COURT OF JUSTICE 168 (Mark Dawson, Bruno de Witte & Elise Muir eds., 2013). 97See Harbo, supra note 13, at 172; CRAIG, supra note 95, at 653–54. 98ECJ, Case C-24/00, Commission v. France, ECLI:EU:C:2004:70 (Feb. 5, 2004), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-24/00. 99ECJ, Case C-34/79, Regina v. Henn and Darby, ECLI:EU:C:1979:295 (Dec. 14, 1979), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-34/79. 100ECJ, Case C-379/98, PreussenElektra v. Schleswag AG, ECLI:EU:C:2001:160 (Mar. 13, 2001), http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-379/98. 101See, e.g., Schmidberger, Case C-112/00. 102See, e.g., ECJ, Case C-313/94, Graffione v. Ditta Fransa, ECLI:EU:C:1996:450 (Nov. 26, 1996), http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-313/94. 103For instance, in PreussenElektra, Case C-379/98, the national measure appeared discriminatory but was still justified as a mandatory requirement (environmental protection), which in the Court’s case law had hitherto only been available only to non-discriminatory measures. 104See, e.g., ECJ, Case C-4/73, Nold KG v. Comm’n, ECLI:EU:C:1975:114 (Sep. 24, 1975), para. 14, http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-4/73; ECJ, Case C-11/70, Internationale Handelsgesellschaft, ECLI:EU:C:1970:114 (Dec. 17, 1970), para. 24, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-11/70; ECJ, Case C-44/79, Hauer v. Land Rheinland-Pfalz, ECLI:EU:C:1979:290 (Dec. 13, 1979), para. 23, http://curia.europa.eu/juris/liste.jsf?language=en&num= C-44/79. lai, The European Court of Justice and the Duty to Respect Sensitive National Interests, in JUDICIAL ACTIVISM AT N COURT OF JUSTICE 168 (Mark Dawson, Bruno de Witte & Elise Muir eds., 2013). 105ECJ, Case C-544/10, Deutsches Weintor eG v. Land Rheinland-Pfalz, ECLI:EU:C:2012:526 (Sep. 6, 2012), http://curia. europa.eu/juris/liste.jsf?language=en&num=C-544/10. 106Société Arcelor Atlantique et Lorraine and Others, Case C-127/07. 107Rainer Nickel, From Integration Through Law to Integration Through Conflict, in ‘INTEGRATION THROUGH LAW’ REVISITED: THE MAKING OF THE EUROPEAN POLITY 121 (Daniel Augenstein ed., 2012). 108See, e.g., ECJ, Case C-292/97, Karlsson and Others, ECLI:EU:C:2000:202 (Apr. 13, 2000), para. 45, http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-292/97; ECJ, Case C–280/93, Ger. v. Council, ECLI:EU:C:1994:367 (Oct. 5, 1994), para. 73, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-280/93; ECJ, Case C-265/87, Schräder v. Hauptzollamt Gronau, ECLI:EU:C:1989:303 (July 11, 1989), para. 15, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-265/87; ECJ, Case C-5/88, Wachauf v. Bundesamt für Ernährung und Forstwirtschaft, ECLI:EU:C:1989:321 (July 13, 1989), para 18, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-5/88. 105ECJ, Case C-544/10, Deutsches Weintor eG v. Land Rheinland-Pfalz, ECLI:EU:C:2012:526 (Sep. 6, 2012), http://curia. europa.eu/juris/liste.jsf?language=en&num=C-544/10. 106Société Arcelor Atlantique et Lorraine and Others, Case C-127/07. 107Rainer Nickel, From Integration Through Law to Integration Through Conflict, in ‘INTEGRATION THROUGH LAW’ REVISITED: THE MAKING OF THE EUROPEAN POLITY 121 (Daniel Augenstein ed., 2012). 108See, e.g., ECJ, Case C-292/97, Karlsson and Others, ECLI:EU:C:2000:202 (Apr. 13, 2000), para. 45, http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-292/97; ECJ, Case C–280/93, Ger. v. Council, ECLI:EU:C:1994:367 (Oct. 5, 1994), para. 73, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-280/93; ECJ, Case C-265/87, Schräder v. Hauptzollamt Gronau, ECLI:EU:C:1989:303 (July 11, 1989), para. 15, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-265/87; ECJ, Case C-5/88, Wachauf v. Bundesamt für Ernährung und Forstwirtschaft, ECLI:EU:C:1989:321 (July 13, 1989), para 18, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-5/88. 109Steve Peers, Taking Rights Away? Limitations and Derogations, in THE EU CHARTER OF FUNDAMENTAL RIGHTS: POLITICS, LAW AND POLICY 143 (Steve Peers & Angela Ward eds., 2004). 110PAUL CRAIG, EU ADMINISTRATIVE LAW 680 (1st ed. 2006). 111Mattias Kumm, Internationale Handelsgesellschaft, Nold and the New Human Rights Paradigm, in THE PAST AND FUTURE OF EU LAW: THE CLASSICS OF EU LAW REVISITED ON THE 50TH ANNIVERSARY OF THE ROME TREATY 107 (Miguel Poiares Maduro & Loïc Azoulai eds., 2010). See particularly Deutsches Weintor eG, Case C-544/10. 112Marton Varju European Union Human Rights Law: The Dynamics of Interpretation and Context (Edward Elgar 2014) IV. Proportionality Applied to Member State Action 14, http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-4/73; ECJ, Case C-11/70, Internationale Handelsgesellschaft, ECLI:EU:C:1970:114 (Dec. 17, 1970), para. 24, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-11/70; ECJ, Case C-44/79, Hauer v. Land Rheinland-Pfalz, ECLI:EU:C:1979:290 (Dec. 13, 1979), para. 23, http://curia.europa.eu/juris/liste.jsf?language=en&num= C-44/79. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 569 German Law Journal even public health—105and equal treatment has been balanced against environmental protec- tion.106 The necessity of balancing is inherent in the postulation of multiple norms at the same level—EU treaty and general principle—which means that the Court frequently must “strike a ‘balance’” between competing “integrated” values.107 This reality marks the decline of hierarchy and the corresponding rise of proportionality in internal case law. This also affects the nature of the norms themselves and, by extension, the EU legal system in which norms are accorded relative—rather than intrinsic—weight. Accordingly, restrictions to fundamental rights must “not constitute, with regard to the aim pursued, disproportionate and unreasonable interference undermining the very substance of that right.”108 This means that such rights are not “absolute prerogatives,”109 but may have an “essence” or “very substance,” which suggests a hardening at their center, although the precise scope of this varies and is unclear.110 This flexibility is perhaps appropriate given the proliferation of fundamental rights— some of vital importance, but some covering even “the most mundane matters.”111 Ultimately, albeit with stricter review from the Court, fundamental rights have a “relational character”112 and the Court readily balances them against other norms,113 essentially seeking a “fair balance between them.”114 Thus, “[t]here is no distinction and hence no hierarchical relationship being posited by the European Court between the basic human rights . . . and the free market rights.”115 Instead, human rights are subject to “the interpretative practice of balancing to reconcile the com- peting rights and interests.”116 Similarly, concerning trade liberalization and consumer protection, Weatherill observes “many delicate cases” in which “[j]udges must decide which interest prevails – and why,”117 with pro- portionality analyses facilitating the “sensitive task of adjudication without any intricate set of guidance or hierarchy mapped by the founding Treaties.”118 Ultimately, “the Court makes a radical choice on a case-by-case basis . . . IV. Proportionality Applied to Member State Action radical in the sense that there are no overarching criteria that provide a rational basis for preferring one value to another.”119 Properly applied, however, it has also been suggested that “[b]alancing offers the best possible predictability in a flexible 111Mattias Kumm, Internationale Handelsgesellschaft, Nold and the New Human Rights Paradigm, in THE PAST AND FUTURE OF EU LAW: THE CLASSICS OF EU LAW REVISITED ON THE 50TH ANNIVERSARY OF THE ROME TREATY 107 (Miguel Poiares Maduro & Loïc Azoulai eds., 2010). See particularly Deutsches Weintor eG, Case C-544/10. (Miguel Poiares Maduro & Loïc Azoulai eds., 2010). See particularly Deutsches Weintor eG, Case C-544/10. 112Marton Varju, European Union Human Rights Law: The Dynamics of Interpretation and Context (Edward Elgar 2014) 114See, e.g., Deutsches Weintor eG, Case C-544/10, at para. 47. J pp p J g g y ( ) ( ) Law Review 669 cited in Sionaidh Douglas-Scott, Constitutional Law of the European Union (Pearson 2002) 457. 116MARTON VARJU, EUROPEAN UNION HUMAN RIGHTS LAW: THE DYNAMICS OF INTERPRETATION AND CONTEXT 264 (2014). 117STEPHEN WEATHERILL, EU CONSUMER LAW AND POLICY 308 (2d ed. 2014). 118Id. 117STEPHEN WEATHERILL, EU CONSUMER LAW AND POLICY 308 (2d ed. 2014). 118Id. 119See BECK, supra note 1, at 178. 119See BECK, supra note 1, at 178. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 570 jurisdiction. [As] [a]ll conflicting interests are taken into account clearly and openly in every single case.”120 Schmidberger—concerning protests limiting free movement of goods—is a helpful example of this. There, the Court stated that “the interests involved must be weighed having regard to all the circumstances of the case in order to determine whether a fair balance was struck between those interests.”121 In comparing the protest in Schmidberger to the protest in Spanish Strawberries122— which was found to have “serious and repeated disruptions to public order”123—the Court was able to indicate the factors that contributed to the Schmidberger protest being justified, identifying the “various administrative and supporting measures [that] were taken by the competent author- ities in order to limit as far as possible the disruption to road traffic.”124 120MATTHIAS KLATT & MORITZ MEISTER, THE CONSTITUTIONAL STRUCTURE OF PROPORTIONALITY 32 (2012). 121Id. at para. 81. 122ECJ, Case C-265/95, Comm’n v. France, ECLI:EU:C:1997:595 (Dec. 9, 1997), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-265/95. 123Id. at para. 88. 124Id. at para. 87 125But see Harbo, supra note 13, at 158–85. 126Sauter, supra note 88, at 464. 127CRAIG, supra note 95, at 647. 128See, e.g., TFEU arts. 258, 259, 263, 267 when questioning the validity of EU legislation. 129Environmental protection is a notable example and causes scholars to question whether prominent EU environmental law case law from the 1980s would be decided similarly today. See STUART BELL, DONALD MCGILLIVRAY & OLE PEDERSEN, ENVIRONMENTAL LAW 225 (8th ed. 2013). See also ELINA PAUNIO, LEGAL CERTAINTY IN MULTILINGUAL EU LAW: LANGUAGE, DISCOURSE AND REASONING AT THE EUROPEAN COURT OF JUSTICE 87–94 (2013). 130For exceptions, see infra, Section D(III)(3). D. Emergence of Discretion by Means Other than Proportionality in External Relations Case Law Due to its Significant Absence I. Inevitability of Discretion: Proportionality’s Shadow in External Relations Case Law As noted at the introduction, proportionality analyses are very largely absent in external rela- tions case law.130 Instead, there is the purport of a formalistic hierarchy, in which international law is mechanically applied in external relations case law in situations where it has direct effect. However, having had a prominent role in developing norms incrementally in internal 120MATTHIAS KLATT & MORITZ MEISTER, THE CONSTITUTIONAL STRUCTURE OF PROPORTIONALITY 32 (2012). 121Id. at para. 81. 122ECJ, Case C-265/95, Comm’n v. France, ECLI:EU:C:1997:595 (Dec. 9, 1997), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-265/95. 123Id. at para. 88. 124Id. at para. 87 125But see Harbo, supra note 13, at 158–85. 126Sauter, supra note 88, at 464. 127CRAIG, supra note 95, at 647. 128See, e.g., TFEU arts. 258, 259, 263, 267 when questioning the validity of EU legislation. 129Environmental protection is a notable example and causes scholars to question whether prominent EU environmental law case law from the 1980s would be decided similarly today. See STUART BELL, DONALD MCGILLIVRAY & OLE PEDERSEN, ENVIRONMENTAL LAW 225 (8th ed. 2013). See also ELINA PAUNIO, LEGAL CERTAINTY IN MULTILINGUAL EU LAW: LANGUAGE, DISCOURSE AND REASONING AT THE EUROPEAN COURT OF JUSTICE 87–94 (2013). 130For exceptions see infra Section D(III)(3) 126Sauter, supra note 88, at 464. 130For exceptions, see infra, Section D(III)(3). V. Reflections on Proportionality Internally In spite of many variables—based on actor, subject matter, nature of right, and changing weight of norm—harsh critics of proportionality within the EU remain few in number.125 Instead, there is recognition by most that proportionality can mitigate the harshness of supremacy coupled with direct effect from the Member State point of view,126 in addition to providing meaningful review of EU action.127 It can also be seen that even where loosely or partially followed, the common steps of proportionality will prompt the Court—in what is undeniably a complex and developing legal system—to at least be somewhat forthcoming concerning its reasoning, and to engage with the facts and circumstances of the case in a concrete way. y With direct effect invariably granted—and often not being required128—it is evident that dis- cretion in internal case law manifests itself primarily through proportionality, that the EU legal order is emerging as non-hierarchical in practice, and that the Court has a key role in incremen- tally developing the relevant norms.129 These factors carry implications for external relations case law, as can now be explored. D. Emergence of Discretion by Means Other than Proportionality in External Relations Case Law Due to its Significant Absence 131On the collaborative development of norms by the Court and legislature, see Phil Syrpis, Theorising the Relationship Between the Judiciary and the Legislature in the EU Internal Market, in THE JUDICIARY, THE LEGISLATURE AND THE EU INTERNAL MARKET (Phil Syrpis ed., 2012). 132Opinion 1/91, Re Agreement on the European Economic Area, 1994 ECR I-6079. 133Id. at paras. 51–52. 134Antonio Cassese, Remarks on Scelle’s Theory of “Role Splitting” (dédoublement fonctionnel) in International Law, 1 EUR. J. INT’L L. 210, 213 (1990). 135Halberstam, supra note 38, at 197. 136See NOLLKAEMPER, supra note 27, at 47. See also Aust, Rodiles & Staubach, supra note 38, at 111. 137LON L. FULLER, THE MORALITY OF LAW 112 (1969). 138Compare, for example, the narrow, hierarchical, mechanical approach in MAX WEBER, ECONOMY AND SOCIETY 886 (Guenther Ross & Claus Wittich eds., 1978), with the Herculean, all-encompassing, but less certain approach in RONALD DWORKIN, LAW’S EMPIRE, 245 (1986). 139KLATT & MEISTER, supra note 120, at 32. 140See infra, Section D(V). https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 571 German Law Journal case law,131 the following question remains: Is it to be expected that the Court will eschew the norms developed in its internal case law when applying international law in external relations cases? The Court’s refusal to permit its members to sit on any newly created international court in Opinion 1/91132 was based on its view that “it will be very difficult, if not impossible, for those judges, when sitting in the Court of Justice, to tackle questions with completely open minds where they have taken part in determining those questions as members of the EEA Court.”133 However, the converse must hold, and even more strongly. The Court cannot be expected to disavow its normative conceptions and mechanically apply international provisions to invalidate EU law. Indeed, the idea that courts have the capacity to “split roles,” so that “any time a domestic court deals with a conflict of law question, it acts qua an international body,”134 has recently been described as “overly optimistic”135 and “shaky.”136 The external relations case law analyzed below is strong evidence that the Court is not able to divide its internal function from its role in applying international law. Instead, modified rules of reliance and application seek to alleviate the absence of proportionality in external relations case law so as to protect EU norms from international law. Setting aside the fact that “it is by no means clear that a neatly defined hierarchy is always the best way of resolving conflicts within a legal system,”137 it is true that a strict hierarchy, mechanically enforced, would give rise to greater legal certainty than would balancing competing norms through proportionality.138 However, as we shall now see, this is no longer the case in external relations case law. Unsurprisingly, normative concerns have permeated the strict hierarchy. We will see that the direct effect test is modified relentlessly, autonomy is claimed—on unclear bases and with varied effects, and interpretative methods emerge as questionable concerning international treaty and customary international law (CIL). This though, it will be seen, falls a long way short of propor- tionality, in which “[a]ll conflicting interests are taken into account clearly and openly in every single case.”139 Instead, we have the worst of both worlds: Neither a strict hierarchy nor propor- tionality, but rather discretion exercised covertly through supposedly mechanical rules. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 141This also reflects the suggestions of Eeckhout and Mendez concerning reform of external relations case law, considered above, supra Section B(IV). 142See ECJ, Joined Cases C-21 to 24/72, International Fruit Company and Others v. Produktschap voor Groenten en Fruit, ECLI:EU:C:1972:115 (Dec. 12, 1972), http://curia.europa.eu/juris/liste.jsf?language=en&num=C-21/72. 143Haegeman, Case C-181/73. 144Id. at para. 5. 145See, e.g., Germany v. Council, Case C–280/93; ECJ, Case C-149/96, Portugal v. Council, ECLI:EU:C:1999:574 (Nov. 23, 1999), http://curia.europa.eu/juris/liste.jsf?language=en&num=C-149/96. 146ECJ, Case C-61/94, Comm’n v. Germany, ECLI:EU:C:1996:313 (Sep. 10, 1996), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-61/94. 147ECJ, Case C-377/98, Netherlands v. Comm’n, ECLI:EU:C:2001:523 (Oct. 9, 2001), paras. 54–56, http://curia.europa.eu/ juris/liste.jsf?language=en&num=C-377/98. 148EECKHOUT, supra note 53, at 297–98. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press The result is a lack of nuanced balance and loss of legal certainty. Whilst proportionality would not overcome the reality that domestic judges, either through issues of competence, confidence, or allegiance, are more likely to favor domestic methods or viewpoints, the benefits of its application to this area would be significant.140 Following the analy- sis on external relations case law we will be better placed to understand the modest means by which proportionality could be deployed. Indeed, in some cases it already has been. It should also be mentioned that the proposal to deploy proportionality in external relations case law is not lim- ited to those agreements which have direct effect currently. Instead, it would be anticipated that access to proportionality for the Court will lessen the need for narrow distinctions within direct https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 572 Rupert Dunbar effect, thus mirroring more fully the internal approach with a more ready acceptance of direct effect where needed and subsequent application of proportionality.141 II. Manifestations of Discretion in External Relations Case Law I: Modifications to Direct Effect-Related Rules in the Absence of Proportionality 1. Discretion Through Wider Application of Direct Effect Tests in External Relations Case Law In spite of distinguishing EU law as a “new legal order of international law” uniquely capable of direct effect in Van Gend en Loos, in International Fruit,142 the Court confirmed that provisions of “old” international law could also have direct effect in the EU. Though relatively receptive of international law, this case already marked a divergence from the equivalent scope of application of internal direct effect. As above, we have seen that in Article 267 TFEU cases calling into ques- tion validity of EU legislation, direct effect of relevant rules is not required, whereas in International Fruit it was. However, in Haegeman II,143 the Court reviewed the Commission’s imposition of a “counter- vailing charge” on the basis of an Association Agreement with Greece. The case was also an Article 267 TFEU referral, and the Court utilized for the first time the oft-cited phrase that “[t]he pro- visions of [an international] agreement, from the coming into force thereof, form an integral part of Community law.”144 This expression was particularly fitting as the Court did not enquire into direct effect at all, which matched with the internal approach. 149R. Pavoni, Controversial Aspects of the Interaction Between International and EU Law in Environmental Matters: Direct Effect and Member States’ Unilateral Measures, in THE EXTERNAL ENVIRONMENTAL POLICY OF THE EUROPEAN UNION 351 (Elisa Morgera ed., 2012). 150Opinion of Advocate General Jääskinen at para. 10, ECJ, Joined Cases C-404 & 405/12 P, Council and Comm’n v. Stichting Natuur en Milieu and Pesticide Action Network Europe, ECLI:EU:C:2014:309 (May 8, 2014), http://curia. europa.eu/juris/liste.jsf?language=en&num=C-404/12. 156The debate over the merits of this position has been intense. For a defense of the jurisprudence of the Court, see, for example, S. Griller, Judicial Enforceability of WTO Law in the European Union: Annotation to Case C-149/96, Portugal v Council, 3 J. INT’L ECON. L. 441 (2000); Piet Eeckhout, Judicial Enforcement of WTO Law in the European Union – Some Further Reflections, 5 J. INT’L ECON. L. 91 (2002); MENDEZ, supra note 4, at 174–249. For criticism, see, for example, Ernst-Ulrich Petersmann, Can the EU’s Disregard for “Strict Observance of International Law” (Article 3 TEU) Be Constitutionally Justified?, in TRADE AND COMPETITION LAW IN THE EU AND BEYOND (Inge Govaere, Reinhard Quick & Marco Bronckers eds., 2011); Nikos Lavranos, The ECJ’s Relationship with Other International Courts and Tribunals, in EUROPE: THE NEW LEGAL REALISM - ESSAYS IN HONOUR OF HJALTE RASMUSSEN (Henning Koch et al. eds., 2010). 154Szilárd Gáspár-Szilágyi, EU International Agreements Through a US Lens: Different Methods of Interpretation, Tests and the Issue of “Rights”, 39 EUR. L. REV. 601, 615 (2014). 155See ECJ, Case C-12/86, Demirel v. Stadt Schwäbisch Gmünd, ECLI:EU:C:1987:400 (Sep. 30, 1987), para 14, http://curia. europa.eu/juris/liste.jsf?language=en&num=C-12/86. See also ECJ, Case C-213/03, Syndicat Professionnel Coordination des Pêcheurs de l'Etang de Berre et de la Region v. Électricité de France, ECLI:EU:C:2004:464 (July 15, 2004), para. 39, http://curia. europa.eu/juris/liste.jsf?language=en&num=C-213/03; ECJ, Case C-171/01, Wählergruppe "Gemeinsam Zajedno/Birlikte Alternative und Grüne GewerkschafterInnen/UG", and Bundesminister für Wirtschaft und Arbeit and Others, ECLI:EU: C:2003:260 (May 8, 2003), para. 54, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-171/01. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 151International Fruit Company, Joined Cases C-21 to 24/72. 152Id. at para. 8. 149R. Pavoni, Controversial Aspects of the Interaction Between International and EU Law in Environmental Matters: Direct Effect and Member States’ Unilateral Measures, in THE EXTERNAL ENVIRONMENTAL POLICY OF THE EUROPEAN UNION 351 (Elisa Morgera ed., 2012). 150Opinion of Advocate General Jääskinen at para. 10, ECJ, Joined Cases C-404 & 405/12 P, Council and Comm’n v. Stichting Natuur en Milieu and Pesticide Action Network Europe, ECLI:EU:C:2014:309 (May 8, 2014), http://curia. europa.eu/juris/liste.jsf?language=en&num=C-404/12. 151International Fruit Company, Joined Cases C-21 to 24/72. 152Id. at para. 8. 153Id. at para. 20. 154Szilárd Gáspár-Szilágyi, EU International Agreements Through a US Lens: Different Methods of Interpretation, Tests and the Issue of “Rights”, 39 EUR. L. REV. 601, 615 (2014). 155See ECJ, Case C-12/86, Demirel v. Stadt Schwäbisch Gmünd, ECLI:EU:C:1987:400 (Sep. 30, 1987), para 14, http://curia. europa.eu/juris/liste.jsf?language=en&num=C-12/86. See also ECJ, Case C-213/03, Syndicat Professionnel Coordination des Pêcheurs de l'Etang de Berre et de la Region v. Électricité de France, ECLI:EU:C:2004:464 (July 15, 2004), para. 39, http://curia. europa.eu/juris/liste.jsf?language=en&num=C-213/03; ECJ, Case C-171/01, Wählergruppe "Gemeinsam Zajedno/Birlikte Alternative und Grüne GewerkschafterInnen/UG", and Bundesminister für Wirtschaft und Arbeit and Others, ECLI:EU: C:2003:260 (May 8, 2003), para. 54, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-171/01. 156The debate over the merits of this position has been intense. For a defense of the jurisprudence of the Court, see, for example, S. Griller, Judicial Enforceability of WTO Law in the European Union: Annotation to Case C-149/96, Portugal v Council, 3 J. INT’L ECON. L. 441 (2000); Piet Eeckhout, Judicial Enforcement of WTO Law in the European Union – Some Further Reflections, 5 J. INT’L ECON. L. 91 (2002); MENDEZ, supra note 4, at 174–249. For criticism, see, for example, Ernst-Ulrich Petersmann, Can the EU’s Disregard for “Strict Observance of International Law” (Article 3 TEU) Be Constitutionally Justified?, in TRADE AND COMPETITION LAW IN THE EU AND BEYOND (Inge Govaere, Reinhard Quick & Marco Bronckers eds., 2011); Nikos Lavranos, The ECJ’s Relationship with Other International Courts and Tribunals, in EUROPE: THE NEW LEGAL REALISM - ESSAYS IN HONOUR OF HJALTE RASMUSSEN (Henning Koch et al. eds., 2010). 151International Fruit Company, Joined Cases C-21 to 24/72. p 154Szilárd Gáspár-Szilágyi, EU International Agreements Through a US Lens: Different Methods of Interpretation, Tests and the Issue of “Rights”, 39 EUR. L. REV. 601, 615 (2014). 155See ECJ, Case C-12/86, Demirel v. Stadt Schwäbisch Gmünd, ECLI:EU:C:1987:400 (Sep. 30, 1987), para 14, http://curia. europa.eu/juris/liste.jsf?language=en&num=C-12/86. See also ECJ, Case C-213/03, Syndicat Professionnel Coordination des Pêcheurs de l'Etang de Berre et de la Region v. Électricité de France, ECLI:EU:C:2004:464 (July 15, 2004), para. 39, http://curia. europa.eu/juris/liste.jsf?language=en&num=C-213/03; ECJ, Case C-171/01, Wählergruppe "Gemeinsam Zajedno/Birlikte Alternative und Grüne GewerkschafterInnen/UG", and Bundesminister für Wirtschaft und Arbeit and Others, ECLI:EU: C:2003:260 (May 8, 2003), para. 54, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-171/01. 156The debate over the merits of this position has been intense. For a defense of the jurisprudence of the Court, see, for example, S. Griller, Judicial Enforceability of WTO Law in the European Union: Annotation to Case C-149/96, Portugal v Council, 3 J. INT’L ECON. L. 441 (2000); Piet Eeckhout, Judicial Enforcement of WTO Law in the European Union – Some Further Reflections, 5 J. INT’L ECON. L. 91 (2002); MENDEZ, supra note 4, at 174–249. For criticism, see, for example, Ernst-Ulrich Petersmann, Can the EU’s Disregard for “Strict Observance of International Law” (Article 3 TEU) Be Constitutionally Justified?, in TRADE AND COMPETITION LAW IN THE EU AND BEYOND (Inge Govaere, Reinhard Quick & Marco Bronckers eds., 2011); Nikos Lavranos, The ECJ’s Relationship with Other International Courts and Tribunals, in EUROPE: THE NEW LEGAL REALISM - ESSAYS IN HONOUR OF HJALTE RASMUSSEN (Henning Koch et al. eds., 2010). https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press However, the case has become an anomaly, and direct effect appears to be required before international law can be relied upon to invalidate EU legislation in Article 267 TFEU actions. g It also emerged that even privileged applicants under Article 263 TFEU—such as the Member States—cannot rely on international agreements to which the EU is party to challenge EU legis- lative action, unless that provision is directly effective.145 In this light, it is worthy of mention that the Commission’s capacity to rely on international agreements to which the EU is party in 258 TFEU enforcement actions against Member States is uncircumscribed—matching the approach in internal case law.146 This gives rise to some asymmetry. However, in Biotechnological Inventions, an Article 263 TFEU case, the Court stated that147: Even if . . . the [Convention on Biological Diversity] contains provisions which do not have direct effect . . . that fact does not preclude review by the courts of compliance with the obli- gations incumbent on the Community as a party to that agreement. This matches with the internal approach in which challenges to EU action are readily enter- tained. Eeckhout has speculated that the reason for departure from previous external relations case law may be “that the Court felt embarrassed by the incapacity of the Member States to seek judicial review [of EU legislation] on grounds of violation of certain [international] agreements.”148 However, Eeckhout forewarned that this approach may not continue, and Pavoni notes that https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 573 German Law Journal “it appears that the Court of Justice is willing to duck the Biotech Patents decision, which has never been endorsed in its subsequent jurisprudence on external relations.”149 And, in spite of encouragement by Advocate General Jääskinen, the Court was not inclined to utilize it more recently.150 The above demonstrates oscillations concerning the requirement for direct effect externally in Article 263 and 267 TFEU cases; a requirement which is not present in internal case law. Accordingly, direct effect, at the Court’s discretion, has a potentially wider application than in internal case law. There are also significant oscillations in the application of the direct effect test itself externally, which will now be considered. 2. Discretion Through Elusive Terms: Broad Scope and Logic/Spirit or Nature of Agreement In International Fruit,151 the Court stated that152: 2. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Discretion Through Elusive Terms: Broad Scope and Logic/Spirit or Nature of Agreement In International Fruit,151 the Court stated that152: Before invalidity can be relied upon before a national court, that provision of international law must also be capable of conferring rights on citizens of the Community which they can invoke before the courts. The Court then explained that “the spirit, the general scheme and the terms of the general agreement must be considered” in order to answer this question.153 The words “spirit” or “nature” provide potentially elusive benchmarks, and therefore “the CJEU’s discretion in applying these criteria is high and requires an amount of subjectivity.”154 Similar formulations have been used by the Court since. These tended not to utilize the conferral of rights criteria expressly, instead replacing it with analysis of the “wording and the purpose and nature”155 of agreements, which is equally elusive. The Court then explained that “the spirit, the general scheme and the terms of the general agreement must be considered” in order to answer this question.153 The words “spirit” or “nature” provide potentially elusive benchmarks, and therefore “the CJEU’s discretion in applying these criteria is high and requires an amount of subjectivity ”154 Similar formulations have been used p p y , J pp y g criteria is high and requires an amount of subjectivity.”154 Similar formulations have been used by the Court since. These tended not to utilize the conferral of rights criteria expressly, instead replacing it with analysis of the “wording and the purpose and nature”155 of agreements, which is equally elusive. by the Court since. These tended not to utilize the conferral of rights criteria expressly, instead replacing it with analysis of the “wording and the purpose and nature”155 of agreements, which is equally elusive. It is well known that the Court’s case law concerning, first, General Agreement on Tariffs and Trade (GATT), and second, World Trade Organization (WTO) agreements, resulted in the blan- ket exclusion of direct effect of these treaties based on their “broad scope and logic.”156 In International Fruit, the Court refused direct effect of GATT due to its flexible nature. The 153Id. at para. 20. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 574 subsequent WTO agreement was viewed as carrying more concrete obligations. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press However, in Portuguese Textiles, the Court refused to accept that it could have direct effect.157 Whilst the Court accepted that the WTO agreement did “differ significantly . . . in particular by reason of the strengthening of the system of safeguards and the mechanism for resolving disputes.”158 It added immediately that “the system resulting from those agreements nevertheless accords con- siderable importance to negotiation between the parties.”159 The Court made reference, in passing, to the preamble and to annexes too,160 the former of which led the Court to focus on aspects of reciprocity. It noted that it was unlike agreements between the EU and other non-member coun- tries “which introduce a certain asymmetry of obligations or create special relations of integration with the Community.”161 It also went on to observe that “some of the contracting parties, which are among the most important commercial partners of the community” had not considered the rules to be “applicable by their judicial organs when reviewing the legality of their rules of domes- tic law.”162 However, in Kupferberg, the Court broadly asserted that a “framework for consultations and negotiations between the parties inter se . . . is not in itself sufficient to exclude all judicial appli- cation of it.”163 This was because case concerned a Free Trade Agreement with Portugal,164 and may therefore be thought to be rather different from WTO law. Indeed, Eeckhout notes that con- cerning “direct effect of free-trade, co-operation, and association agreements the Court has never decided against direct effect on the basis of the structure and nature of the agreement as such.”165 Klabbers similarly observes of Kupferberg that “[d]irect effect within one of the parties’ legal sys- tem but not within the other’s would not, e.g., constitute a fatal lack of reciprocity.”166 g g Klabbers similarly observes of Kupferberg that “[d]irect effect within one of the parties’ legal sys- tem but not within the other’s would not, e.g., constitute a fatal lack of reciprocity.”166 g p y Of course, the reality is that reciprocity is being used as an inconvenient proxy, enabling covert differentiation on the basis of the trading partner. But this creates confusion. 157Portugal v. Council, Case C-149/96. 158Id. at para. 47. 159Id. at para. 47. 160Id. at para. 42. 161Id. 162Id. at para. 44. 163ECJ C C 104/81 H t ll t M 157Portugal v. Council, Case C-149/96. 158Id. at para. 47. 159Id. at para. 47. 160Id. at para. 42. 161Id. 162Id. at para. 44. 163ECJ, Case C-104/81, Hauptzollamt Mainz v. CA Kupferberg, ECLI:EU:C:1982:362 (Oct. 26, 1982), para. 20, http://curia. europa.eu/juris/liste.jsf?language=en&num=C-104/81. 164Agreement Concluded on 22 July 1972 Between the European Economic Community and the Portuguese Republic, 1972 O.J. SPEC. ED. 167. 165EECKHOUT, supra note 53, at 338. 166Jan Klabbers, International Law in Community Law: The Law and Politics of Direct Effect, 21 Y.B. EUR. L. 263, 282 (2001). 167Opinion of Advocate General Rozès, Case C-104/81, Hauptzollamt Mainz v. CA Kupferberg (Oct. 26, 1982). 168Marc Maresceau, Bilateral Agreements Concluded by the European Community, 309 COLLECTED COURSES HAGUE ACAD. INT’L L. 125, 295 (2004). 169ECJ, Case C-192/89, S.Z. Sevince v. Staatssecretaris Van Justitie, ECLI:EU:C:1990:322 (Sept. 20, 1990), https://curia. europa.eu/juris/liste.jsf?language=en&num=C-192/89. 170Council Directive of December 29, 1964, Establishing an Association between the European Economic Community and Turkey, 1977 O.J. (L. 361) 29 (EC). https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 575 German Law Journal “programme . . . not sufficiently precise and unconditional to be capable of governing directly the free movement of workers.”171 However, in Demirel, the Court referred to Decision 1/80 of the Council of Association, which had been given “exclusive powers to lay down detailed rules” for the functioning of the agreement.172 The Decision was not relevant to the case at hand, but in referring to it, the Court implied that such Decisions would be determinative in future cases. In Sevince, the Court found that the programmatic nature of the EU-Turkey Association Agreement,173 which gave the Council of Association power to lay down detailed rules for its func- tioning, “does not prevent . . . decisions . . . which give effect in specific respects to the programmes envisaged . . . from having direct effect.”174 Accordingly, Decision 2/76 and 1/80 could have direct effect, a finding that was said to “merely lay emphasis on the obligation to implement in good faith an international Agreement.”175 g The conditionality of WTO obligations will be recalled as being a central tenet in the precluding of direct effect in Portuguese Textiles.176 The WTO dispute settlement procedure’s binding nature is also debated.177 Article 19 of the Dispute Settlement Understanding (DSU) says that the panel (DSB) or Appellate Body “shall recommend that a Member concerned bring the measure into conformity” in the case of a breach or that the relevant body “may suggest ways in which the Member could implement the recommendations.” Article 22(2) DSU provides that in the event of non-compliance a “Member shall . . . enter into negotiations with any party having invoked the dispute settlement procedures, with a view to developing mutually acceptable compensation.” Article 21 DSU though speaks of “prompt compliance” being “essential” to the system. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Indeed, in Kupferberg itself, Advocate General Rozès, unlike the Court, thought that direct effect should not be granted as it would be to the Community’s “disadvantage” where Portugal did not “guarantee that an indi- vidual may rely on the provision in Portugal on the same terms and with the same results.”167 Once more, discretion emerges. 3. Discretion Through Selective Adherence to Binding Decisions of Bodies Established Under Agreements The “remarkable phenomenon”168 in which decisions of bodies established by international agree- ments could have direct effect was established in Sevince.169 This case concerned the EU-Turkey Association Agreement,170 which was found in Demirel not to have direct effect as it set out a The “remarkable phenomenon”168 in which decisions of bodies established by international agree- ments could have direct effect was established in Sevince.169 This case concerned the EU-Turkey Association Agreement,170 which was found in Demirel not to have direct effect as it set out a p j j g g 164Agreement Concluded on 22 July 1972 Between the European Economic Community and the Portuguese Republic, 1972 O.J. SPEC. ED. 167. p j j g g 164Agreement Concluded on 22 July 1972 Between the European Economic Community and the Portuguese Republic, 1972 O.J. SPEC. ED. 167. 165E t 53 t 338 , p , 166Jan Klabbers, International Law in Community Law: The Law and Politics of Direct Effect, 21 Y.B. EUR. L. 263, 282 (2001). , p , 166Jan Klabbers, International Law in Community Law: The Law and Politics of Direct Effect, 21 Y.B. EUR. L. 263, 282 (2001). 167Opinion of Advocate General Rozès, Case C-104/81, Hauptzollamt Mainz v. CA Kupferberg (Oct. 26, 1982). 167Opinion of Advocate General Rozès, Case C-104/81, Hauptzollamt Mainz v. CA Kupferberg (Oct. 26, 1982). 168Marc Maresceau, Bilateral Agreements Concluded by the European Community, 309 COLLECTED COURSES HAGUE ACAD. INT’L L. 125, 295 (2004). 168Marc Maresceau, Bilateral Agreements Concluded by the European Community, 309 COLLECTED COURSES HAGUE ACAD. INT’L L. 125, 295 (2004). e C-192/89, S.Z. Sevince v. Staatssecretaris Van Justitie, ECLI:EU:C:1990:322 (Sept. 20, 1990), https://curia. s/liste.jsf?language=en&num=C-192/89. p j j g g 170Council Directive of December 29, 1964, Establishing an Association between the European Economic Community and Turkey, 1977 O.J. (L. 361) 29 (EC). p j j g g 170Council Directive of December 29, 1964, Establishing an Association between the European Economic Community and Turkey, 1977 O.J. (L. 361) 29 (EC). 178See DSU art. 22. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press “Compensation [by the Member in breach] and suspension of other obligations [by Members harmed]” are considered to be “temporary” alternatives in the event of non-compliance and “neither compensation nor the suspension of concessions or other obligations is preferred to full implementation of a recommendation to bring a measure into conformity with the covered agree- ments.”178 Article 3(7) DSU supports this further by stating that “compensation should be resorted to only if the immediate withdrawal of the measure is impracticable and as a temporary measure.” Could this clear postulation of a hierarchical preference for compliance179 render a DSB or Panel recommendation directly effective in the EU legal system given the “good faith” in imple- mentation of international law referred to in Sevince? The Biret180 case appeared to leave the door open to such a possibility.181 A negative DSB find- ing was made in 1997 concerning the EU’s banning of certain substances being administered to farm animals.182 Though the case was unsuccessful, the reasoning of the Court was very much 177See Understanding on Rules and Procedures Governing the Settlement of Disputes, art. 1, Apr. 15, 1994, Marrakesh Agreement Establishing the World Trade Organization, Annex 2, 1869 U.N.T.S. 401 [hereinafter DSU]. For a close analysis of the WTO case law see Petros C. Mavroidis, Remedies in the WTO Legal System: Between a Rock and a Hard Place, 11(4) EUR. J. INT’L. L. 763 (2000). 179Eeckhout suggests that, after a breach has been “authoritatively” found by the Dispute Settlement Body, there is “a vio- lation of the DSU provisions requiring compliance” and that, “[a]ll this is quite serious.” See Eeckhout, supra note 57, at 27. 180ECJ, Case C-93/02P, Biret International SA v. Council, ECLI:EU:C:2003:517, (Sept. 30, 2003), https://curia.europa.eu/ 179Eeckhout suggests that, after a breach has been “authoritatively” found by the Dispute Settlement Body, there is “a vio- lation of the DSU provisions requiring compliance” and that, “[a]ll this is quite serious.” See Eeckhout, supra note 57, at 27. 180ECJ, Case C-93/02P, Biret International SA v. Council, ECLI:EU:C:2003:517, (Sept. 30, 2003), https://curia.europa.eu/ juris/liste.jsf?language=en&jur=C,T,F&num=c-93/02&td=ALL. 171ECJ, Case 12/86, Meryem Demirel v. Stadt Schwäbisch Gmünd, ECLI:EU:C:1987:400 (Sept. 30, 1987), para. 23, https:// curia.europa.eu/juris/liste.jsf?language=en&jur=C,T,F&num=C-12/86&td=ALL. 172See Meryem Demirel, Case 12/86 at para. 21. 173Council Directive of 29 December 1964, Establishing an Association between the European Economic Community and Turkey, 1977 O.J. (L. 361) 29 (EC). 174See Sevince, Case C-192/89 at para. 21. 175Id. at para. 23. 176ECJ Case C 149/96 Port gal Co ncil ECLI EU C 1999 574 (No 23 1999) http //c ria e ropa e /j ris/liste jsf? 171ECJ, Case 12/86, Meryem Demirel v. Stadt Schwäbisch Gmünd, ECLI:EU:C:1987:400 (Sept. 30, 1987), para. 23, https:// curia.europa.eu/juris/liste.jsf?language=en&jur=C,T,F&num=C-12/86&td=ALL. 172See Meryem Demirel, Case 12/86 at para. 21. 173Council Directive of 29 December 1964, Establishing an Association between the European Economic Community and Turkey, 1977 O.J. (L. 361) 29 (EC). 174See Sevince, Case C-192/89 at para. 21. 175Id. at para. 23. 176ECJ, Case C-149/96, Portugal v. Council, ECLI:EU:C:1999:574 (Nov. 23, 1999), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-149/96. 177See Understanding on Rules and Procedures Governing the Settlement of Disputes, art. 1, Apr. 15, 1994, Marrakesh Agreement Establishing the World Trade Organization, Annex 2, 1869 U.N.T.S. 401 [hereinafter DSU]. For a close analysis of the WTO case law see Petros C. Mavroidis, Remedies in the WTO Legal System: Between a Rock and a Hard Place, 11(4) EUR. J. INT’L. L. 763 (2000). 178See DSU art. 22. 179Eeckhout suggests that, after a breach has been “authoritatively” found by the Dispute Settlement Body, there is “a vio- lation of the DSU provisions requiring compliance” and that, “[a]ll this is quite serious.” See Eeckhout, supra note 57, at 27. 180ECJ, Case C-93/02P, Biret International SA v. Council, ECLI:EU:C:2003:517, (Sept. 30, 2003), https://curia.europa.eu/ juris/liste.jsf?language=en&jur=C,T,F&num=c-93/02&td=ALL. 181Mario Mendez, The Impact of WTO Rulings in the Community Legal Order, 29 EUR. L. REV. 517, 529 (2004); ANNE THEIS, INTERNATIONAL TRADE DISPUTES AND EU LIABILITY 27 (2013). 182The substance ban included certain hormonal supplements and all substances having thyrostatic action. Turkey, 1977 O.J. (L. 361) 29 (EC). 174See Sevince, Case C-192/89 at para. 21. 175Id. at para. 23. 176ECJ, Case C-149/96, Portugal v. Council, ECLI:EU:C:1999:574 (Nov. 23, 1999), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-149/96. 177S U d t di R l d P d G i th S ttl t f Di t t 1 A 15 1994 M k h p 176ECJ, Case C-149/96, Portugal v. Council, ECLI:EU:C:1999:574 (Nov. 23, 1999), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-149/96. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 183ECJ, Case C-69/89, Nakajima v. Council, ECLI:EU:C:1991:186 (July 5, 1991) https://curia.europa.eu/juris/documents.jsf? num=C-69/89. See also supra notes 229–36 and accompanying text. 184See Biret International, Case C-93/02P at para. 74. 185Id. at para. 67. 186In addition to the already noted passages, the Court added, “[t]he purpose of the WTO agreements is to govern relations between States or regional organisations for economic integration and not to protect individuals.” See id. at para. 72. 187ECJ, Case C-377/02, Léon van Parys NV v. Belgisch Interventieen Restitutiebureau, ECLI:EU:C:2005:121 (Mar. 1, 2005), https://curia.europa.eu/juris/liste.jsf?language=en&num=C-377/02. 188Council Regulation 1637/98 of July 20, 1998, amending Regulation (EEC) No. 404/93 On the Common Organisation of the Market in Bananas, 1998 O.J. (L 210) 28 (EC). 189EECKHOUT, supra note 53, at 364. 190Leon van Parys, Case C-377/02 at para. 50. 191Nakajima, Case C-69/89. Considered below, at Part 4.2.6. 192Id. at para. 43. 193Peter-Tobias Stoll & Arthur Steinmann, WTO Dispute Settlement: The Implementation Stage, 3 MAX PLANCK Y.B. ( ) https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 179Eeckhout suggests that, after a breach has been “authoritatively” found by the Dispute Settlement Body, there is “a vio lation of the DSU provisions requiring compliance” and that, “[a]ll this is quite serious.” See Eeckhout, supra note 57, at 27 Eeckhout suggests that, after a breach has been authoritatively found by the Dispute Settlement Body, there is a vio- lation of the DSU provisions requiring compliance” and that, “[a]ll this is quite serious.” See Eeckhout, supra note 57, at 27. 180ECJ, Case C-93/02P, Biret International SA v. Council, ECLI:EU:C:2003:517, (Sept. 30, 2003), https://curia.europa.eu/ juris/liste.jsf?language=en&jur=C,T,F&num=c-93/02&td=ALL. p q g p , [ ] q , p , 180ECJ, Case C-93/02P, Biret International SA v. Council, ECLI:EU:C:2003:517, (Sept. 30, 2003), https://curia.europa.eu/ juris/liste.jsf?language=en&jur=C,T,F&num=c-93/02&td=ALL. 180ECJ, Case C-93/02P, Biret International SA v. Council, ECLI juris/liste.jsf?language=en&jur=C,T,F&num=c-93/02&td=ALL. j j g g j 181Mario Mendez, The Impact of WTO Rulings in the Community Legal Order, 29 EUR. L. REV. 517, 529 (2004); ANNE THEIS, INTERNATIONAL TRADE DISPUTES AND EU LIABILITY 27 (2013). j j g g j 181Mario Mendez, The Impact of WTO Rulings in the Community Legal Order, 29 EUR. L. REV. 517, 529 (2004); ANNE THEIS, INTERNATIONAL TRADE DISPUTES AND EU LIABILITY 27 (2013). 182The substance ban included certain hormonal supplements and all substances having thyrostatic action 182The substance ban included certain hormonal supplements and all substances having thyrostatic ac https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 576 focused on the “timeline.” As will be seen below, where the EU intends to implement WTO law, a review of secondary legislation on its basis can occur.183 However, because the relevant Union legislation was adopted “several years before” the WTO rules came into force, it was “not logically possible for them either to give rise to a specific obligation entered into under that agreement or to refer expressly to some of its provisions.”184 As to the breach of the DSB recommendation, the Court observed that a grace period had been provided until May 13, 1999. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press As the applicant’s losses fell within this period, the Court found that the EU “cannot, on any view, have incurred liabil- ity.”185 Though the tone of the judgment could hardly be considered generous to the possible effects of WTO law before EU courts, by engaging with detailed claims rather than dismissing them in “broad logic,” the question remained concerning the possible direct effect of WTO law after DSB or Appellate Body decisions.186 The Van Parys case followed.187 After a negative 1997 DSB finding on EU banana imports, the EU implemented a new Regulation188 “expressly” referring to the aim of bringing EU rules into compliance with the WTO system.189 This was found not to have been successful by the WTO in 1999, and the Court acknowledged that at that point the time limit had expired.190 Van Parys challenged the Regulation, but the combined considerations of expired time limit and possible Nakajima-type implementation principle191 were not sufficient for WTO rules to be relied upon. The Court instead placed great emphasis on Article 22(2) DSU, allowing for negotiation over compensation. The Court did acknowledge that the main aim under Article 3(7) DSU was com- pliance, but also that there is anticipation of continued breach “where the immediate withdrawal of the measures is impracticable.”192 Though it is suggested that, at WTO level, “the exception [of impracticability] has become the rule,”193 the argument is at least engaged with. In Van Parys, there was no questioning at all of whether impracticability was present, let alone a reference to the fact that the relevant time period, even in the presence of impracticability, “should not exceed 15 months.”194 This was so in spite of the reference from the Belgian court being lodged in 2002, some three years after the negative WTO finding. The Court’s refusal to acknowledge the shift from political discretion to legal obligation in WTO proceedings at any stage and its avoid- ance of Nakajima when it is “hard to envisage” a more relevant case, has been criticized by scholars.195 What is notable for our purposes concerning this WTO case law is the Court’s disinclination to pick a clear course and stick to it. Instead, discretion is retained—at the expense of legal 183ECJ, Case C-69/89, Nakajima v. Council, ECLI:EU:C:1991:186 (July 5, 1991) https://curia.europa.eu/juris/documents.jsf? num=C-69/89. See also supra notes 229–36 and accompanying text. 184See Biret International, Case C-93/02P at para. 74. 185Id. at para. 67. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 186In addition to the already noted passages, the Court added, “[t]he purpose of the WTO agreements is to govern relations between States or regional organisations for economic integration and not to protect individuals.” See id. at para. 72. 187ECJ, Case C-377/02, Léon van Parys NV v. Belgisch Interventieen Restitutiebureau, ECLI:EU:C:2005:121 (Mar. 1, 2005), https://curia.europa.eu/juris/liste.jsf?language=en&num=C-377/02. 188Council Regulation 1637/98 of July 20, 1998, amending Regulation (EEC) No. 404/93 On the Common Organisation of the Market in Bananas, 1998 O.J. (L 210) 28 (EC). 189EECKHOUT, supra note 53, at 364. 190Leon van Parys, Case C-377/02 at para. 50. 191Nakajima, Case C-69/89. Considered below, at Part 4.2.6. 192Id. at para. 43. 193Peter-Tobias Stoll & Arthur Steinmann, WTO Dispute Settlement: The Implementation Stage, 3 MAX PLANCK Y.B. p 193Peter-Tobias Stoll & Arthur Steinmann, WTO Dispute Settlement: The Implementation Stage, 3 MAX PLANCK Y.B. UNITED NATIONS L. 407, 411 (1999). 194 p p g UNITED NATIONS L. 407, 411 (1999). 194DSU art. 21(3). 195 UNITED NATIONS L. 407, 411 (1999) 194DSU art. 21(3). UNITED NATIONS L. 407, 411 (1999). 194DSU art. 21(3). 195See EECKHOUT, supra note 53, at 364. See also Piet Eeckhout, The Appellate Bodies and the Courts, in WTO LAW AND PROCESS: THE PROCEEDINGS OF THE 2005 AND 2006 ANNUAL WTO CONFERENCES (Federico Ortino & Sergey Ripinsky eds., 2007); Pieter Jan Kuijper, It Shall contribute to . . . the Strict Observance and Development of International Law . . . The Role of the Court of Justice, in THE COURT OF JUSTICE AND THE CONSTRUCTION OF EUROPE: ANALYSES AND PERSPECTIVES ON SIXTY YEARS OF CASE-LAW (Springer ed., 2013). 195See EECKHOUT, supra note 53, at 364. See also Piet Eeckhout, The Appellate Bodies and the Courts, in WTO LAW AND PROCESS: THE PROCEEDINGS OF THE 2005 AND 2006 ANNUAL WTO CONFERENCES (Federico Ortino & Sergey Ripinsky eds., 2007); Pieter Jan Kuijper, It Shall contribute to . . . the Strict Observance and Development of International Law . . . The Role of the Court of Justice, in THE COURT OF JUSTICE AND THE CONSTRUCTION OF EUROPE: ANALYSES AND PERSPECTIVES ON SIXTY YEARS OF CASE-LAW (Springer ed., 2013). https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 577 German Law Journal German Law Journal certainty and applicants—even in circumstances where the Court appears to have backed itself into a corner.196 certainty and applicants—even in circumstances where the Court appears to have backed itself into a corner.196 g p y g Building from experience, rather than principle, one may then simply need to think that WTO agreements are of a different ilk concerning their non-conferral of rights, among other things, precluding reliance. Indeed, commenting on Simutenkov,197 Jacobs notes that “it seems as if the nature of the Agreement is no longer an obstacle to direct effect, so long as the Agreement contains provisions which . . . directly govern the position of individuals.”198 This case concerned a Partnership and Cooperation agreement, which creates a more limited relationship between parties than Association Agreements.199 In case law concerning the latter, it has been noted that the Court will often simply proceed to consider whether the specific provision a hand is sufficiently “clear, precise and unconditional” before then reflecting on the nature of the agree- ment. As Jacobs puts it, it would make little sense to consider a specific provision’s direct effect only to refuse direct effect on the basis of the broad scope and logic of the agreement; accordingly, the Court has not done so.200 Therefore, there is a presumption towards direct effect; “[s]uch a reference [to the nature of the agreement] is perhaps a relic of an earlier period.”201 202 g p y g Building from experience, rather than principle, one may then simply need to think that WTO agreements are of a different ilk concerning their non-conferral of rights, among other things, precluding reliance. Indeed, commenting on Simutenkov,197 Jacobs notes that “it seems as if the nature of the Agreement is no longer an obstacle to direct effect, so long as the Agreement contains provisions which . . . 196The uncertainty has prompted others to try—unsuccessfully—again. See ECJ, Case C-120/06, FIAMM and Others v. Council, ECLI:EU:C:2008:476 (Sept. 9, 2008), https://curia.europa.eu/juris/liste.jsf?num=C-120/06&language=en; ECJ, Case C-306/13 LVP NV v. Belgische Staat, ECLI:EU:C:2014:2465 (Dec. 18, 2014), https://curia.europa.eu/juris/liste.jsf? num=C-306/13&language=EN. 196The uncertainty has prompted others to try—unsuccessfully—again. See ECJ, Case C-120/06, FIAMM and Others v. Council, ECLI:EU:C:2008:476 (Sept. 9, 2008), https://curia.europa.eu/juris/liste.jsf?num=C-120/06&language=en; ECJ, Case C-306/13 LVP NV v. Belgische Staat, ECLI:EU:C:2014:2465 (Dec. 18, 2014), https://curia.europa.eu/juris/liste.jsf? num=C-306/13&language=EN. 197ECJ, Case C-265/03, Simutenkov v. Ministerio de Educacion y Cultura and Others, ECLI:EU:C:2005:213, (Apr. 12, 2005), https://curia.europa.eu/juris/showPdf.jsf;jsessionid=9ea7d2dc30db1450f0974b954b61bb23bc1b656c831a.e34KaxiLc3qMb40Rch0 SaxuKaNr0?docid=66313&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4341448. 198Francis G. Jacobs, The Internal Effects of the EU’s International Agreements and the Protection of Individual Rights, in, A CONSTITUTIONAL ORDER OF STATES?: ESSAYS IN EU LAW IN HONOUR OF ALAN DASHWOOD 536 (Anthony Arnull et. al. eds., 2011). 199Maresceau, supra note 168, at 426. 200See Jacobs, supra note 198, at 532. 201Id. at 536. 202ECJ, Case C-308/06, Intertanko and Others, ECLI:EU:C:2008:312 (June 3, 2008), https://curia.europa.eu/juris/liste.jsf? language=en&num=C-308/06. 203Convention on the Law of the Sea, Dec. 19, 1982, 1833 U.N.T.S. 3, [hereinafter, UNCLOS]. 204Id. para. 59. 205Id. para. 64. The Court defensively added that doubt was not cast on this by Part XI of UNCLOS—addressing natural and legal persons exploration, use and exploitation of the ocean floor. See, id. 206See MENDEZ, supra note 4, at 319. See also Jan Wouters & Philip de Man, International Association of Independent Tanker Owners (Intertanko), International Association of Dry Cargo Shipowners (Intercargo), Greek Shipping Cooperation Committee, Lloyd's Register and International Salvage Union V. Secretary of State for Transport, 103 AM. J. INT’L. L. 555 (2009). 207MENDEZ, supra note 4, at 275. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 196The uncertainty has prompted others to try—unsuccessfully—again. See ECJ, Case C-120/06, FIAMM and Others v. Council, ECLI:EU:C:2008:476 (Sept. 9, 2008), https://curia.europa.eu/juris/liste.jsf?num=C-120/06&language=en; ECJ, Case C-306/13 LVP NV v. Belgische Staat, ECLI:EU:C:2014:2465 (Dec. 18, 2014), https://curia.europa.eu/juris/liste.jsf? num=C-306/13&language=EN. 197ECJ, Case C-265/03, Simutenkov v. Ministerio de Educacion y Cultura and Others, ECLI:EU:C:2005:213, (Apr. 12, 2005), https://curia.europa.eu/juris/showPdf.jsf;jsessionid=9ea7d2dc30db1450f0974b954b61bb23bc1b656c831a.e34KaxiLc3qMb40Rch0 SaxuKaNr0?docid=66313&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4341448. 198Francis G. Jacobs, The Internal Effects of the EU’s International Agreements and the Protection of Individual Rights, in, A CONSTITUTIONAL ORDER OF STATES?: ESSAYS IN EU LAW IN HONOUR OF ALAN DASHWOOD 536 (Anthony Arnull et. al. eds., 2011). 199M 168 426 https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Secretary of State for Transport, 103 AM. J. INT’L. L. 555 (2009). 207MENDEZ, supra note 4, at 275. 206See MENDEZ, supra note 4, at 319. See also Jan Wouters & Philip de Man, International Association of Independent Tanker Owners (Intertanko), International Association of Dry Cargo Shipowners (Intercargo), Greek Shipping Cooperation Committee, Lloyd's Register and International Salvage Union V. Secretary of State for Transport, 103 AM. J. INT’L. L. 555 (2009). 207MENDEZ, supra note 4, at 275. 197ECJ, Case C-265/03, Simutenkov v. Ministerio de Educacion y Cultura and Others, ECLI:EU:C:2005:213, (Apr. 12, 2005), https://curia.europa.eu/juris/showPdf.jsf;jsessionid=9ea7d2dc30db1450f0974b954b61bb23bc1b656c831a.e34KaxiLc3qMb40Rch0 SaxuKaNr0?docid=66313&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4341448. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press directly govern the position of individuals.”198 This case concerned a Partnership and Cooperation agreement, which creates a more limited relationship between parties than Association Agreements.199 In case law concerning the latter, it has been t d th t th C t ill ft i l d t id h th th ifi i i h d Building from experience, rather than principle, one may then simply need to think that WTO agreements are of a different ilk concerning their non-conferral of rights, among other things, precluding reliance. Indeed, commenting on Simutenkov,197 Jacobs notes that “it seems as if the nature of the Agreement is no longer an obstacle to direct effect, so long as the Agreement contains provisions which . . . directly govern the position of individuals.”198 This case However, in Intertanko,202 which concerned the United Nations Convention on the Law of the Sea (UNCLOS),203 the Court reasoned that “individuals are in principle not granted independent rights and freedoms by virtue of UNCLOS”204 and that “UNCLOS does not establish rules intended to apply directly and immediately to individuals and to confer upon them rights or free- doms capable of being relied upon against States, irrespective of the attitude of the ship’s flag State.”205 The Court has been criticized heavily for its “curt” assertions and defensive stance con- cerning direct effect.206 It is also notable that in refusing direct effect, the Court also expressly closes the door on any possible argument concerning reciprocity in application of the agreement, unlike in WTO case law. In Intertanko, Advocate General Kokott is suggested to have been “wrongfooted”207 by the con- clusion which the Court reached concerning the lack of direct effect. The confusion was still greater because, in Poulsen, a case concerning an EU Regulation seeking to control fishing on 202ECJ, Case C-308/06, Intertanko and Others, ECLI:EU:C:2008:312 (June 3, 2008), https://curia.europa.eu/juris/liste.jsf? language=en&num=C-308/06. g g 203Convention on the Law of the Sea, Dec. 19, 1982, 1833 U.N.T.S. 3, [hereinafter, UNCLOS]. 204Id. para. 59. p 205Id. para. 64. The Court defensively added that doubt was not cast on this by Part XI legal persons exploration, use and exploitation of the ocean floor. See, id. 206See MENDEZ, supra note 4, at 319. See also Jan Wouters & Philip de Man, International Association of Independent Tanker Owners (Intertanko), International Association of Dry Cargo Shipowners (Intercargo), Greek Shipping Cooperation Committee, Lloyd's Register and International Salvage Union V. SaxuKaNr0?docid=66313&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4341448. 198Francis G. Jacobs, The Internal Effects of the EU’s International Agreements and the Protection of Individual Rights, in, A CONSTITUTIONAL ORDER OF STATES?: ESSAYS IN EU LAW IN HONOUR OF ALAN DASHWOOD 536 (Anthony Arnull et. al. eds., 2011). 199Maresceau, supra note 168, at 426. 200See Jacobs, supra note 198, at 532. num=C-306/13&language=EN. 197ECJ, Case C-265/03, Simutenkov v. Ministerio de Educacion y Cultura and Others, ECLI:EU:C:2005:213, (Apr. 12, 2005), https://curia.europa.eu/juris/showPdf.jsf;jsessionid=9ea7d2dc30db1450f0974b954b61bb23bc1b656c831a.e34KaxiLc3qMb40Rch0 SaxuKaNr0?docid=66313&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4341448. 198Francis G. Jacobs, The Internal Effects of the EU’s International Agreements and the Protection of Individual Rights, in, A CONSTITUTIONAL ORDER OF STATES?: ESSAYS IN EU LAW IN HONOUR OF ALAN DASHWOOD 536 (Anthony Arnull et. al. eds., 208Wouters & de Man, supra note 206, at 557 adding that the “lapse is striking in view of the Court's express acknowl- edgment in Poulsen that many provisions of the LOS Convention were ‘considered to express the current state of customary international maritime law’”. See id. at para. 10. 209ECJ, Case C-213/03, Syndicat Professionnel Coordination des Pêcheurs de l'Etang de Berre et de la Region v. EDF, ECLI: EU:C:2004:464 (July 15, 2004), https://curia.europa.eu/juris/liste.jsf?language=en&num=C-213/03. 210Convention for the Protection of the Mediterranean Sea Against Pollution (Barcelona Convention), art. 6(3), Aug. 14, 1978, 27 U.N.T.S. 1102. 211ECJ, Case C-239/03, Commission v. France (Étang de Berre), ECLI:EU:2004:598, (Oct. 7,2004), para. 85, https://curia. europa.eu/juris/liste.jsf?language=en&num=C-239/03. 212See Pavoni, supra note 149, at 353. See similarly, Pal Wannerås, THE ENFORCEMENT OF EC ENVIRONMENTAL LAW 25 (2007). 213Mendez, supra note 4, at 276. 214Holdgaard opts not to analyze it in his monograph on the basis that it “is largely identical to the internal direct effect test.” See Holdgaard, supra note 15, at 273. See similarly Maresceau, supra note 168, at 295. 215Ghazaryan, supra note 24, at 54. 216ECJ, Case C-65/77, Razanatsimba, ECLI:EU:C:1977:193, (Nov. 24, 1977), https://curia.europa.eu/juris/liste.jsf? language=en&num=C-65/77. 217Convention of Lomé, art. 62, Feb. 28, 1975, O.J. (L 25/2). 208Wouters & de Man, supra note 206, at 557 adding that the “lapse is striking in view of the Court's express acknowl- edgment in Poulsen that many provisions of the LOS Convention were ‘considered to express the current state of customary international maritime law’”. See id. at para. 10. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 578 the high seas, some rules within UNCLOS—which the EU was not yet party to—were applied analogously as CIL rules. Of course, not all UNCLOS rules will be CIL, but attention has been drawn to the fact that the Court did not even engage with this potential,208 let alone the ironic sequitur: Making UNCLOS formally binding on the EU simultaneously made it less enforceable. q g y g y In contrast to Intertanko, in EDF,209 the Court found direct effect of the Barcelona Convention for the Protection of the Mediterranean Sea against Pollution. Article 6(3) of the Convention pro- vided that “discharges shall be strictly subject to the issue, by the competent national authorities, of an authorisation.”210 However, a Commission action against France three months later con- firmed that France had provided only “operating instructions” to EDF but that it was in breach of the Convention in doing so.211 This did not preclude a private individual from being able to rely on the Convention as against EDF in the interim, a conclusion which Pavoni notes the Court reached “without enquiring into whether the pertinent treaty norms on the regulation of pollution from land-based sources attributed rights to individuals.”212 Mendez suggests that the explanation for the previous approach in EDF when compared to Intertanko was “the important distinction— a challenge to domestic rather than EU action.”213 This is a hidden factor, as it is one with which the Court has never been willing to engage in its case law, and suggests rules which vary according to latent discretion. One may also speculate that in both cases the environment was protected, meaning that normative concerns may have led to two very different interpretations of what amounts to con- ferring rights. Ultimately, externally the conferral of rights test’s resurgence has meant an addi- tional means of utilizing discretion for the Court, particularly as it remains ill-defined. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press p se C-65/77, Razanatsimba, ECLI:EU:C:1977:193, (Nov. 24, 1977), https://curia.europa.eu/juris/liste.jsf? &num=C-65/77. 5. Discretion in Determining Whether a Specific Provision Within a Directly Effective Agreement May be Relied Upon 5. Discretion in Determining Whether a Specific Provision Within a Directly Effective Agreement May be Relied Upon The “clear, precise and unconditional” requirement of the test has been less problematic than that concerning “broad scope and logic.” It has thus received less coverage in prominent accounts of the case law.214 A recent exception, though, notes flexibility and subjectivity in application of this element.215 p The “clear, precise and unconditional” requirement of the test has been less problematic than that concerning “broad scope and logic.” It has thus received less coverage in prominent accounts of the case law.214 A recent exception, though, notes flexibility and subjectivity in application of this element.215 Razanatsimba216 marked divergence from the internal rule concerning the specific wording of a provision, thereby providing a further locus for the Court to utilize its discretion. The case concerned the Lomé Convention, a former international trade and aid agreement between the EU and African Caribbean and Pacific countries. Article 62 of that agreement provided for non-discrimination concerning establishment unless “a Member is unable to provide such treatment.”217 Mr. Razanatsimba was noted by the Court as seeking “to rely upon the principles 217Convention of Lomé, art. 62, Feb. 28, 1975, O.J. (L 25/2). https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 579 German Law Journal which form the basis of the judgment of the Court . . . [in] Reyners.”218 The outcome of that case essentially found “that direct effect could apply even where the Member States possessed discre- tion, because the exercise thereof could be judicially controlled.”219 Here though, the Court adopted a more restrictive reading by which a Member State or ACP State not adhering to non-discrimination “is not guilty of any infringement” but “merely exposes itself to the risk of a retaliatory measure by the other party.”220 y y y The Court’s conclusion and referral to what it termed “negative reciprocity” is interesting. In case law concerning agreements outside of GATT/WTO law, it has not often made reference to reciprocity, except often to note—as with this agreement—that the EU has undertaken asymmet- rical obligations, and that accordingly the Court should enforce them.221 Perhaps then, the con- ditionality of the requirement here was the key to the Court’s conclusion? 218Razanatsimba, Case C-65/77, para. 10, citing ECJ, Case C-2/74 Reyners v. Belgian State, ECLI:EU:C:1974:68, (June 21, 1974), https://curia.europa.eu/juris/liste.jsf?language=en&jur=C,T,F&num=2-74&td=ALL. 219Paul Craig and Gráinne de Búrca, EU LAW: TEXT, CASES AND MATERIALS 190 (6th ed. 2015). 220Razanatsimba, Case C-65/77. 221See e.g., ECJ, Case C-87/75, Conceria Daniele Bresciani v. Amministrazzione Italiana delle Finanze dello Stato, ECLI:EU: C:1976:18, (Feb. 5, 1976), para. 23, https://curia.europa.eu/juris/liste.jsf?language=en&jur=C,T,F&num=87/75&td=ALL. 222EECKHOUT, supra note 53, at 333. 223See e.g., Alexander Somek, Monism: A Tale of the Undead, in CONSTITUTIONAL PLURALISM IN THE EUROPEAN UNION AND BEYOND 358 (Matej Abevlj & Jan Komárek eds., 2012) (applying this principle to EU law itself). 224See e.g., Mendez, supra note 4, at 128, there Mendez describes Case C-18/90 ONEM v Kziber [1991] ECR I-199 as “the fountain from which nearly all later jurisprudential developments pertaining to social security provisions in Community Agreements have stemmed.”; Mario Mendez, The legal effect of Community agreements: maximalist treaty enforcement and judicial avoidance techniques, 21 EUR. J. INT’L. L. 83, 92 (2010) (highlighting that “[t]he direct effect finding [in Kziber] bore a stark resemblance, unmentioned by the ECJ, to the approach to internal Community law as evinced most famously in the Reyners ruling where the absence of explicitly textually envisaged implementation measures was not permitted to stand in the way of the direct effect holding.”). 225Not least due to the criticisms that often follow such a blanket finding, for example, regarding WTO law. 226ECJ Case C-240/09 Lesoochranárske zoskupenie VLK v Ministerstvo životného prostredia Slovenskej republiky 227For the argument that it should have, see Pavoni, supra note 149, at 357. 228See id. at 360. See also EJC, Joined Cases 404 & 405/12, Council and Commission v. Stichting Natuur en Milieu and Pesticide Action Network Europe, ECLI:EU:C:2015:5, (Jan. 13, 2015), https://curia.europa.eu/juris/liste.jsf?language=en& num=C-404/12%20P. Not least due to the criticisms that often follow such a blanket finding, for example, regarding WTO law. 226ECJ, Case C-240/09, Lesoochranárske zoskupenie VLK v. Ministerstvo životného prostredia Slovenskej republiky (‘Slovak Brown Bear’), ECLI:EU:C:2011:125, (Mar. 8, 2011), https://curia.europa.eu/juris/liste.jsf?language=en&num=c- 240/09. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 5. Discretion in Determining Whether a Specific Provision Within a Directly Effective Agreement May be Relied Upon Eeckhout considered that the Court “did not expressly rule on direct effect” and that “as the provision at issue contained an open-ended reservation, granting participating States a wide discretionary power, it was impos- sible to consider its direct application.”222 But this does mark a significant divergence from Reyners.223 The case certainly evidences that the “specific provision” test is not, as is often sug- gested, equivalent to the internal test and that there is complexity within it. g q p y This case may remain an anomaly,224 but the sheer presence of this case creates concerns where the “broad scope and logic” of an agreement has been found not to be problematic yet the Court may wish to limit the effects of it in a future case. Equally, the Court may be less inclined to pre- clude direct effect based on “broad scope and logic”225—as this limits its discretion in the future— and so may make increased use of the “specific provision” test. The Slovak Brown Bear case is perhaps evidence of the dexterity and usefulness of the “specific provision” test compared to the “broad scope and logic” test moving forward.226 Here, the Court was not inclined to overcome conditionality inherent in Article 9(3) of the Aarhus Convention, concerning access to justice, in order to require a Slovakian court to apply it directly so as to enable an organization to have greater access to justice in judicial review proceedings concerning environmental matters.227 Judicial review at EU level is notoriously narrow in scope for private individuals’ standing. On this basis, it was inevitable that a case would arrive concerning not Member State rules on access to justice but EU rules.228 This may explain the fact that the p 223See e.g., Alexander Somek, Monism: A Tale of the Undead, in CONSTITUTIONAL PLURALISM IN THE EUROPEAN UNION AND BEYOND 358 (Matej Abevlj & Jan Komárek eds., 2012) (applying this principle to EU law itself). 224See e.g., Mendez, supra note 4, at 128, there Mendez describes Case C-18/90 ONEM v Kziber [1991] ECR I-199 as “the fountain from which nearly all later jurisprudential developments pertaining to social security provisions in Community Agreements have stemmed.”; Mario Mendez, The legal effect of Community agreements: maximalist treaty enforcement and judicial avoidance techniques, 21 EUR. J. INT’L. L. y g 25Not least due to the criticisms that often follow such a blanket finding, for example, regarding WTO law. 229See supra notes 142–150 and accompanying text. 230ECJ, Case C-366/10, Air Transport Association of America and Others, ECLI:EU:C:2011:864 (Dec. 21, 2011), para. 110, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-366/10. 231Id. at para 123; ECJ, Case C-162/96, Racke v. Hauptzollamt Mainz, ECLI:EU:C:1998:293, (June 16, 1998), para. 45, https:// curia.europa.eu/juris/liste.jsf?language=en&jur=C,T,F&num=C-162/96&td=ALL; ECJ, Case C-286/90, Anklagemindigheden v. Poulsen and Diva Navigation, ECLI:EU:C:1992:453, (Nov. 24, 1992), para. 9, https://curia.europa.eu/juris/liste.jsf?num=C- 286/90. 232Air Transport Association of America and Others, Case C-366/10. 233Id. at para. 107. 234Id. 235Directive 2008/101/EC, of the European Parliament and of the Council of 19 November 2008 amending Directive 2003/ 87/EC so as to include aviation activities in the scheme for greenhouse gas emission allowance trading within the Community, 2009 O.J. (L 8/3). 236For a criticism of the Court’s divergent approaches to international treaties and customary international law, see Jan Wouters & Dries Van Eeckhoutte, The Enforcement of Customary International Law through EC Law, in DIRECT EFFECT: RETHINKING A CLASSIC OF EC LEGAL DOCTRINE (J. M. Prinssen & A. Schrauwen eds., 2004). 237ECJ, Case C-69/89, Nakajima v. Council, ECLI:EU:C:1991:186 (July 5, 1991) https://curia.europa.eu/juris/documents.jsf? num=C-69/89. 238ECJ, Case C-70/87, Fediol v. Commission, ECLI:EU:C:1989:254, (June 22, 1989), https://curia.europa.eu/juris/ documents.jsf?num=70/87. 239KOUTRAKOS, supra note 21, at 262. 6. Limited Discretion Through Presumed “Direct Effect” of Customary International law 6. Limited Discretion Through Presumed “Direct Effect” of Customary International law It will be discussed below229 that relative strength with which CIL is applied in the Court may be weaker than that of treaties to which the EU is a party. This is so as the Court today appears to limit its review of the legislator’s action to “manifest errors in assessment.”230 Conversely, and likely because of this state of affairs, the potential for an individual to rely upon CIL has been more liberal. The case law has tended towards a presumption of direct effect, with the Court fre- quently simply stating that the EU “must respect international law in the exercise of its powers.”231 q y p y g p p Air Transport232 saw a somewhat different approach to previous case law. In order to establish that the claimant could rely upon the international rule the Court focused on EU law. The international rule only had to be “capable of calling into question the competence of the European Union to adopt that act,”233 however, the Union act had to be “liable to affect rights which the individual derives from European Union law or to create obligations under European law.”234 Though odd—given that this was a preliminary reference and direct effect or quasi-direct effect of the EU provision is not typically a requirement—the latter test would frequently be met with ease. This test was indeed met in Air Transport as the claimant would, as an association of commercial airline operators, have been required to comply with the provi- sions of the directive.235 The approach to reliance on CIL still remains starkly more generous when compared with the labyrinth developments in international treaties to which the EU is a party;236 although we will see below that application of CIL is often weaker. 7. Discretion Through Selective Application of Reference and Implementation Principles A variation to direct effect has manifested itself in the “implementation” and “reference” princi- ples founded in the Nakajima237 and Fediol238 cases respectively. Koutrakos notes of the Fediol and Nakajima cases that to create this exception the court there viewed “the duties undertaken by GATT as ‘internalised’ in the Community legal order.”239 This raises questions regarding the 232Air Transport Association of America and Others, Case C-366/10. 233Id t 107 Id. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 239KOUTRAKOS, supra note 21, at 262. 5. Discretion in Determining Whether a Specific Provision Within a Directly Effective Agreement May be Relied Upon 83, 92 (2010) (highlighting that “[t]he direct effect finding [in Kziber] bore a stark resemblance, unmentioned by the ECJ, to the approach to internal Community law as evinced most famously in the Reyners ruling where the absence of explicitly textually envisaged implementation measures was not permitted to stand in the way of the direct effect holding.”). g p g g 226ECJ, Case C-240/09, Lesoochranárske zoskupenie VLK v. Ministerstvo životného prostredia Slovenskej republiky (‘Slovak Brown Bear’), ECLI:EU:C:2011:125, (Mar. 8, 2011), https://curia.europa.eu/juris/liste.jsf?language=en&num=c- 240/09. g p 228See id. at 360. See also EJC, Joined Cases 404 & 405/12, Council and Commission v. Stichting Natuur en Milieu and Pesticide Action Network Europe, ECLI:EU:C:2015:5, (Jan. 13, 2015), https://curia.europa.eu/juris/liste.jsf?language=en& num=C-404/12%20P. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 580 Rupert Dunbar Court declined to grant direct effect of this specific provision despite direct effect of other Aarhus provisions being possible. The “specific provision” test here then provided delicacy in discretion that “broad scope and logic’” tests cannot. 6. Limited Discretion Through Presumed “Direct Effect” of Customary International law 232Air Transport Association of America and Others, Case C-366/10. 233Id. at para. 107. 234Id. 235Directive 2008/101/EC, of the European Parliament and of the Council of 19 November 2008 amending Directive 2003/ 87/EC so as to include aviation activities in the scheme for greenhouse gas emission allowance trading within the Community, 2009 O.J. (L 8/3). 38ECJ, Case C-70/87, Fediol v. Commission, ECLI:EU:C:1989:254, (June 22, 1989), https://curia.europ uments.jsf?num=70/87. 6. Limited Discretion Through Presumed “Direct Effect” of Customary International law 235Directive 2008/101/EC, of the European Parliament and of the Council of 19 November 2008 amending Directive 2003/ 87/EC so as to include aviation activities in the scheme for greenhouse gas emission allowance trading within the Community, 2009 O.J. (L 8/3). 236For a criticism of the Court’s divergent approaches to international treaties and customary international law, see Jan Wouters & Dries Van Eeckhoutte, The Enforcement of Customary International Law through EC Law, in DIRECT EFFECT: RETHINKING A CLASSIC OF EC LEGAL DOCTRINE (J M Prinssen & A Schrauwen eds 2004) 236For a criticism of the Court’s divergent approaches to international treaties and customary international law, see Jan Wouters & Dries Van Eeckhoutte, The Enforcement of Customary International Law through EC Law, in DIRECT EFFECT: RETHINKING A CLASSIC OF EC LEGAL DOCTRINE (J. M. Prinssen & A. Schrauwen eds., 2004). 237ECJ, Case C-69/89, Nakajima v. Council, ECLI:EU:C:1991:186 (July 5, 1991) https://curia.europa.eu/juris/documents.jsf? num=C-69/89. 237ECJ, Case C-69/89, Nakajima v. Council, ECLI:EU:C:1991:186 (July 5, 1991) https://curia.europa.eu/juris/documents.jsf? num=C-69/89. 238ECJ, Case C-70/87, Fediol v. Commission, ECLI:EU:C:1989:254, (June 22, 1989), https://curia.europa.eu/juris/ documents.jsf?num=70/87. 238ECJ, Case C-70/87, Fediol v. Commission, ECLI:EU:C:1989:254, (June 22, 1989), https://curia.europa.eu/juris/ documents.jsf?num=70/87. 239KOUTRAKOS, supra note 21, at 262. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 581 German Law Journal German Law Journal assertion in Haegeman II which appeared to internalize all international treaties as an “integral part of [EU] law.”240 Nakajima and Fediol would seem to provide that, notwithstanding a lack of direct effect, such agreements can provide a benchmark against which to review the legislature’s actions. This poten- tially undermines the Court’s reasons that such agreements preclude review generally due to their nature or lack of precision. Moreover, the fact that “there is no evidence that other WTO members have a similar exception [to Nakajima and Fediol]”241 undermines equally the rationale of reci- procity in the Court’s WTO law more generally. Confusion in this area is exacerbated by the Intertanko judgment. As the Court acknowledged, the directive provided that it would apply “in accordance with international law,” with specific reference made to UNCLOS.242 But in spite of clear reference in the legislation to UNCLOS, the Court simply went on to highlight a lack of direct effect. Thus, in Intertanko the Court did not engage with the reference principle all, with no explanation of why this should be so. 240ECJ, Case 181/73, Haegeman v. Belgium, ECLI:EU:C:1974:41, (Apr. 30, 1973), paras. 4-6, https://curia.europa.eu/juris/ liste.jsf?num=C-181/73. 241MENDEZ, supra note 4, at 238. 242See Intertanko and Others, Case C-308/06, at para. 24 (citing Council Directive 2005/35/EC of the European Parliament and of the Council of 7 September 2005 on ship-source pollution and on the introduction of penalties, particularly criminal penalties, for infringements, 2005 O.J. (L322/8), art. 3(1)). 243Doris König, The EU Directive on Ship-Source Pollution and on the Introduction of Penalties for Infringements: Development or Breach of International Law?, in LAW OF THE SEA, ENVIRONMENTAL LAW AND SETTLEMENT OF DISPUTES: LIBER AMICORUM JUDE THOMAS A. MENSAH (Tafsir Malick Ndiaye & Rüdiger Wolfrum eds., 2007). See also, ECJ, Case C-308/06, Interanko and Others, ECLI:EU:C:2007:689 (Nov. 20, 2007), http://curia.europa.eu/juris/liste.jsf?language=en& num=C-308/06. 244MENDEZ, supra note 4, at 100. 245Id. at 302. 246ECJ, Case C-76/00P, Petrotub v. Council, ECLI:EU:C:2003:4, (Jan. 9, 2003), https://curia.europa.eu/juris/liste.jsf? language=en&num=C-76/00%20P. 247“In any event the case law has gradually reduced the scope for invoking the principle . . . [except] in the field of anti- dumping.” See EECKHOUT, supra note 53, at 361. See similarly, Szilard Gáspár-Szilágyi, The Relationship Between EU Law And International Agreements: Restricting The Application Of The Fediol And Nakajima Exceptions In Vereniging Milieudefensie, 52 COMMON MKT. L. REV. 1059 (2015); KOUTRAKOS, supra note 21, at 306. 248ECJ, Case C-341/95, Bettati v. Safety Hi-Tech Srl, ECLI:EU:C:1998:353, (July 14, 1998), https://curia.europa.eu/juris/liste. jsf?language=en&num=C-341/95. 249Id. at para. 20. 240ECJ, Case 181/73, Haegeman v. Belgium, ECLI:EU:C:1974:41, (Apr. 30, 1973), paras. 4-6, https://curia.europa.eu/juris/ liste.jsf?num=C-181/73. 241MENDEZ, supra note 4, at 238. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 247“In any event the case law has gradually reduced the scope for invoking the principle . . . [except] in the field of anti- dumping.” See EECKHOUT, supra note 53, at 361. See similarly, Szilard Gáspár-Szilágyi, The Relationship Between EU Law And International Agreements: Restricting The Application Of The Fediol And Nakajima Exceptions In Vereniging Milieudefensie, 52 COMMON MKT. L. REV. 1059 (2015); KOUTRAKOS, supra note 21, at 306. 244MENDEZ, supra note 4, at 100. 6. Limited Discretion Through Presumed “Direct Effect” of Customary International law Though König considers that the directive in Intertanko could have remained intact were UNCLOS to be fully applied,243 others disagree.244 One cynical suggestion might be that in Fediol and Nakajima the challenges were ultimately unsuccessful. j g y However, though such a simplistic “meta-narrative” may provide a degree of legal certainty, Mendez draws attention to the fact that the General Court has annulled regulations and decisions on the basis of international law245 and that the Court has also done so in Petrotub.246 These cases all concerned anti-dumping rules and today scholars suggest that the reference and implementa- tion principles are limited to GATT/WTO law, and probably only to anti-dumping within that;247 however, until recently the case law gave a very different impression. y g y p In Bettati248 an EU regulation was adopted in order to fulfil the commitments entered into under the Vienna Convention of 22 March 1985 for the Protection of the Ozone Layer. The Court stated that “[i]t is settled law that Community legislation must, so far as possible, be inter- preted in a manner that is consistent with international law, in particular where its provisions are intended specifically to give effect to an international agreement concluded by the Community.”249 Though conflation of indirect effect—a mere interpretative tool—and the implementation principle—a basis for review—rendered the Court’s position less clear, there was an implication that the implementation principle could be relevant beyond anti-dumping and even WTO case law. 243Doris König, The EU Directive on Ship-Source Pollution and on the Introduction of Penalties for Infringements: Development or Breach of International Law?, in LAW OF THE SEA, ENVIRONMENTAL LAW AND SETTLEMENT OF DISPUTES: LIBER AMICORUM JUDE THOMAS A. MENSAH (Tafsir Malick Ndiaye & Rüdiger Wolfrum eds., 2007). See also, ECJ, Case C-308/06, Interanko and Others, ECLI:EU:C:2007:689 (Nov. 20, 2007), http://curia.europa.eu/juris/liste.jsf?language=en& num=C-308/06. 246ECJ, Case C-76/00P, Petrotub v. Council, ECLI:EU:C:2003:4, (Jan. 9, 2003), https://curia.europa.eu/juris/liste.jsf? language=en&num=C-76/00%20P. 247“In any event the case law has gradually reduced the scope for invoking the principle . . . [except] in the field of anti- dumping.” See EECKHOUT, supra note 53, at 361. See similarly, Szilard Gáspár-Szilágyi, The Relationship Between EU Law And International Agreements: Restricting The Application Of The Fediol And Nakajima Exceptions In Vereniging Milieudefensie, 52 COMMON MKT. L. REV. 1059 (2015); KOUTRAKOS, supra note 21, at 306. 247“In any event the case law has gradually reduced the scope for invoking the principle . . . g g 249Id. at para. 20. 246ECJ, Case C-76/00P, Petrotub v. Council, ECLI:EU:C:2003:4, (Jan. 9, 2003), https://curia.europa.eu/juris/liste.jsf? language=en&num=C-76/00%20P. jsf?language=en&num=C-341/95. 6. Limited Discretion Through Presumed “Direct Effect” of Customary International law [except] in the field of anti- dumping.” See EECKHOUT, supra note 53, at 361. See similarly, Szilard Gáspár-Szilágyi, The Relationship Between EU Law And International Agreements: Restricting The Application Of The Fediol And Nakajima Exceptions In Vereniging Milieudefensie, 52 COMMON MKT. L. REV. 1059 (2015); KOUTRAKOS, supra note 21, at 306. p 248ECJ, Case C-341/95, Bettati v. Safety Hi-Tech Srl, ECLI:EU:C:1998:353, (July 14, 1998), https://curia.europa.eu/juris/liste. jsf?language=en&num=C-341/95. 248ECJ, Case C-341/95, Bettati v. Safety Hi-Tech Srl, ECLI:EU:C:1998:353, (July 14, 1998), https://curia.europa.eu/juris/liste. jsf?language=en&num=C-341/95. j g g 249Id. at para. 20. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 582 Rupert Dunbar Rupert Dunbar Racke250 confirmed this further by applying the principle to CIL. The Court identified that the challenged regulation, which suspended an international agreement with the Socialist Federal Republic of Yugoslavia, had been adopted so as to comply with CIL governing the suspension of such agreements.251 The Court drew direct analogies between invocation of CIL and the Nakajima principle in the judgment252 and conducted a review of EU secondary legislation in light of international rules, ostensibly on the basis of that principle.253 g y p p Commune de Mesquer254 saw the Court return to the principle in passing—although without a direct reference to Fediol or Nakajima—concerning various conventions providing for liability caps in the event of oil pollution. Ultimately the precise directive at hand did not, rather conven- iently for the Court, refer to international conventions on liability, leaving it free to impose a higher level of liability in total. But the clear prospect of applying the implementation principle beyond WTO law once more rendered Intertanko all the more questionable, particularly as Commune de Mesquer was decided after Intertanko. q The above has so far shown little clarity as to who exactly is in the driving seat regarding the reference and implementation exceptions: Is it the legislature? The Court on an overtly ad hoc basis? Or is it dependent on the type of international agreement or customary rule? Stichting saw the General Court and Court disagree profoundly. Though the General Court found that the regulation “was adopted to meet the European Union’s international obligations under Article 9(3) of the Aarhus Convention,”255 the Court ruled that “it cannot be considered that, by adopting Regulation No 1367/2006 . . . 250ECJ, Case C-162/96, Racke v. Hauptzollamt Mainz, ECLI:EU:C:1998:293, (June 16, 1998), https://curia.europa.eu/juris/ liste.jsf?language=en&jur=C,T,F&num=C-162/96&td=ALL. 251Id. at para. 19. 252Racke is invoking fundamental rules of customary international law against the disputed regulation, which was taken pursuant to those rules, and deprives Racke of the rights to preferential treatment granted to it by the Cooperation Agreement. For a comparable situation in relation to basic rules of a contractual nature, see ECJ, Case C-69/89, Nakajima v. Council, ECLI: EU:C:1991:186 (July 5, 1991), paras. 31 & 48, https://curia.europa.eu/juris/documents.jsf?num=C-69/89. 253Concerning the controversy over the Court’s interpretation of CIL, see below at 4.3.1. 254ECJ, Case C-188/07, Commune de Mesquer v. Total France SA and Total International Ltd, ECLI:EU:C:2008:359, (June 24, 2008), https://curia.europa.eu/juris/liste.jsf?language=en&num=C-188/07. 255ECJ, Case T-338/08, Stichting Natuur en Milieu and Pesticide Action Network Europe v. European Commission, ECLI: EU:T:2012:300, (June 14, 2012), para. 58, https://curia.europa.eu/juris/liste.jsf?num=T-338/08. 256EJC, Joined Cases 404 & 405/12, Council and Commission v. Stichting Natuur en Milieu and Pesticide Action Network Europe, ECLI:EU:C:2015:5, (Jan. 13, 2015), https://curia.europa.eu/juris/liste.jsf?language=en&num=C-404/12%20P. 257Gáspár-Szilágyi, supra note 246, at 1063. 258EJC, Joined Cases 404 & 405/12, Council and Commission v. Stichting Natuur en Milieu and Pesticide Action Network Europe, ECLI:EU:C:2015:5, (Jan. 13, 2015), para. 49, https://curia.europa.eu/juris/liste.jsf?language=en&num=C-404/12% 20P. 259See ECJ, Stichting Natuur en Milieu, Case T-338/08 at para. 56. 260For example, the conferral of rights test. j g g 251Id. at para. 19. 250ECJ, Case C-162/96, Racke v. Hauptzollamt Mainz, ECLI:EU:C:1998:293, (June 16, 1998), https://curia.europa.eu/juris/ liste.jsf?language=en&jur=C,T,F&num=C-162/96&td=ALL. 251Id t 19 6. Limited Discretion Through Presumed “Direct Effect” of Customary International law the European Union was intended to implement the obligations [of the Aarhus Convention].”256 Gáspár-Szilágyi notes that this was held despite “the more than obvious title which specifically refers to the Aarhus Convention [and the fact that] almost every recital of the preamble of the Regulation makes references to the Convention.”257 Ultimately the Court “came clean” and stated of Nakajima and Fediol, “those two exceptions were justified solely by the particularities of the agreements that led to their application.”258 This overturned the more liberal approach of the General Court, which had relied on the Nakajima principle and ultimately annulled the regulation on the basis of the Convention. The fact that the Court neglected even to mention Racke, which formed a cornerstone in the General Court’s reasoning in evidencing the principle’s application beyond GATT/WTO law,259 is also a strong indication of the principle’s limitations today. However, caution is warranted given the Court’s tendency to repurpose seemingly dormant judicial devices.260 252Racke is invoking fundamental rules of customary international law against the disputed regulation, which was taken pursuant to those rules, and deprives Racke of the rights to preferential treatment granted to it by the Cooperation Agreement. For a comparable situation in relation to basic rules of a contractual nature, see ECJ, Case C-69/89, Nakajima v. Council, ECLI: EU:C:1991:186 (July 5, 1991), paras. 31 & 48, https://curia.europa.eu/juris/documents.jsf?num=C-69/89. 254ECJ, Case C-188/07, Commune de Mesquer v. Total France SA and Total International Ltd, ECLI:EU:C:2008:359, (June 24, 2008), https://curia.europa.eu/juris/liste.jsf?language=en&num=C-188/07. 254ECJ, Case C-188/07, Commune de Mesquer v. Total France SA and Total International Ltd, ECLI:EU:C:2008:359, (June 24, 2008), https://curia.europa.eu/juris/liste.jsf?language=en&num=C-188/07. p p j j g g 255ECJ, Case T-338/08, Stichting Natuur en Milieu and Pesticide Action Network Europe v. European Commission, ECLI: EU:T:2012:300, (June 14, 2012), para. 58, https://curia.europa.eu/juris/liste.jsf?num=T-338/08. p p j j g g 255ECJ, Case T-338/08, Stichting Natuur en Milieu and Pesticide Action Network Europe v. European Commission, ECLI: EU:T:2012:300, (June 14, 2012), para. 58, https://curia.europa.eu/juris/liste.jsf?num=T-338/08. 256EJC, Joined Cases 404 & 405/12, Council and Commission v. Stichting Natuur en Milieu and Pesticide Action Network Europe, ECLI:EU:C:2015:5, (Jan. 13, 2015), https://curia.europa.eu/juris/liste.jsf?language=en&num=C-404/12%20P. 257Gáspár-Szilágyi, supra note 246, at 1063. p gy p 258EJC, Joined Cases 404 & 405/12, Council and Commission v. Stichting Natuur en Milieu and Pesticide Action Network Europe, ECLI:EU:C:2015:5, (Jan. 13, 2015), para. 49, https://curia.europa.eu/juris/liste.jsf?language=en&num=C-404/12% 20P. p gy p 258EJC, Joined Cases 404 & 405/12, Council and Commission v. 261Holdgaard, supra note 15, at 245. 262Id. 263ECJ, Joined Cases 89 & 104/85, Ahlström Osakeyhtiö and Others v. Commission of the European Communities, ECLI: EU:C:1994:12, (Jan. 20, 1994), https://curia.europa.eu/juris/showPdf.jsf?text=&docid=93657&pageIndex=0&doclang=en& mode=lst&dir=&occ=first&part=1&cid=1607495. 264Id. at para 18. 265ECJ, Case C-286/90, Anklagemindigheden v. Poulsen and Diva Navigation, ECLI:EU:C:1992:453, (Nov. 24, 1992), para. 10, https://curia.europa.eu/juris/liste.jsf?num=C-286/90. 266See ECJ, Case C-308/06, Intertanko and Others, ECLI:EU:C:2008:312 (June 3, 2008), para. 51, https://curia.europa.eu/ juris/liste.jsf?language=en&num=C-308/06. 267Id. at para. 55. 268Wouters & de Man, supra note 206, at 557. 269ECJ, Case T-115/94, Opel Austria v. Council, ECLI:EU:T:1997:3, (July, 15, 1998), ECR II-39, https://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=CELEX:61994TJ0115:EN:PDF 6. Limited Discretion Through Presumed “Direct Effect” of Customary International law Stichting Natuur en Milieu and Pesticide Action Network Europe, ECLI:EU:C:2015:5, (Jan. 13, 2015), para. 49, https://curia.europa.eu/juris/liste.jsf?language=en&num=C-404/12% 20P. 259See ECJ, Stichting Natuur en Milieu, Case T-338/08 at para. 56. 259See ECJ, Stichting Natuur en Milieu, Case T-338/08 at para. 56. 260For example, the conferral of rights test. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 583 German Law Journal Holdgaard observes “[t]he hierarchical status of international agreements concluded by the Community has never been much disputed,”261 sitting above secondary legislation but below the Treaty. CIL is ranked similarly. Of course, “the practical effect of a hierarchically superior rule of international law depends on whether or not it can be directly invoked.”262 If granted, hierarchy would simply dictate mechanical application of a superior rule to invalidate a lower ranked one. However, the multifaceted discretion accruing to the Court through modifications to direct effect above has not proven sufficient to redress the absence of proportionality. Accordingly, we can now chart some further modifications which have also enhanced the Court’s discretion at the application stage. Holdgaard observes “[t]he hierarchical status of international agreements concluded by the Community has never been much disputed,”261 sitting above secondary legislation but below the Treaty. CIL is ranked similarly. Of course, “the practical effect of a hierarchically superior rule of international law depends on whether or not it can be directly invoked.”262 If granted, hierarchy would simply dictate mechanical application of a superior rule to invalidate a lower ranked one. However, the multifaceted discretion accruing to the Court through modifications to direct effect above has not proven sufficient to redress the absence of proportionality. Accordingly, we can now chart some further modifications which have also enhanced the Court’s discretion at the application stage. 1. Discretion Through Opportunistic Interpretations of Provisions of International Agreem and CIL The case concerned the adoption of a regulation which was argued to be in breach of a signed, but not yet in force, EEA treaty.270 The General Court held that “the principle of good faith is a rule of customary international law.”271 a case at the General Court (then CFI), is notable in that it saw the rare occurrence of EU sec- ondary law being invalidated where international law was invoked. However, on closer inspection, the extent to which international law formed the basis for invalidation, rather than the EU prin- ciple of legitimate expectations, emerges as questionable. The case concerned the adoption of a regulation which was argued to be in breach of a signed, but not yet in force, EEA treaty.270 The General Court held that “the principle of good faith is a rule of customary international law.”271 However, it found it to be a “corollary . . . of the [EU general] principle of protection of legitimate expectations.”272 Ultimately the General Court referred to the regulation as having “infringed the applicant’s legitimate expectations”273 rather than the principle of good faith. Eeckhout notes that the principle was “channeled”274 throughout EU law. Konstadinides takes a sterner approach in stating that “despite the custom-friendly outcome,”275 in reality “[i]t makes more sense to argue that the customary principle of good faith was not the real ground for annulment.”276 This sug- gestion is further supported by the Air Transport277 case, concerning airline emissions, which fur- ther limited the extent to which CIL—in and of itself—is a genuine restraint on EU action. The Court found that278: [S]ince a principle of customary international law does not have the same degree of precision as a provision of an international agreement, judicial review must necessarily be limited to the question whether, in adopting the act in question, the institutions of the European Union made manifest errors of assessment concerning the conditions for applying those principles.279 Accordingly, a “manifest errors” approach, which was not present in the review on the basis of EU law in Opel Austria, is present here where CIL alone is applied, with Racke being cited as justification. However, it is submitted that the reference to Racke is misleading. p 283S.S. Lotus (Fr. V. Turk.), Judgement, 1927 P.C.I.J. (ser. A) No. 10, at para. 45 (Sept. 7). 270The Court noted that “the Communities were aware of the date on which the EEA Agreement would enter into force” seven days before conclusion of the contested regulation as they had been the final signatory to approve it. Id. at para. 92. 271Id. at para. 90. 272Id. at para. 93. 273Id. at para. 123. 274EECKHOUT, supra note 53, at 387. 275Theodore Konstadinides, When in Europe: Customary International Law and EU Competence in the Sphere of External Action, 13 GERMAN. L. J. 1177, 1187 (2012). 276Id. at 1188. 277ECJ, Case C-366/10, Air Transport Association of America and Others, ECLI:EU:C:2011:864 (Dec. 21, 2011), para. 110, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-366/10. 278Id. at para. 110. 279See ECJ, Case C-162/96, Racke v. Hauptzollamt Mainz, ECLI:EU:C:1998:293, (June 16, 1998), para. 52, https://curia. europa.eu/juris/liste.jsf?language=en&jur=C,T,F&num=C-162/96&td=ALL. 280Id. 281Id. at para. 46. 282Wouters & Van Eeckhoutte, supra note 235, at 206–07. 283S.S. Lotus (Fr. V. Turk.), Judgement, 1927 P.C.I.J. (ser. A) No. 10, at para. 45 (Sept. 7). 1. Discretion Through Opportunistic Interpretations of Provisions of International Agreem and CIL 1. Discretion Through Opportunistic Interpretations of Provisions of International Agreements and CIL This Article has identified above that the Court has taken a more liberal view towards the invo- cation of CIL than it has to international agreements. However, it appears the Court has limited its view of what CIL demands. In Woodpulp,263 the Court dealt with a challenge that Article 101 TFEU was being applied extraterritorially, as an agreement to restrict competition had been concluded outside of the EU. However, its effect was to restrict competition within the EU. Therefore the Court asserted that “the Community’s jurisdiction to apply its competition rules to such conduct is covered by the territoriality principle as universally recognised in public international law.”264 The implication though was that EU action, and even the EU Treaty, would be appropriately constrained by CIL. However, Intertanko demonstrates that the Court will not necessarily be quick to accept that international rules reflect custom. In Poulsen the Court said of UNCLOS that “many of its pro- visions are considered to express the current state of customary international maritime law.”265 In Intertanko the Court stated: Admittedly, as is clear from settled case-law, the powers of the Community must be exercised in observance of international law, including provisions of international agreements in so far as they codify customary rules of general international law . . . .266 The Court cited Poulsen, but it did not clearly address UNCLOS in this regard. Even the later statement that “UNCLOS’s main objective is to codify, clarify and develop the rules of general international law”267 did not prompt the Court to do so. The lack of analysis in Intertanko in this regard has been said to be striking.268 Where accepted as relevant, case law has also shown the complexities which can arise concern- ing interpretations of CIL. The decisions have been met with some criticism. Opel Austria,269 https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 584 a case at the General Court (then CFI), is notable in that it saw the rare occurrence of EU sec- ondary law being invalidated where international law was invoked. However, on closer inspection, the extent to which international law formed the basis for invalidation, rather than the EU prin- ciple of legitimate expectations, emerges as questionable. p 282Wouters & Van Eeckhoutte, supra note 235, at 206–07. 270The Court noted that “the Communities were aware of the date on which the EEA Agreement would enter into force” seven days before conclusion of the contested regulation as they had been the final signatory to approve it. Id. at para. 92. 271Id. at para. 90. 272Id at para 93 274EECKHOUT, supra note 53, at 387. 281Id. at para. 46. , p , 275Theodore Konstadinides, When in Europe: Customary International Law and EU Competence in the Sphere of External Action, 13 GERMAN. L. J. 1177, 1187 (2012). 276Id. at 1188. 1. Discretion Through Opportunistic Interpretations of Provisions of International Agreem and CIL In Racke the Court did conclude that “the complexity of the rules in question and the imprecision of some of the concepts to which they refer”280 meant that only a marginal review of the legislator’s actions could be conducted. However, the reference was not to all rules of customary law but to “the rules of customary international law concerning the termination and the suspension of treaty relations by reason of a fundamental change of circumstances.”281 It is notable that even concerning Racke this approach was criticized.282 pp Notwithstanding Racke, the Court’s approach to interpreting CIL in Air Transport itself can be questioned. Territorial sovereignty is arguably the precursor to the proper functioning of international law.283 The extension of the concept to airspace is both essential for the proper y 271Id. at para. 90. 272Id. at para. 93. p 279See ECJ, Case C-162/96, Racke v. Hauptzollamt Mainz, ECLI:EU:C:1998:293, (June 16, 1998), para. 52, https://curia. europa.eu/juris/liste.jsf?language=en&jur=C,T,F&num=C-162/96&td=ALL. 280 d https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 585 German Law Journal functioning of aviation and uncontroversial. The ICJ describes the rule concerning sovereignty over airspace as a “basic legal concept,”284confirming “no doubt that . . . prescriptions of treaty-law285 merely respond to firmly established and longstanding tenets of customary international law.”286 This does not sit well with the Court’s assertion that the rule lacked precision.287 p Arguably, the outcome simply evidences the problem by which environmental protection has developed in importance in more recent years, a development with which external relations case law, based in hierarchy and formalism, has struggled to keep up. Advocate General Kokott appeared to acknowledge this problem and encourage change in stating: [The Court’s] judgment will be of fundamental importance not only to the future shaping of European climate change policy but also generally to the relationship between European Union (EU) law and international law. In particular it will be necessary to consider whether and to what extent individuals are entitled to rely in court on certain international agree- ments and principles of customary international law in order to defeat an act of the European Union.288 Though the Court was not so colorful, it did refer to the importance of “environmental pro- tection objectives which it has set for itself.”289 But, as Koutrakos points out, this point was irrel- evant in the current constellation of the case law.290 Although few would question that it did, in fact, influence the Court. 284Military And Paramilitary Activities In And Against Nicaragua (Nicar. v. U.S.) Judgment, 1986 I.C.J. 14, para. 212 (June 27). 285The ICJ made reference inter alia to the Chicago Convention on International Civil Aviation, Dec. 7, 1944, 15 U.N.T.S. 295. 286Military and Paramilitary Activities in and against Nicaragua (Nicar. v U.S.) Judgment, 1986 I.C.J. 14, para. 212. Shaw notes that the principle of territorial sovereignty over airspace developed “very quickly in the years immediately before and during the First World War.” See, MALCOLM N. SHAW, INTERNATIONAL LAW 55 (7th ed. 2014). 287ECJ, Case C-366/10, Air Transport Association of America and Others, ECLI:EU:C:2011:864 (Dec. 21, 2011), para. 110, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-366/10. 288See Opinion of Advocate General Kokott at para. 4, Case C-366/10, Air Transport Association of America and Others (Oct. 6, 2011), http://curia.europa.eu/juris/liste.jsf?language=en&num=C-366/10. 289See Air Transport, Case C-366/10 at para. 128. 290KOUTRAKOS, supra note 21, at 318. 291ECJ, Case C-127/07, Société Arcelor Atlantique et Lorraine and Others v. Premier Minister, ECLI:EU:C:2008:728 (Dec. 16, 2008), para. 57, http://curia.europa.eu/juris/liste.jsf?language=en&num=C-127/07. 292On complexity of international affairs requiring a “wide discretion” for the legislature generally, see KOUTRAKOS, supra note 21, at 183. On the “climate change regime complex” specifically, see Joanne Scott & Lavanya Rajamani, EU Climate Change Unilateralism, 23 EUR. J. INT’L. L. 469, 470 (2012). 293ECJ, Case C-344/04, IATA and ELFAA, ECLI:EU:C:2006:10 (Jan. 10, 2006), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-344/04. 286Military and Paramilitary Activities in and against Nicaragua (Nicar. v U.S.) Judgment, 1986 I.C.J. 14, para. 212. Shaw notes that the principle of territorial sovereignty over airspace developed “very quickly in the years immediately before and during the First World War.” See, MALCOLM N. SHAW, INTERNATIONAL LAW 55 (7th ed. 2014). 284Military And Paramilitary Activities In And Against Nicaragua (Nicar. v. U.S.) Judgment, 1986 I.C.J. 14, para. 212 (June 27). 285The ICJ made reference inter alia to the Chicago Convention on International Civil Aviation Dec 7 1944 15 U N T S https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 285The ICJ made reference inter alia to the Chicago Convention on International Civil Aviation, Dec. 7, 194 295. 88See Opinion of Advocate General Kokott at para. 4, Case C-366/10, Air Transport Association of America a ct. 6, 2011), http://curia.europa.eu/juris/liste.jsf?language=en&num=C-366/10. 1. Discretion Through Opportunistic Interpretations of Provisions of International Agreem and CIL Moreover, if we reconsider Air Transport, which concerned the expansion of the EU emissions trading scheme to airlines, in light of Arcelor, which also featured the expansion of the emissions trading scheme to steel producers, then a further impact of the absence of proportionality is ap- parent. In Arcelor the Court provided a margin to the legislature as it should have a “broad dis- cretion where its action involves political, economic and social choices.”291 From the legislature’s perspective though, it is clear which expansion was more complex,292 but the Court’s rules gov- erning reception and application of international law could not accommodate this, hence the interpretative strain to find CIL uncertain. It is not just CIL which has seen strained interpretation. A prominent example of opportunistic interpretation of an international treaty is ELFAA.293 The case saw a challenge to an EU regulation on the basis that it breached the Montreal Convention. The Montreal Convention provides for the 292On complexity of international affairs requiring a “wide discretion” for the legislature generally, see KOUTRAKOS, supra note 21, at 183. On the “climate change regime complex” specifically, see Joanne Scott & Lavanya Rajamani, EU Climate Change Unilateralism, 23 EUR. J. INT’L. L. 469, 470 (2012). g 293ECJ, Case C-344/04, IATA and ELFAA, ECLI:EU:C:2006:10 (Jan. 10, 2006), http://curia.europa.eu/juris/liste.jsf? language=en&num=C-344/04. language=en&num=C-344/04. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 586 circumstances and amount of compensation to which passengers are entitled. The EU is a party to the Montreal Convention and the Court found the relevant articles of the Convention to have direct effect.294 It then referred to Article 31 of the Vienna Convention on the Law of Treaties (VCLoT) and directed itself that an international treaty “must be interpreted . . . in good faith in accordance with the ordinary meaning to be given to its terms in their context and in the light of its object and purpose.”295 j p p However, in spite of the Court acknowledging the Montreal Convention limiting liability of carriers to set amounts,296 the Court reasoned that “[t]he system prescribed in Article 6 [of the regulation] simply operates at an earlier stage than the system which results from the Montreal Convention.”297 Thus the challenge was unfounded; the EU legislature was free to impose liability, even in extraordinary circumstances, as the Court felt that it was regulating a distinct aspect of compensation not covered by the Convention. 294Id. at para. 39. 295Id. at para. 40. 296Id. at para. 42. 297Id. at para. 46. 298Id. at para. 43. 299Id. at para. 47. 300MENDEZ, supra note 4, at 270. 301See e.g., id. at 269–270; Aust, Rodiles & Staubach, supra note 38, at 75; B. Harris, The ‘Force Of Law’ Of International Carriage Conventions In The EU Internal Market, 25 INT’L COMPANY & COM. L. REV. 98, 105–6 (2014). 302ECJ, Joined Cases 402 & 415/05P, Kadi and Al Barakaat International Foundation v. Council and Commission (Kadi I), ECLI:EU:C:2008:461, Judgement of Sept. 3, 2008, at 316, https://curia.europa.eu/juris/liste.jsf?num=C-402/05. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 300MENDEZ, supra note 4, at 270. 1. Discretion Through Opportunistic Interpretations of Provisions of International Agreem and CIL The Court reasoned that the Convention covered individual damage sustained by passengers— who could then bring a claim—but that the regulation covered “standardised and immediate assistance.”298 The Court implicitly acknowledges the potential for some overlap with the Convention, stating “[t]he standardised and immediate assistance and care measures do not themselves prevent the passengers concerned, should the same delay also cause them damage conferring entitlement to compensation, from being able to bring in addition actions to redress that damage under the conditions laid down by the Montreal Convention.”299 In spite of passengers not being able to claim twice for the same damage, there is the problem that the 4 150 SDRs cap is now artificially raised. Mendez also draws attention to the fact that the Convention contains an exclusivity provision, allowing for passengers to seek damages only through it.300 Overall there is heavy criticism of the reasoning adopted by the Court.301 It is submitted that Air Transport and ELFAA ultimately appear as contorted interpretative efforts deployed to pursue environmental protection and consumer protection, respectively. The norms of consumer and environmental protection are indeed important, but their accom- modation through the finding of interpretative room within international provisions that appear to allow none is problematic for legal certainty. Proportionality analysis would have provided for richer argumentation and engagement with the facts and, given the heavy criticism of both cases, could not have damaged perceptions of the EU’s respectfulness towards EU law any further. 2. Discretion Through Recourse to the Concept of an Autonomous Legal System The EU legal system “cannot be prejudiced by an international agreement.”302 This infamous phrase in fact belies the outcome of the Kadi case law. As is well known, in Kadi the Court was confronted with an EU regulation which transposed a UN Security Council Resolution aimed at combatting terrorism by listing terrorist suspects and suspected financiers. The potential infringement on fundamental rights was subjected to a limited review on the basis of jus cogens by the General Court, in deference to the international origin of the EU regulation. The Court instead reviewed the EU Regulation on the basis of EU law and made that clear through the lan- guage of autonomy. But on closer inspection, the autonomous legal system is quite permeable. 294Id. at para. 39. 295Id. at para. 40. 296Id. at para. 42. 297Id. at para. 46. 298Id. at para. 43. 299Id. at para. 47. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press p 301See e.g., id. at 269–270; Aust, Rodiles & Staubach, supra note 38, at 75; B. Harris, The ‘Force Of Law’ Of International Carriage Conventions In The EU Internal Market, 25 INT’L COMPANY & COM. L. REV. 98, 105–6 (2014). 302ECJ, Joined Cases 402 & 415/05P, Kadi and Al Barakaat International Foundation v. Council and Commission (Kadi I), ECLI:EU:C:2008:461, Judgement of Sept. 3, 2008, at 316, https://curia.europa.eu/juris/liste.jsf?num=C-402/05. 1. Discretion Through Opportunistic Interpretations of Provisions of International Agreem and CIL 300MENDEZ, supra note 4, at 270. 301See e.g., id. at 269–270; Aust, Rodiles & Staubach, supra note 38, at 75; B. Harris, The ‘Force Of Law’ Of International Carriage Conventions In The EU Internal Market, 25 INT’L COMPANY & COM. L. REV. 98, 105–6 (2014). 302ECJ, Joined Cases 402 & 415/05P, Kadi and Al Barakaat International Foundation v. Council and Commission (Kadi I), ECLI:EU:C:2008:461, Judgement of Sept. 3, 2008, at 316, https://curia.europa.eu/juris/liste.jsf?num=C-402/05. 294Id. at para. 39. 295Id. at para. 40. 296Id. at para. 42. 297Id. at para. 46. 298Id. at para. 43. 299Id. at para. 47. 300MENDEZ, supra note 4, at 270. 301See e.g., id. at 269–270; Aust, Rodiles & Staubach, supra note 38, at 75; B. Harris, The ‘Force Of Law’ Of International Carriage Conventions In The EU Internal Market, 25 INT’L COMPANY & COM. L. REV. 98, 105–6 (2014). 302ECJ, Joined Cases 402 & 415/05P, Kadi and Al Barakaat International Foundation v. Council and Commission (Kadi I), ECLI:EU:C:2008:461, Judgement of Sept. 3, 2008, at 316, https://curia.europa.eu/juris/liste.jsf?num=C-402/05. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 587 German Law Journal “The Court must determine whether a fair balance has been struck between the demands of the public interest and the interest of the individuals concerned” and that public interest must be judged “[w]ith reference to an objective of general interest as fundamental to the international community as the fight by all means . . . against the threats to international peace and security posed by acts of terrorism.”303 Accordingly the international political and legal context permeated the “autonomous” EU legal system and was balanced against the “very foundations” of that legal system.304 However, due to the significant shortcomings in the listing process the Court’s appli- cation of this balance is less discernible. It is notable though that while the Court noted the multiple flaws in the regulation305 it con- cluded “annulment to that extent of the contested regulation with immediate effect would be capable of seriously and irreversibly prejudicing the effectiveness of the restrictive measures imposed by the regulation and which the Community is required to implement . . . .”306 The Court accordingly allowed the regulation to maintain its effects for three months, within which time a new regulation was concluded. 303Id. at para. 360. 304Id. at para. 344. 305Id. at para 348; Id. at paras. 369–370. 306Id. at para 373. 307Pasquale De Sena & Maria Chiara Vitucci, The European courts and the Security Council: Between Dedoublement Functionnel and Balancing of Values, 20 EUR. J. INT’L. L. 193, 224 (2009). 308ECJ, Case C-459/03, Commission v. Ireland, ECLI:EU:C:2006:345, (May 30, 2006), https://curia.europa.eu/juris/liste.jsf? num=C-459/03. 309ECJ, Case C-2/13, Opinion re Accession of the European Union to the European Convention for the Protection of Human Rights and Fundamental Freedoms, ECLI:EU:C:2014:2454, (Dec. 14, 2014), https://curia.europa.eu/juris/document/document. jsf?docid=160882&doclang=EN. 310See e.g., ECJ, Case C-584/10, Commission and others v. Kadi (‘Kadi II’), ECLI:EU:C:2013:518, (July 18, 2013), https:// curia.europa.eu/juris/liste.jsf?num=C-584/10; ECJ, Case C-364/10, Hungary v. Slovakia, ECLI:EU:C:2012:630, (Oct. 16, 2012), https://curia.europa.eu/juris/liste.jsf?num=C-364/10&language=EN stating that fundamental freedom of movement of the Hungarian President was curtailed based on the international treaty and CIL. Id. at para 373. 307Pasquale De Sena & Maria Chiara Vitucci, The European courts and the Security Council: Between Dedoublement Functionnel and Balancing of Values, 20 EUR. J. INT’L. L. 193, 224 (2009). 308ECJ, Case C-459/03, Commission v. Ireland, ECLI:EU:C:2006:345, (May 30, 2006), https://curia.europa.eu/juris/liste.jsf? num=C-459/03. 309ECJ, Case C-2/13, Opinion re Accession of the European Union to the European Convention for the Protection of Human Rights and Fundamental Freedoms, ECLI:EU:C:2014:2454, (Dec. 14, 2014), https://curia.europa.eu/juris/document/document. jsf?docid=160882&doclang=EN. 310See e.g., ECJ, Case C-584/10, Commission and others v. Kadi (‘Kadi II’), ECLI:EU:C:2013:518, (July 18, 2013), https:// curia.europa.eu/juris/liste.jsf?num=C-584/10; ECJ, Case C-364/10, Hungary v. Slovakia, ECLI:EU:C:2012:630, (Oct. 16, 2012), https://curia.europa.eu/juris/liste.jsf?num=C-364/10&language=EN stating that fundamental freedom of movement of the Hungarian President was curtailed based on the international treaty and CIL. p 307Pasquale De Sena & Maria Chiara Vitucci, The European courts and the Security Council: Between Functionnel and Balancing of Values, 20 EUR. J. INT’L. L. 193, 224 (2009). 1. Discretion Through Opportunistic Interpretations of Provisions of International Agreem and CIL External Relations Cases Which Have Seen the Deployment of Proportionality It is appropriate to end our analysis of the case law with an anomaly or two: The Court’s deploy- ment of proportionality analysis in the application of international law. It is appropriate to end our analysis of the case law with an anomaly or two: The Court’s deploy- ment of proportionality analysis in the application of international law. Omega Air311 concerned secondary legislation which sought to limit noise pollution of aircraft. The Court engaged with the principle of proportionality and stated that “with respect to judicial review . . . the Community legislature has wide legislative powers in the field of the common trans- port policy as regards the adoption of appropriate common rules.”312 In favorably reviewing the legislature’s actions, the Court even invoked ICAO assembly resolutions, which it claimed endorsed the approach adopted by the EU.313 pp p y Though the Court would later expressly recall the fact that WTO law does not have direct effect, the passages concerning proportionality appear implicitly to disentangle international pro- visions in both WTO law and the Chicago Convention—to which the EU is not even party—from the need for direct effect for the purposes of that analysis.314 Overall, the balance between respect for international law and protection of the environment appeared to be entertained. p pp Commission v. Italy also saw deployment of proportionality.315 The case concerned a Free Trade Agreement with Norway. In finding Italy in breach of the agreement, the Court reasoned that due to provisions analogous to Article 34 and 36 TFEU, Italy’s requirement for health inspec- tions of fish had “infringed the principle of proportionality.”316 This application of proportionality is interesting as it concerns a balance within the international agreement rather than between protection of EU law and respect for international law. However, Eeckhout points out that in Commission v. Italy the deployment of proportionality “[found] little basis in the actual agreement.”317 The international treaty provision allowed for measures to protect, inter alia, health, provided they do “not constitute a means of arbitrary dis- crimination or a disguised restriction on trade between the Contracting Parties,” whereas here the Court balanced the health imperative against the trade imperative, as it would do in internal case law. 1. Discretion Through Opportunistic Interpretations of Provisions of International Agreem and CIL The effort to conclude trade agreements which more appropriately reflect non-trade concerns within them means that this approach may be available to the Court more often, meaning at least a limited role for proportionality moving forward.318 311ECJ, Joined Cases 27 & 122/00, Omega Air and Others, ECLI:EU:C:2002:161, (Mar. 12, 2002), https://curia.europa.eu/ juris/liste.jsf?language=en&num=C-27/00. 312Id. at para. 63. 313Id. at para. 70. 314The Advocate General also applied the proportionality test. Opinion of Advocate General Alber at paras. 61-87, Cases 27 & 122/00, Omega Air and Others (Mar. 12, 2002). 315ECJ, Case C-228/91, Commission v. Italy, ECLI:EU:C:1993:206, (May 25, 1993), https://eur-lex.europa.eu/legal-content/ EN/TXT/?uri=CELEX%3A61991CJ0228#SM 316Id. at para. 49. 317EECKHOUT, supra note 53, at 318. 318See e.g., Lonel Zamfir, European Parliamentary Research Service, Human Rights in EU Trade Agreements: The Human Rights Clause And Its Application, (July 2019), https://www.europarl.europa.eu/RegData/etudes/BRIE/2019/637975/EPRS_ BRI(2019)637975_EN.pdf. 319TFEU arts. 258, 259, 263; TFEU art. 267. 320TFEU art. 267. 1. Discretion Through Opportunistic Interpretations of Provisions of International Agreem and CIL Thus, in spite of the fact that multiple infringements of fundamental rights were found, the remedy was simply to provide the legislature with a further three months in which to remedy the shortcomings. This is markedly deferential to international law and, of course, recognizes the importance of combatting terrorism, which it would be artificial to suggest is purely an EU, and not international, concern. The consequence was that a new Security Council Resolution was passed, transposed into EU law, and then challenged once more in Kadi II. Five years had passed since the Court’s Kadi I judgment in which the EU legal system had been declared to be unable to be prejudiced by international law. p j y Far from “autonomy,” Sena and Vitucci’s argument that “the findings reached in its judgment can, ultimately, be regarded as the outcome of a direct balancing of the EC principles on funda- mental rights with the value of cooperation of the EU states with the UN in the fight against terrorism”307 seems correct. Autonomy here was simply proportionality by another name. However, autonomy has meant autonomy in cases such as MoxPlant, precluding Member State submission of a dispute to an international court where EU law is concerned,308 and Opinion 2/13, refusing accession to the ECHR.309 Equally though it has not been mentioned in cases where it appeared relevant.310 Though Kadi I shows that autonomy can have a softer and more proportionate side, the deployment of autonomy is guaranteed to garner much attention and for this reason the term may have been eschewed in Kadi II. Interestingly, though, the Court returned to autonomy in Opinion 2/13. Ultimately then the concept itself is elusive and allows for discretion in both deploy- ment and application, yet falling short of that which proportionality would offer. The label of autonomy is unhelpful, but the concept of autonomy is confirmatory of the discretion which a domestic court holds when applying international law. The manifestation of that discretion, though, would be better served through proportionality. 310See e.g., ECJ, Case C-584/10, Commission and others v. Kadi (‘Kadi II’), ECLI:EU:C:2013:518, (July 18, 2013), https:// curia.europa.eu/juris/liste.jsf?num=C-584/10; ECJ, Case C-364/10, Hungary v. Slovakia, ECLI:EU:C:2012:630, (Oct. 16, 2012), https://curia.europa.eu/juris/liste.jsf?num=C-364/10&language=EN stating that fundamental freedom of movement of the Hungarian President was curtailed based on the international treaty and CIL. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 588 3. IV. Reflections on Diffuse Discretion in External Relations Case Law In internal case law the Court’s discretion does not reside exclusively within the proportionality test, but it is its primary residence. This is the consequence of accepting reliance on rules without requiring direct effect in many instances.319 Equally, where direct effect is required320 we have seen a remarkably liberal approach in finding it to be present. With EU law readily engaged, the Court’s 311ECJ, Joined Cases 27 & 122/00, Omega Air and Others, ECLI:EU:C:2002:161, (Mar. 12, 2002), https://curia.europa.eu/ juris/liste.jsf?language=en&num=C-27/00. 312Id. at para. 63. 313Id. at para. 70. 314The Advocate General also applied the proportionality test. Opinion of Advocate General Alber at paras. 61-87, Cases 27 & 122/00, Omega Air and Others (Mar. 12, 2002). 315ECJ, Case C-228/91, Commission v. Italy, ECLI:EU:C:1993:206, (May 25, 1993), https://eur-lex.europa.eu/legal-content/ EN/TXT/?uri=CELEX%3A61991CJ0228#SM 316Id. at para. 49. 317EECKHOUT, supra note 53, at 318. 318See e.g., Lonel Zamfir, European Parliamentary Research Service, Human Rights in EU Trade Agreements: The Human Rights Clause And Its Application, (July 2019), https://www.europarl.europa.eu/RegData/etudes/BRIE/2019/637975/EPRS_ BRI(2019)637975_EN.pdf. 319TFEU arts. 258, 259, 263; TFEU art. 267. 320TFEU art. 267. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 589 German Law Journal practice is to then postulate competing interests at the same level and to resolve them with con- sideration of the unique facts of the given case in a proportionality analysis. Externally, direct effect has been more actively deployed as a gatekeeper of the EU legal order. Perhaps this is to be expected given that direct effect was justified by the EU being, in the Court’s view, “a new legal order of international law.”321 However, complexity in external relations cases arises due to the sheer breadth of the rules, not to mention their variation over time. A further confounding feature is that the rules of direct effect seem to be heavily influenced by unexpressed normative concerns to the point where one wonders “whether . . . the actual review which the judgment avoids, is influencing the answer . . . as to the capacity of the Agreement itself to form a review criterion vis-à-vis Community law.”322 y Moreover, overburdening direct effect as a locus for discretion clearly lacks the refinement of proportionality analyses. IV. Reflections on Diffuse Discretion in External Relations Case Law Pavoni notes this problem in Intertanko; by refusing direct effect of a significant number of UNCLOS provisions, the case led to higher environmental protection but would, in theory, be equally applicable to EU and Member State conduct in a subsequent case and accordingly may result in “lower EU standards of environmental protection” in the future.323 Similarly, interpreting away the demands of international law, through eminently subjective readings of international treaties or claimed uncertainties of CIL which few international scholars would recognize,324 may ultimately protect EU norms, but it does so by mutating the international norm itself rather than balancing interests in a given case. This too renders it hard to recant from in a subsequent case with fresh competing interests. Ultimately, where proportionality has become dominant in resolving internal complexity, external recourse to it—in name at least—is a statistical outlier. This results in fragmented case law which can at times seem capricious, at least until one considers normative factors hidden behind judgments. Legal certainty is clearly affected by the continued purport of neutrality and a formal hierarchy, with normative concerns permeating without a clear rationale or mea- sured resolution and in stark contrast to internal case law. 321van Gend en Loos, Case C-26/62 at 12. 322MENDEZ, supra note 4, at 100. 323Pavoni, supra note 149, at 356. 324Wouters & Van Eeckhoutte, supra note 235, at 230–31. 325But see ECJ, Case C-308/06, Intertanko and Others, ECLI:EU:C:2008:312 (June 3, 2008), para. 51, https://curia.europa. eu/juris/liste.jsf?language=en&num=C-308/06. 326As occurred in Air Transport and ELFAA. 327See d'Aspremont & Dopagne, supra note 31, at 371. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press V. How Could Proportionality in External Case Law be Expanded in the Future? From the above we can see a significant fragmentation in approaches and even divergences within individual approaches. It has been argued that these seemingly narrow and often technical dis- tinctions are best understood through a normative lens. Proportionality is the purest expression of that normative lens available within EU law. The sheer presence of proportionality will lessen the pressure on direct effect related rules and the uncertainty it creates. For example, a finding of direct effect would no longer be fatal to a piece of EU secondary legislation as it may be justified at the proportionality stage.325 For a similar reason, international law would also be less susceptible to being imputed with questionable mean- ing as an avoidance strategy.326 This is particularly important given that although application of international law—through direct effect tests—is readily accepted as being within the discretion of domestic courts, interpretation of international law is not. Accordingly, though d’Aspremont and Dopagne suggest there is an “elementary divide”327 between the application of international law at https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar 590 the international and domestic levels, d’Aspremont is more nuanced concerning interpretation; “when applying the rules of another legal order, judges should heed the rules of interpretation of that legal order . . . [otherwise] judges would simply apply another rule than the rule originating in that foreign legal order.”328 With proportionality present the allure for the Court of opportunistic interpretations of international law and oscillating tests for direct effect as a means to control international law’s impact would be lessened in external case law. The logic for retaining structural differences com- pared to internal case law concerning access to justice would also be removed. Consequently, in the future, approaches in external case law could more closely reflect those already present in internal case law discussed above. For example, in judicial review cases under Article 263 TFEU, privileged applicants such as Member States should not be dependent on direct effect of the international provision. Under Article 267 TFEU, preliminary reference questions arising concerning the legality of EU secondary legislation do not require direct effect internally, nor should they externally. Direct effect would be required to challenge Member State legislation under 267 TFEU, as is the case internally: However, direct effect of international law in Member State cases has often been found and is less uncertain as a result. 328See Jean d’Aspremont, The Systemic Integration of International Law by Domestic Courts: Domestic Judges as Architects of the Consistency of the International Legal Order, in THE PRACTICE OF INTERNATIONAL AND NATIONAL COURTS AND THE DE- FRAGMENTATION OF INTERNATIONAL LAW 152 (Ole Kristian Fauchald & André Nollkaemper eds. 2012). See also Helmut Philipp Aust, Between Universal Aspiration and Local Application: Concluding Observations, in THE INTERPRETATION OF INTERNATIONAL LAW BY DOMESTIC COURTS: UNITY, DIVERSITY AND CONVERGENCE 339 (Helmut Phillip Aust & Georg Nolte eds., 2016); Wouters & Van Eeckhoutte, supra note 235, at 207. 329KOUTRAKOS, supra note 21, at 183. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press V. How Could Proportionality in External Case Law be Expanded in the Future? These changes would helpfully restore a consistent approach to the balance between actors across both internal and external case law. This is not to say that these changes will result in overbearing review of EU action in all instances, which should also alleviate concerns over floodgates opening to claimants. It should be remembered that the intensity in proportionality review of EU and Member State action differs. Member States often need to adopt the least restrictive path, whereas more lenience is afforded to the EU institutions. The practice of allowing a margin of appreciation to the EU legislature would seem appropriate here, given that its basis tends to be the complexity of judgements which the legislature must undertake, and International affairs are generally recognized to be an area in which the executive or legislature has wide discretion.329 Moreover, the odd potential divergence between cases such as Arcelor, concerning internal expansion of the emissions trading scheme, and Air Transport, concerning expansion of the same scheme with an international context, would be remedied. In Air Transport a margin was provided, but not based on the complexity of judgement required. Instead, unconvincingly, the uncertainty of customary international law was invoked; were the case to have concerned international treaty law binding on the EU, rather than customary law, no margin would have been provided to the legislature. k l f h ’ f f h A key limitation to any margin of appreciation arrives concerning the EU’s infringement of the essence of fundamental rights, and to the extent that this principle is present internally, this would continue to be the case externally. It is also submitted that the reference and implementation prin- ciples could appropriately be applied, in and beyond anti-dumping law, through proportionality, and could also affect the legislature’s margin of appreciation. This seems logical as statements that EU legislation has been passed “in accordance with” or to implement international law would mean that, were legislation not to meet this aim, then it would be disproportionate, except argu- ably in the most complex of instances. V. How Could Proportionality in External Case Law be Expanded in the Future? y p It can be seen that these changes would remove the need for direct effect in some cases and, in all cases, would lessen uncertainty within direct effect related rules and the need for opportunistic https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 591 German Law Journal interpretations of international law by providing a release valve in the form of proportionality. The question then becomes whether it is helpful to replace multiple small problems with one large problem; namely, what weight should be afforded to respect for international law within the case law? It is submitted that this single question was both already present and, indeed, driving the multiple oscillations and divergences present in the case law, but through a series of smaller syn- thetic proxies. Instead, by focusing more closely on the large question in scholarship and openly in the Court, clarity will emerge in a similar fashion to that which occurs internally concerning EU norms. Incremental arguments based within proportionality concerning respect for international law in differing contexts will inform future action in a meaningful way and will allow litigants to argue their case in alignment with the Court’s underlying rationales. 331It is submitted that the Court’s vision of “trade” in external relations case law is particularly inconsistent/limited at cur- rent as is its monosyllabic tendency to find Member States in breach of international agreements. On the latter see, Mendez, supra note 4. 332See supra discussion accompanying notes 23–38. 333B t it d p t 39 53 https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press 330See supra discussion accompanying notes 23–38. 330See supra discussion accompanying notes 23–38. 331It is submitted that the Court’s vision of “trade” in external relations case law is particularly inconsistent/limited at cur- rent as is its monosyllabic tendency to find Member States in breach of international agreements. On the latter see, Mendez, supra note 4. 332See supra discussion accompanying notes 23–38. 333But see sources cited supra notes 39–53. 332See supra discussion accompanying notes 23–38. 333 333But see sources cited supra notes 39–53. Cite this article: Dunbar R (2021). The Application of International Law in the Court of Justice of the European Union: Proportionality Rising. German Law Journal 22, 557–592. https://doi.org/10.1017/glj.2021.25 https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press E. Conclusion What is missing from external relations case law is the capacity for the Court to engage with the facts of a case in their specific context. Though proportionality has only appeared briefly in external relations case law and scholarship to date, it does appear to be a point upon which there is gradual convergence.330 Proportionality provides a convenient means by which to distinguish specific cases without the need to avoid or mutate international law on specious bases, which has occurred in its absence. Fully embracing proportionality in external relations case law would see international law being more readily invoked. However, respect for it would be balanced against other EU norms, with the consequence that—like all EU norms—respect for international law would be extrinsically defined, through applying proportionality in specific cases, rather than intrinsically defined through vacillating assertions about international law’s capability to be relied upon. g p y p When and which EU norms should trump respect for international law? These are clearly ques- tions for future research, and they would be facilitated by scholars openly acknowledging and promoting proportionality’s relevance rather than multiplying the range of nomenclatures. Then meaningful debate on substance can begin. For instance, many would argue that a margin ought to be provided to the legislature in fields related to international law, and yet others would argue that there should be heavy presumption in favor of respecting international law. Initially, it is clear that significant fundamental rights, such as Kadi, should be more likely to be favorably weighed against international law than more minor issues within consumer protection, such as ELFAA. But there are more challenging cases, and ones which call for very delicate consideration. The balance will also necessarily change over time, as all norms develop331 and cases differ. Proportionality can accommodate this debate and the complexity, which is already inherent in the case law, whilst simultaneously providing greater legal certainty. https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press respect for international law. However, it currently does so through the more crude and less legally certain means of oscillating direct effect tests, opportunistic interpretations, and an incoherent, autonomous legal system. So, though it does seem that there are embryonic or sporadic references to proportionality in aspects of both external relations case law and literature, it is also hoped that there is more to come on both counts. E. Conclusion Balancing respect for international law against other EU norms is eminently justifiable from the perspective of international law—as states retain discretion concerning application of international law domestically.332 More generally, the diminishing esteem in which the Court’s case law is held for its respectfulness towards international law means change is now opportune.333 Finally, it should be remembered that the Court is already balancing EU norms such as fun- damental rights, consumer protection, environmental protection, and trade interests against https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press Rupert Dunbar Rupert Dunbar 592 respect for international law. However, it currently does so through the more crude and less legally certain means of oscillating direct effect tests, opportunistic interpretations, and an incoherent, autonomous legal system. So, though it does seem that there are embryonic or sporadic references to proportionality in aspects of both external relations case law and literature, it is also hoped that there is more to come on both counts. respect for international law. However, it currently does so through the more crude and less legally certain means of oscillating direct effect tests, opportunistic interpretations, and an incoherent, autonomous legal system. So, though it does seem that there are embryonic or sporadic references to proportionality in aspects of both external relations case law and literature, it is also hoped that there is more to come on both counts. Cite this article: Dunbar R (2021). The Application of International Law in the Court of Justice of the European Union: Proportionality Rising. German Law Journal 22, 557–592. https://doi.org/10.1017/glj.2021.25 https://doi.org/10.1017/glj.2021.25 Published online by Cambridge University Press
W3201563208.txt	https://www.e3s-conferences.org/articles/e3sconf/pdf/2021/73/e3sconf_iccsre21_01041.pdf	en		Application analysis of ANFIS strategy for greenhouse climate parameters prediction: Internal temperature and internal relative humidity case of study	E3S web of conferences	2,021	cc-by	3,543	E3S Web of Conferences 297, 01041 (2021) ICCSRE’2021 https://doi.org/10.1051/e3sconf/202129701041 Application analysis of ANFIS strategy for greenhouse climate parameters prediction: Internal temperature and internal relative humidity case of study Hafsa Hamidane1∗ , Samira El Faiz1 , Iliass Rkik1 , Mohamed El Khayat1 , Mohammed Guerbaoui1 , Abdelali Ed-Dhhak1 and Abdeslam Lachhab1 1 Modelling, Materials and Control of Systems Team, High School of Technology, Moulay Ismail University, Meknes, Morocco Abstract. The present paper, introduces Adaptive Neuro Fuzzy Inference System (ANFIS) as one of the most mature and intelligent methods to predicte internal temperature and relative humidity of a greenhouse system. To conduct the application of the proposed strategy, an experimenntal greenhouse equipied with several sensors and actuators is engaged. In this sense a data base was collected during a period of day time where the temperature and relative humidity dynamics were observed inpresence of others climatic parameters and the actuators’ actions. The results demonstrate that by using ANFIS method, the predictions match the target points with a good accuracy . Therefore, the effectiveness of the strategy in term of both inside climate parameters’ prediction is guaranteed. 1 Introduction Nowadays, the agriculture domain faces several challenges, especially in term of hard climate conditions and the water deficit for any outdoor cultivation type. Hence, it has been admitted that greenhouses take a leading role in facing such obstacles, in fact, greenhouses have the ability to maintain the adequate micro climate for cultivated indoor crops, in addition to shield those latests from any climate weather excess and unwanted pests and leaks [1]. From a modelisation point of view, greenhouses are considered to be nonlinear complex systems, the dynamic coupling between different parameters such as, the inside air temperature, the outside temperature, the air velocity, the relative humidity, in addition to the outdoor meteorological conditions, make the modelling and control of inside climate parameters more computationally difficult to be ensured. For an adequate control and system management regarding such conditions, it is necessary to create and identify models that guarantee the system stability and that maintain its own properties. Modelling greenhouse’s comportement and dynamics is an interesting aspect since it represents the main task for any choosen control strategy. In fact, the comlexity and nonlinearity of greenhouses as buildings have encouraged researchers to think about more accurate identification technique in this sense, three major types of identifaction paradigms are commonly proposed: White box models, gray box model and black box models [2]. The first type is mostly based on mass/energy consevation principles and describes the systems from a physical knowledge, where a large set of parameters are included, hence, resulted models are inacurate and the implemetation of such ∗ e-mail: Hamidanehafsa@gmail.com boxes are difficult to be acheived. Grey box models in there part are based on sets of differential equations presented in form of a state space presntation derived from the physical laws that describe the system dynamics. In addition this technique of identification uses estimation techniques to estimate the unknown parameters included in the model. However, a major challenge of gray box modeling is that it relies on robust parameter estimation methods, which require more care related to the training process of data sets. The third modelling paradigm is black box method, this type of modelling is a data driven model based completely on measured data without incorporating any equation of physical dynamics regarding the system. They can be presented as parametric linear models such as Auto Regressive (AR), Auto Regressive with eXogenous inputs (ARX), Auto Regressive with Moving Average and eXogenous inputs (ARMAX) etc..., or as nonparametric/parametric nonlinear ones such as kNearest Neighbors (kNN), Gaussian Processes (GP), Random Forest (RF), Artificial Neural Network (ANN), Genetic Algorithm (GA),Adaptative Neuro-Fuzzy Inference System (ANFIS) and many other techniques for more details the reader is referd to [4]. ANFIS is now considered to be among the most commonly used AI models, therfore, recentely many are the areas that have emphasized the improvement of ANFIS performance by implementing it within multiple projects and domains. For instance, in buildings such as greenhouse’s and especially for climate prediction aims. From the greenhouse perspective, inside temperature and relative humidity take a leading role in the process of identification depending on the type of cultivation and crops, however these parameters are mostly affected; in a way or another; by a number of factors and phenomena such as the © 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/). E3S Web of Conferences 297, 01041 (2021) ICCSRE’2021 https://doi.org/10.1051/e3sconf/202129701041 greenhouse external climatic conditions, heating, cooling, moisutrizing, etc.. [3]. In this sense, the present paper aimed to investigate the prediction of internal temperature and internal humidity of the greenhouse as a complexe system, using both, ANFIS strategy as an intelligent alternative and historical data for external climatic parameters and actuators’ operation regarding the Schefflera cultivation planted in the real greenhouse. The remainder of this paper is organized as follows, Some preliminaries regarding ANFIS strategy will be presented in section 2. Section 3 will step through materials and methods including the experimental greenhouse presentation and data preparation. The results of this paper will be discussed in section 4. In section 5, conclusions will be provided. and the membership function of Ai. The standard form of µAi (x) is as follows: − µAi (x) = e (x−Ci )2 2σ2 i (2) σ and C present the parameters of the Gaussian function. - The second layer: Every node is presented to fix and its role is to calculate the weights ωi of a generated rule. The weightt of each node is given by: O2i = ωi = µAi (x) × µBi (y) f or i = 1, 2 (3) - The third layer: For every ith node in this layer, the average of the ith rule’s weight to the sum of weights of all the rules is calculated. The formula is presented as follows: 2 Preliminaries O3i = ω̄i = 2.1 Theory behind ANFIS 2.1.1 Brief insight: ωi ω1 + ω2 f or i = 1, 2 (4) - The fourth layer: The ratio of every weight calculated in layer 3 , is then multiplied by its associated function in the fourth layer: An ANFIS structure: ANFIS network is a data driven technique that engages Neural Network strategy to solve approximation problems of complexe systems. In addition a set of input and output variables are incorporated to generate fuzzy inference based on fuzzy reasoning with the IfThen rules proposed by Takagi and Sugeno, its structure is generally composed of five main layers [5]. Its principle is based on fuzzy logic and neural network method where inputoutput variables, a set of rules, and fuzzy inference system take a part in the process of systems’ behavior forcasting. For simlicity a structure of two inputs and single output is depicted in Figure 1 O4i = ω̄i f i = ω̄i (pi x + qi y + ri ) f or i = 1, 2 (5) where pi , qi and ri , present the resulted parameters set. - The fifth layer: Herein, the sum of the previous outputs in the last layer, the resulted output is given by: ωi f i O5i = ω̄i f i = i (6) i ωi i It is worth mentionnig that the application of ANFIS models has been considered in various areas we can cite health, industry, finance etc... Its different paradigms utilities [6] are: Pattern classification, time series prediction, regression, fault detection and industrial problems. 2.1.2 Learning/ training algorithms: For any ANFIS, the training of models is a compulsory step to take into account, for this aim, various are the algorithms that are commonly employed in training process models based on neural networks and fuzzy systems. In general, the utility of the mentionned algorithms is that they allow the resulted answer to be adapted to the objective for which they are aimed. Among these algorithms we can find: Figure 1. General structure of Anfis model Backpropagation: A widely used training algorithm regarding ANFIS models, this method permits to determine a vector of weights, that optimizes(minimize) the model’s training error repeatedely. In a simple way, the aim of this technique is to find a set of weights that minimizes the output of the network error, in a way to map the inputs to the outputs in a correct manner. This tehcnique is based on the so-called gradient descent optimization algorithm, the reader can be refered to [7]. - The first layer: In this layer, each incorporated node i is adaptative with a function as: O1i = µAi (x) (1) where x, Ai and µAi present respectivelly the input to node i, the variable associated with the node function 2 E3S Web of Conferences 297, 01041 (2021) ICCSRE’2021 https://doi.org/10.1051/e3sconf/202129701041 Table 1. The components of the experimental greenhouse. Genetic Fuzzy neural networks: Genetic Algorithms are considered to be as optimization algorithms that solve a given problem based on natural selection. The search strategy adopted in genetic algorithms is considered to be structured and random and works using a group of points instead of a single point. They mostly have the ability to identify environmental factors which results on optimal solutions [8]-[10]. Component LM35DZ HIH-4000-001 Heater Securia 10" BOX FAN NI USB-6009 Deep learning: Beeing one of the sub-categories of machine learning, deep learning is considered to be one of the automatic learning techniques that gives the computers the opportunity to learn based on examples [11][12]. The advantage of this algorithm, is that for traditional neural networks, three or less hidden layers are supported by the network, while for the case of deep learning, the network can contain an enormous number of hidden layers. Role Sensor Sensor Actuator Actuator Acquisition card Measurements Temperature Relative humidity Heating Ventilation Data logging and measurements 3 Materials and Methods 3.1 Prototype of the Experimental Greenhouse The greenhouse engaged in the present article is dipected in Figure 2, it is an experimental greenhouse prototype, located at the department of physics, Faculty of Sciences, Meknes, Morocco. The system is a single wall polyethylene construction, equipped with two temperature sensors and two humidity sensors that provide respectivelly indoor and outdoor temperature and relative humidity measurements. Aditionally, two actuators presented as a heating system and a ventilator are installed as well in order to insure an adequate inside climate for the greenhouse plants. Hybrid optimization: Hybrid is generaly based on backpropagation for the parameters with the input membership functions, and the least squares estimation method for the parameters in association with the output membership functions[13][14] More algorithms and methods are engaged in this sence,we mention: • Evolving and on-line algorithm • Evolutionary algorithms • Extreme learning machine (ELM) • etc... 2.1.3 ANFIS metrics and fitness functions: In order to validate the efficiency and the prediction performance of the propesed strategy, some fitness functions named also statistical variables [15][16], are generally used to determine how well the predicted values and observed values match each others. These functions are namely, Mean Square Error (MSE), Root Mean Square Error (RMSE) and the correlation factor R2 , that are described in Equations (7)-(9). MS E = RMS E = 1 N N i=1 N 1 (xobs,i − x pred,i )2 N i=1 N R = 1 − i=1 N 2 (xobs,i − x pred,i )2 (xobs,i − x pred,i )2 i=1 (xobs,i − xobs,i ¯ )2 Figure 2. Experimental Greenhouse System. Data acquisition and control tasks are insured by the means of an acquisition cards attached to a personal computer where the mentionned sensors and actuators are connected. This card is installed to manage the different actuator orders. In addition, a protection and signal conditionning cards are also installed to guarantee the hole system protection. In a second place; the tasks of supervision of measured indoor and outdoor climate variables; are provided in form of historical database sets using Matlab/Simulink as software. More details about the different components and their roles are provided in Table 1. (7) (8) (9) ¯ refer respectivelly to, Where N, xobs,i , x pred,i and xobs,i the number of data sets, the observed or actual output, the predicted or aproximated output and the average of the present points. 3.2 Data setting and preparation In the first stage,the historical data set used for temperature and relative humidity prediction was collected and 3 E3S Web of Conferences 297, 01041 (2021) ICCSRE’2021 https://doi.org/10.1051/e3sconf/202129701041 obtained from the real greenhouse prototype as detailled in subsection 3.1 . Data were collected for two hours and a half successively during the early afternoon, 5 seconds was used as sample time Ts. In total, the whole experimental data set was 2200 samples that was devided into two categories: training part samples that represent 70% of the data and the testing part which describe 30% of the set. For internal temperature prediction case, and for both, training and testing processes, the inputs were respectively chosen to be: the external temperature (T ex ), the external relative humidity (Rhex ), the internal relative humidity (Rhin ), the fan and the heater actions (F), (H), while the output was selected to be the internal temperature (T in ). For internal relative humidity prediction case, and for both processes as well, the inputs were: the external temperature (T ex ), the internal temperature (T in ), the external relative humidity (Rhex ), the fan and the heater actions (F), (H) respectively, and the output was set to be the internal relative humidity (Rhin ). Figure 3 and Figure 4 show the structure of ANFIS models regarding the inside temperature and relative humidity prediction. Herein, the training part was dedicated to generate the best network while the testing part had the functionality to validate the performance of each model concerning the temperature and relative humidity. this study, Data collection and simulation developpement were obtained using a Core 3 personel computer with 4G of RAM. Figure 5 shows the evolution of external temperature and external relative humidity during the same period of data collection, it is noticable that these latests evoluate between a range of 22 ◦ C and 25 ◦ C for temperature and a range of 62% and 70% for humidity. With this end in view, two cases were studied: Figure 5. External temperature and relative humidity 4.1 Temperature case: T in prediction For a period of 2 hours and a half of time and a sample time of 5 seconds, 2200 Data were collected and performed to validate the ANFIS strategy for the case of temperature prediction, As it is seen, Figure 6 shows the training/ testing processes for the internal temperature data in response to the effects of the other climatic parameters and both actuators’ actions, while Figure 7 validates the accuracy of the strategy. We can notice that the predicted internal temperature matchs the observed or real one. It is remarkable Figure 3. ANFIS model for internal temperature Figure 4. ANFIS model for internal relative humidity 4 Results and discusions In order to validate the proposed identification strategy, results were performed in form of simulations. It is worth reminding that the goal of this paper is the identification of time series of the greenhouse inside temperature and relative humidity, taking into account the effect of the other climatic parameters and the actuators’ actions. In Figure 6. Training and testing processes for internal temperature Tin 4 E3S Web of Conferences 297, 01041 (2021) ICCSRE’2021 https://doi.org/10.1051/e3sconf/202129701041 Figure 7. Observed and predicted internal temperature Tin Figure 9. Training and testing processes for internal relative humidity Rhin Figure 8. Anfis model RMSE for temperature case during training that, Figure 8 shows the evolution of the error (RMSE) of the training process, herein the error decreases from 0.3888 to take 0.3872. Figure 10. Observed and predicted internal relative humidity Rhin 4.2 Relative humidity case: Rhin prediction 0.6334 For the same period of time, 2200 Data were collected for the humidity prediction case, As depicted in Figure 9, the training/ testing processes for the internal relative humidity data is similar to the temperature one in response to the same actuators and climatic variations for the other parameters, hence this time, the accuracy for the humidity prediction decreases compared to the temperature case as it is clearely seen from Figure 10, despite this, we can remark that the predicted internal relative humidity fits the observed one in an acceptable manner. In addition to that, Figure 11 shows the evolution of the error (RMSE) regarding the training process, we notice that the error decreases to be setlled to 0.6320. The ANFIS models’ details and fitting metrics for both, internal temperature and internal relative humidity are presented respectively in Table 2, Table 3 and Table 4. 0.6328 0.6332 RMSE 0.633 0.6326 0.6324 0.6322 0.632 0.6318 0 100 200 300 400 500 600 Epoch Figure 11. Anfis model RMSE for humidity case during training 5 E3S Web of Conferences 297, 01041 (2021) ICCSRE’2021 https://doi.org/10.1051/e3sconf/202129701041 Table 2. DETAILS ON THE ANFIS MODEL FOR TEMPERATURE CASE. ANFIS model for Tin prediction Membership function (MF) type Number of (MF) Optimization method Number of training Data points Number of testing Data points Iterations [3] S. Ghani, et al, "Design challenges of agricultural greenhouses in hot and arid environments – A review," Engineering in Agriculture, Environment and Food, vol. 12, no 1, pp. 48-70, 2019. [4] J.Drgoňa, et al, "All you need to know about model predictive control for buildings," [5] A. Guerrero-Santana et al, "Prediction of air temperature and relative humidity in a solar greenhouse dryer using neuro-fuzzy models," 2018 ASABE Annual International Meeting, pp. 1, 2018. [6] S.de Campos and P. Vitor, "Fuzzy neural networks and neuro-fuzzy networks: A review the main techniques and applications used in the literature," Applied Soft Computing, vol. 92, p. 106275, 2020. [7] Y.Shi and M. Mizumoto, "Some considerations on conventional neurofuzzy learning algorithms by gradient descent method," Fuzzy sets and systems, vol. 112, no. 1 pp. 51-63, 2000. [8] H. Wang, et al, "Greenhouse CO2 Control Based on Improved Genetic Algorithm and Fuzzy Neural Network," 2018 2nd IEEE Advanced Information Management, Communicates, Electronic and Automation Control Conference (IMCEC). IEEE, pp. 1537-1540, 2018. [9] M. Outanoute, et al, "A neural network dynamic model for temperature and relative humidity control under greenhouse," 2015 Third International Workshop on RFID And Adaptive Wireless Sensor Networks (RAWSN). IEEE, pp. 6-11, (2015). [10] K. Ozgur, H. Sanikhani, and M. Cobaner, "Soil temperature modeling at different depths using neuro-fuzzy, neural network, and genetic programming techniques," Theoretical and Applied Climatology, vol. 129, no 3, pp. 833-848, (2017). [11] J. Dae-Hyun, et al, "Time-serial analysis of deep neural network models for prediction of climatic conditions inside a greenhouse," Timeserial analysis of deep neural network models for prediction of climatic conditions inside a greenhouse, vol. 173, p. 105402, (2020). [12] Li. Chengdong, et al, "Building energy consumption prediction: An extreme deep learning approach," Energies, vol. 10, no 10, p. 1525,(2017). [13] Qiao, Weibiao, et al, "A hybrid algorithm for carbon dioxide emissions forecasting based on improved lion swarm optimizer," Journal of Cleaner Production, vol. 244, p. 118612, (2020). [14] Yu, Huihui, et al, "Prediction of the temperature in a Chinese solar greenhouse based on LSSVM optimized by improved PSO.," Computers and Electronics in Agriculture, vol. 122, p. 94-102, (2016). [15] R. Daneshfar, et al, "Estimating the Heat Capacity of Non-Newtonian Ionanofluid Systems Using ANN, ANFIS, and SGB Tree Algorithms," Applied Sciences, vol. 10, no 18, p. 6432, (2020). Grid partition 2 Hybrid 1540 660 200 Table 3. DETAILS ON THE ANFIS MODEL FOR RELATIVE HUMIDITY CASE. ANFIS model for Rhin prediction Membership function (MF) type Number of (MF) Optimization method Number of training Data points Number of testing Data points Iterations Grid partition 2 Hybrid 1540 660 600 Table 4. PERFORMANCE OF ANFIS MODELS USING STATISTICAL METRICS ANFIS Temperature Humidity Process Training Testing Total Training Testing Total MSE 0.1499 0.1521 0.1505 0.3994 0.4064 0.4014 RMSE 0.3872 0.3900 0.3880 0.6320 0.6375 0.6336 R² 0.9821 0.8078 0.9298 0.9686 0.6940 0.8862 5 Conclusion In this paper, we have developed two ANFIS models in a way to predict the comportement of the major climate parameters under greenhouses, which are the internal temperature and the internal relative humidity. The statistical metrics of the proposed method showed satisfactory results, temperature case prediction had the highest accuracy with 0.3880 RMSE and 0.9298 R², while the prediction of relative humidity performances was described to be less satisfactory than the temperature case with 0.6336 RMSE and 0.8862 R². One can conclude that overall, the results performed in the present paper demonstrate the advantage of using Anfis based prediction strategy for greenhouse climate prediction and/or for climate control purposes. References [1] A. Guo, et al, "Modeling and Optimization of Environment in Agricultural Greenhouses for Improving Cleaner and Sustainable Crop Production," Journal of Cleaner Production, p. 124843 , (2020). [2] A.Boodi, et al, "Intelligent systems for building energy and occupant comfort optimization: A state of the art review and recommendations," Energies, vol. 11, no 10, pp. 2604, 2018. 6 E3S Web of Conferences 297, 01041 (2021) ICCSRE’2021 https://doi.org/10.1051/e3sconf/202129701041 [16] H. Oubehar, et al. "Design and real time implementation of ANFIS controller for greenhouse climate," 2018 International Conference on Elec- tronics, Control, Optimization and Computer Science (ICECOCS). IEEE, p. 1-4, (2018). 7
https://openalex.org/W2774278057	https://www.pure.ed.ac.uk/ws/files/137652738/Chauche_C_Mammalian_adaptation....pdf	English	null	Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1	Journal of virology	2,018	cc-by	15,770	Edinburgh Research Explorer Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1 Citation for published version: Chauché, C, Nogales, A, Zhu, H, Goldfarb, D, Ahmad Shanizza, AI, Gu, Q, Parrish, CR, Martínez-sobrido, L, Marshall, JF, Murcia, PR & Schultz-Cherry, S (ed.) 2018, 'Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1', Journal of Virology, vol. 92, no. 5, e01875-17. https://doi.org/10.1128/JVI.01875-17 Citation for published version: Chauché, C, Nogales, A, Zhu, H, Goldfarb, D, Ahmad Shanizza, AI, Gu, Q, Parrish, CR, Martínez-sobrido, L, Marshall, JF, Murcia, PR & Schultz-Cherry, S (ed.) 2018, 'Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1', Journal of Virology, vol. 92, no. 5, e01875-17. https://doi.org/10.1128/JVI.01875-17 Citation for published version: Citation for published version: Chauché, C, Nogales, A, Zhu, H, Goldfarb, D, Ahmad Shanizza, AI, Gu, Q, Parrish, CR, Martínez-sobrido, L, Marshall, JF, Murcia, PR & Schultz-Cherry, S (ed.) 2018, 'Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1', Journal of Virology, vol. 92, no. 5, e01875-17. https://doi.org/10.1128/JVI.01875-17 General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Digital Object Identifier (DOI): 10.1128/JVI.01875-17 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Journal of Virology Publisher Rights Statement: Copyright© 2018 Chauché et al. This is anopen-access article distributed under the termsof theCreative Commons Attribution 4.0Internationallicense. Mammalian Adaptation of an Avian Inﬂuenza A Virus Involves Stepwise Changes in NS1 on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from C. Chauché,a A. Nogales,b H. Zhu,a D. Goldfarb,a A. I. Ahmad Shanizza,a Q. Gu,a C. R. Parrish,c L. Martínez-Sobrido,b J F Marshall d P R Murciaa aMRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom bDepartment of Microbiology and Immunology, University of Rochester, Rochester, New York, USA cBaker Institute of Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA dEquine Clinical Sciences Division, Weipers Centre Equine Hospital, School of Veterinary Medicine, University of Glasgow, Glasgow, United Kingdom ABSTRACT Inﬂuenza A viruses (IAVs) are common pathogens of birds that occa- sionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral nonstruc- tural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host species barrier. We characterized the NS1 proteins of equine inﬂuenza vi- rus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionarily distinct NS1 proteins counteract the IFN response using different and mutually ex- clusive mechanisms: while the NS1 proteins of early EIVs block general gene expres- sion by binding to cellular polyadenylation-speciﬁc factor 30 (CPSF30), NS1 proteins from more evolved EIVs speciﬁcally block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are as- sociated with two mutations that appeared sequentially and were rapidly selected for during EIV evolution, highlighting the importance of evolutionary processes in immune evasion mechanisms during IAV adaptation. on March 3, 2020 at UNIVERSITY OF EDINBURGH rg/ 3, 2020 at UNIVERSITY OF EDINBURGH IMPORTANCE Inﬂuenza A viruses (IAVs) infect certain avian reservoir species and oc- casionally transfer to and cause epidemics of infections in some mammalian hosts. However, the processes by which IAVs gain the ability to efﬁciently infect and trans- mit in mammals remain unclear. H3N8 equine inﬂuenza virus (EIV) is an avian-origin virus that successfully established a new lineage in horses in the early 1960s and is currently circulating worldwide in the equine population. Here, we analyzed the mo- lecular evolution of the virulence factor nonstructural protein 1 (NS1) and show that NS1 proteins from different time periods after EIV emergence counteract the host in- nate immune response using contrasting strategies, which are associated with two mutations that appeared sequentially during EIV evolution. Mammalian Adaptation of an Avian Inﬂuenza A Virus Involves Stepwise Changes in NS1 The results shown here indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation. Received 27 October 2017 Accepted 29 November 2017 Accepted manuscript posted online 13 December 2017 Citation Chauché C, Nogales A, Zhu H, Goldfarb D, Ahmad Shanizza AI, Gu Q, Parrish CR, Martínez-Sobrido L, Marshall JF, Murcia PR. 2018. Mammalian adaptation of an avian inﬂuenza A virus involves stepwise changes in NS1. J Virol 92:e01875-17. https://doi.org/10 .1128/JVI.01875-17. Editor Stacey Schultz-Cherry, St. Jude Children's Research Hospital Copyright © 2018 Chauché et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to P. R. Murcia, Pablo.Murcia@glasgow.ac.uk. C.C. and A.N. contributed equally to this article. Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 VIRUS-CELL INTERACTIONS Received 27 October 2017 Accepted 29 November 2017 Accepted manuscript posted online 13 December 2017 Citation Chauché C, Nogales A, Zhu H, Goldfarb D, Ahmad Shanizza AI, Gu Q, Parrish CR, Martínez-Sobrido L, Marshall JF, Murcia PR. 2018. Mammalian adaptation of an avian inﬂuenza A virus involves stepwise changes in NS1. J Virol 92:e01875-17. https://doi.org/10 .1128/JVI.01875-17. Editor Stacey Schultz-Cherry, St. Jude Children's Research Hospital Copyright © 2018 Chauché et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to P. R. Murcia, Pablo.Murcia@glasgow.ac.uk. C.C. and A.N. contributed equally to this article. KEYWORDS NS1, evolution, inﬂuenza, virus-host interactions I nﬂuenza A viruses (IAVs) have caused several epizootics in various animal species and four pandemics in humans in the last hundred years. The main natural reservoir of IAVs is wild water birds of various types, but the viruses also circulate in some mammalian host populations, including humans, pigs, horses, and dogs (1). However, most infections of mammals by avian IAVs result in either spillover infections or isolated outbreaks (1, 2), and very few have resulted in the establishment of novel endemic I n f March 2018 Volume 92 Issue 5 e01875-17 March 2018 Volume 92 Issue 5 e01875-17 Journal of Virology jvi.asm.org 1 Chauché et al. Journal of Virology lineages. The reasons underlying the establishment of an avian-origin IAV in a mam- malian host are still only partially understood. In most cases it appears that emerging IAVs have to overcome multiple barriers to infect and become established in a new host. For example, the presence of particular forms or linkages of sialic acids within the respiratory tract can facilitate or impair binding by the viral hemagglutinin (HA) or the activity of the viral neuraminidase (NA) (3, 4). Other incompatibilities between IAVs and host proteins can also account for the species barrier, as it has been recently shown that ANP32A is a host factor that is required for optimal function of the IAV polymerase complex (5). Molecular incompat- ibilities can sometimes be overcome by adaptive mutations in the virus, and the high mutation rates exhibited by IAVs can facilitate their appearance. The role of mutations in HA in virus-host interaction are among the best characterized (6), but mutations in other viral segments, including those in PB2, nucleoprotein (NP), NA, M, or NS, have also been described (7–9). However, the roles of the latter group in posttransfer adaptation are still incompletely understood. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from IAV nonstructural protein 1 (NS1) is encoded by segment eight and possesses two functionally distinct domains: an N-terminal RNA binding domain (RBD) (amino acids 1 to 73) and a C-terminal effector domain (ED) (amino acid 85 to end of protein) separated by a short and ﬂexible linker region (10, 11). The last 25 residues of NS1 are thought to form a disordered and ﬂexible “tail” (10). Molecular interactions with multiple host proteins allow NS1 to perform a remarkable number of activities (12). KEYWORDS NS1, evolution, inﬂuenza, virus-host interactions One of the best-described functions of NS1 is its ability to antagonize the innate immune response, predominantly the production of type I interferons (IFN). The type I IFN response is an essential component of the host barrier that plays a critical role against emerging viruses, as the recipient hosts usually lack preexisting immunity. A variety of pathogen recognition receptors (PRRs) recognize pathogen-associated molecular pat- terns (PAMPs) and activate a cascade of events that lead to the production and secretion of type I IFN (13). Secreted type I IFN binds to its receptor in both infected and neighboring cells and activates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, which in turn results in the assembly of a protein complex, referred to as interferon-stimulated gene factor 3 (ISGF3), which is composed of phospho-STAT1, phospho-STAT2, and interferon regulatory factor 9 (IRF9). This complex translocates to the nucleus, binds to DNA regulatory sequences containing IFN-stimulated response elements (ISREs), and stimulates the transcription of hundreds of IFN-stimulated genes (ISGs), including ISG15, myxovirus resistance protein 1 (MX1), and 2=-5=-oligoadenylate synthetase 1/2/3 (OAS1/2/3). As a result, IFN-stimulated cells establish an antiviral state to protect against different viruses, including IAVs (13, 14). H3N8 equine inﬂuenza virus (EIV) is an avian-origin virus lineage that has been circulating in horses since at least 1963 (15, 16) and thus provides a model for the long-term mammalian adaptation of avian-derived IAVs. Phylogenetic studies have shown that several amino acid substitutions occurred within each genomic segment during the evolutionary history of EIV, some of which have been associated with host adaptation in other IAVs (17). However, the role of any of those mutations in EIV adaptation to horses is unclear. We hypothesized that the evolution of NS1 would be part of the process of EIV adaptation to horses. To test this hypothesis, we characterized the NS1 genes of a group of evolutionarily distinct EIVs using a combination of approaches that included experimental infections, reverse genetics, site-directed mu- tagenesis, phylogenetics, and transcriptomics. IAV nonstructural protein 1 (NS1) is encoded by segment eight and possesses two functionally distinct domains: an N-terminal RNA binding domain (RBD) (amino acids 1 to 73) and a C-terminal effector domain (ED) (amino acid 85 to end of protein) separated by a short and ﬂexible linker region (10, 11). The last 25 residues of NS1 are thought to form a disordered and ﬂexible “tail” (10). KEYWORDS NS1, evolution, inﬂuenza, virus-host interactions Molecular interactions with multiple host proteins allow NS1 to perform a remarkable number of activities (12). One of the best-described functions of NS1 is its ability to antagonize the innate immune response, predominantly the production of type I interferons (IFN). The type I IFN response is an essential component of the host barrier that plays a critical role against emerging viruses, as the recipient hosts usually lack preexisting immunity. A variety of pathogen recognition receptors (PRRs) recognize pathogen-associated molecular pat- terns (PAMPs) and activate a cascade of events that lead to the production and secretion of type I IFN (13). Secreted type I IFN binds to its receptor in both infected and neighboring cells and activates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, which in turn results in the assembly of a protein complex, referred to as interferon-stimulated gene factor 3 (ISGF3), which is composed of phospho-STAT1, phospho-STAT2, and interferon regulatory factor 9 (IRF9). This complex translocates to the nucleus, binds to DNA regulatory sequences containing IFN-stimulated response elements (ISREs), and stimulates the transcription of hundreds of IFN-stimulated genes (ISGs), including ISG15, myxovirus resistance protein 1 (MX1), and 2=-5=-oligoadenylate synthetase 1/2/3 (OAS1/2/3). As a result, IFN-stimulated cells establish an antiviral state to protect against different viruses, including IAVs (13, 14). H3N8 equine inﬂuenza virus (EIV) is an avian-origin virus lineage that has been on March 3, 2020 at UNIVERSITY OF EDINBURGH H3N8 equine inﬂuenza virus (EIV) is an avian-origin virus lineage that has been circulating in horses since at least 1963 (15, 16) and thus provides a model for the long-term mammalian adaptation of avian-derived IAVs. Phylogenetic studies have shown that several amino acid substitutions occurred within each genomic segment during the evolutionary history of EIV, some of which have been associated with host adaptation in other IAVs (17). However, the role of any of those mutations in EIV adaptation to horses is unclear. We hypothesized that the evolution of NS1 would be part of the process of EIV adaptation to horses. To test this hypothesis, we characterized the NS1 genes of a group of evolutionarily distinct EIVs using a combination of approaches that included experimental infections, reverse genetics, site-directed mu- tagenesis, phylogenetics, and transcriptomics. RESULTS Evolutionarily distinct NS1 proteins of the H3N8 EIV lineage exhibit marked functional differences. To experimentally study the functional evolution of the H3N8 EIV NS1 proteins, we selected 13 H3N8 EIVs that included at least one representative virus per decade since its ﬁrst isolation in 1963 (Table 1) and cloned the coding sequences of their NS1 proteins into a pCAGGS expression plasmid. jvi.asm.org 2 March 2018 Volume 92 Issue 5 e01875-17 Functional Evolution of the EIV NS1 Gene Journal of Virology TABLE 1 Viruses used in this study Virus name Accession no. Abbreviation A/equine/Uruguay/1/1963 ACD85423 U/63 A/equine/Miami/1/1963 ABY81497 M/63 A/equine/SaoPaulo/1/1969 ACD85390 SP/69 A/equine/Fontainebleau/1/1979 ACD85401 F/79 A/equine/Sussex/1/1989 ACD97430 S/89 A/equine/Kentucky/1/1991 ACA24672 K/91 A/equine/LaPlata/1995 MF182460 LP/95 A/equine/Kentucky/1995 MF182451 K/95 A/equine/Kentucky/1999 MF182443 K/99 A/equine/Kentucky/5/2002 ABA42429 K/02 A/equine/Newmarket/5/2003 ACI48802 N/03 A/equine/Ohio/1/2003 ABA42431 O/03 A/equine/Mongolia/3/2013 MF182459 M/13 on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from As the F2/F3 region of CPSF30 involved in interaction with inﬂuenza virus NS1 proteins is conserved between human and equine species (data not shown) and the transfection efﬁciency of 293T cells is highly superior to that of equine dermal ﬁbro- blasts (E-derm cells) (data not shown), we used 293T cells to study the functional evolution of EIV NS1. To compare the abilities of the individual NS1 proteins to limit the production of IFN-, we cotransfected 293T cells with NS1 expression plasmids or with an empty vector (pCAGGS), together with a plasmid expressing ﬁreﬂy luciferase (FF-Luc) under the IFN- promoter (pIFN--FF-Luc) and with a constitutively active Renilla luciferase (REN-Luc) expression plasmid (pREN-Luc) to normalize for transfection efﬁ- cacy. At 24 h posttransfection (hpt), the cells were infected with Sendai virus (SeV), a well-described IFN- inducer. As expected, SeV infection resulted in strong activation of the IFN- promoter in cells cotransfected with the empty vector (set to 100%) (Fig. 1A) compared to uninfected control cells. However, SeV activation of the IFN- promoter was blocked in cells transfected with the different EIV NS1-expressing plasmids used in this study (Fig. 1A). on March 3, 2020 at UNIVERSITY OF EDINBURGH We also compared the abilities of these NS1 proteins to block the induction of ISGs upon IFN treatment. To this end, we cotransfected 293T cells with the NS1 expression plasmids described above, together with a plasmid expressing ﬁreﬂy luciferase under the control of a promoter containing an IFN-stimulated regulatory element (pISRE-FF- Luc), and again used pREN-Luc as an internal control. RESULTS At 24 hpt, we treated the cells with universal type I IFN (uIFN) (PBL Assay Science; 500 units/well) to stimulate the ISRE-containing promoter, and 18 h later, we measured FF-Luc and REN-Luc activities. The NS1 proteins of EIVs isolated in 1963 (so-called “early”: U/63 and M/63) displayed relatively low repression of the ISRE-containing promoter (Fig. 1B), and the antagonistic property of NS1 over this promoter increased in EIVs that circulated between 1969 and 1995 (SP/69, F/79, S/89, K/91, LP/95, and K/95). Furthermore, the NS1 proteins of EIVs isolated between 1999 and 2013 (so-called “late”: K/99, K/02, O/03, N/03, and M/13) showed variable control of the ISRE-containing promoter, depending on the NS1 protein tested (Fig. 1B). To assess the abilities of the NS1 proteins to inhibit general gene expression, we performed cotransfections of the individual NS1 expression plasmids, together with pREN-Luc, and measured luciferase activity 24 h later. The NS1 proteins of EIVs that circulated in the 1960s strongly blocked the otherwise constitutively active Renilla luciferase (Fig. 1C). In contrast, the NS1 proteins of EIVs isolated after 1979 did not block general gene expression, as REN-Luc expression was not strongly reduced. In addition, in some cases, NS1 expression was associated with an increase in luciferase expression (Fig. 1C), as described for other NS1 proteins (18). We also examined the expression levels of NS1 by Western blotting in parallel transfected-cell lysates. As expected, the NS1 proteins that strongly blocked general gene expression (i.e., U/63, M/63, and SP/69) could not be detected, probably because of the ability of these NS1 proteins to inhibit their own synthesis (18, 19) (Fig. 1D). On the other hand, NS1 proteins that did not block jvi.asm.org 3 March 2018 Volume 92 Issue 5 e01875-17 Journal of Virology Chauché et al. FIG 1 Functional characterization of evolutionarily distinct EIV NS1 proteins. (Top) Schematic representation of three versions of the NS1 protein (early, intermediate, and late) found throughout EIV evolution. Early NS1 proteins, full length (230 amino acids) and harboring E186, were found in EIVs circulating between the 1960s and 1970s; intermediate (Interm.) NS1 proteins, full length with K186, were found in EIVs circulating between the late 1970s and late 1990s; and late NS1 proteins, truncated at the C terminus (219 amino acids) with K186, are found in EIVs isolated since the late 1990s. March 2018 Volume 92 Issue 5 e01875-17 jvi.asm.org 4 RESULTS These results suggest a direct effect of these NS1 proteins on the IFN signaling pathway. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from Amino acid 186 and the C-terminal tail affect EIV NS1 protein function. To identify the mutations responsible for the stepwise chronological changes in EIV NS1 function, we ﬁrst examined a multiple-sequence alignment of the NS1 proteins used in our studies (Fig. 1E). While various amino acid changes were observed, the most evident was an 11-amino-acid truncation in the carboxy (C) terminus that appeared in 1999 (20), as well as the appearance of two amino acid substitutions in the 1970s: E186K and A112T. The E186K substitution lies within the putative CPSF30-binding domain, and notably, this substitution took place at the same time as the loss of repression of general gene expression by EIV NS1 (Fig. 1C). To determine the impacts of the E186K substitution and the C-terminal truncation on NS1 function, we introduced mutations in the NS1 gene of A/equine/Ohio/2003 (O/03), a virus isolated after 40 years of continuous EIV circulation in horses. The NS1 protein of O/03 is 219 amino acids long (naturally truncated) and possesses a lysine at position 186 (Fig. 2A, Late). Three NS1 revertant viruses were tested: a K186E substi- tution (O/03-K186E), a C-terminal extension of 11 amino acids (O/03-230), and the double change of the extended NS1 and the K186E substitution together (O/03-K186E- 230) (Fig. 2A). The O/03-230 NS1 revertant would therefore represent the intermediate (Fig. 2A, Interm.) NS1 proteins of EIVs that circulated between 1979 and 1995 (F/79, S/89, K/91, LP/95, and K/95), while the NS1 double revertant represents early NS1 proteins of EIVs that circulated between 1963 and 1969 (U/63, M/63, and SP/69) (Fig. 2A, Early). To determine the impact of a glutamic acid residue at position 186, we intro- duced a K186E mutation in the O/03 NS1 (Fig. 2A, Artiﬁcial). However, it should be noted that such a revertant has not been detected in nature. on March 3, 2020 at UNIVERSITY OF EDINBURGH rg/ The presence of a glutamic acid at position 186 in O/03-K186E and O/03-K186E-230 was associated with decreased ability to control the IFN- promoter (Fig. 2B), decreased capacity to control the activity of the ISRE-containing promoter (Fig. 2C), and strong repression of the constitutively expressed promoter compared to O/03 and an O/03- 230 revertant (Fig. 2D). RESULTS (A and B) Human 293T cells were transiently cotransfected with a pCAGGS expression (Continued on next page) March 2018 Volume 92 Issue 5 e01875-17 on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from on March 3, 2020 at UNIVERSITY OF EDINBURGH rg/ 3, 2020 at UNIVERSITY OF EDINBURGH FIG 1 Functional characterization of evolutionarily distinct EIV NS1 proteins. (Top) Schematic representation of three versions of the NS1 protein (early, intermediate, and late) found throughout EIV evolution. Early NS1 proteins, full length (230 amino acids) and harboring E186, were found in EIVs circulating between the 1960s and 1970s; intermediate (Interm.) NS1 proteins, full length with K186, were found in EIVs circulating between the late 1970s and late 1990s; and late NS1 proteins, truncated at the C terminus (219 amino acids) with K186, are found in EIVs isolated since the late 1990s. (A and B) Human 293T cells were transiently cotransfected with a pCAGGS expression (Continued on next page) FIG 1 Functional characterization of evolutionarily distinct EIV NS1 proteins. (Top) Schematic representation of three versions of the NS1 protein (early, intermediate, and late) found throughout EIV evolution. Early NS1 proteins, full length (230 amino acids) and harboring E186, were found in EIVs circulating between the 1960s and 1970s; intermediate (Interm.) NS1 proteins, full length with K186, were found in EIVs circulating between the late 1970s and late 1990s; and late NS1 proteins, truncated at the C terminus (219 amino acids) with K186, are found in EIVs isolated since the late 1990s. (A and B) Human 293T cells were transiently cotransfected with a pCAGGS expression (Continued on next page) jvi.asm.org 4 jvi.asm.org 4 March 2018 Volume 92 Issue 5 e01875-17 Functional Evolution of the EIV NS1 Gene Journal of Virology host gene expression (F/79 to M/13) were easily detected. Interestingly, the level of expression of NS1 proteins of viruses isolated between 1979 and 1995 (F/79 to LP/95) was lower than that of more recent NS1 proteins (K/95 to M/13). Taken together, these results suggest that the NS1 proteins of the H3N8 EIV lineage maintained strong control over IFN- production throughout evolution while progres- sively increasing their control of IFN-stimulated signal transduction. Such control mechanisms are likely independent of the NS1-mediated control of general gene expression, as the NS1 proteins of EIVs that circulated after 1969 lost that ability. plasmid encoding the indicated NS1 proteins (or with empty plasmid), together with either a ﬁreﬂy luciferase IFN- promoter reporter construct (pIFN--FF-Luc) (A) or a plasmid expressing ﬁreﬂy luciferase under the control of a promoter containing an IFN-stimulated regulatory element (pISRE-FF-Luc) (B), as well as with a constitutively active Renilla luciferase expression plasmid (pREN-Luc). At 24 hpt, the cells were either infected with SeV () or mock infected () (A) or treated with uIFN () or mock treated () (B) for 18 h. The relative activity of FF-Luc in cells transfected with the pIFN--FF-Luc (A) or pISRE-FF-Luc (B) plasmid was determined as the ratio between FF-Luc and REN-Luc in each corresponding sample. The values were normalized to the empty pCAGGS plasmid plus SeV (A) or the empty pCAGGS plasmid plus uIFN (B) (set to 100%). (C) Human 293T cells were transiently cotransfected individually with the indicated NS1 expression plasmids together with pREN-Luc. Total REN-Luc levels were measured 24 h later, and the values were normalized to empty-pCAGGS- transfected cells. (A to C) The bars represent means of the results from three independent experiments, and the error bars represent SEM. Signiﬁcance was calculated as indicated in Materials and Methods. **, P 0.0001 for pCAGGS plus SeV or pCAGGS plus uIFN versus all other conditions. ££, P 0.01; ££, P 0.001; £££, P 0.0001 for the indicated NS1 against all other NS1 proteins. (D) The level of expression of the indicated NS1 was analyzed by Western blotting from parallel transfected lysates using a rabbit polyclonal anti-EIV NS1 antibody. (E) Alignment of NS1 amino acid sequences of the 13 EIVs used in this study. Yellow shading, polymorphism between the 13 NS1 proteins; red shading, NS1 amino acid changes investigated in the study; gray shading, other polymorphisms present at high frequency in the EIV population. RESULTS Consistent with this result, NS1 proteins harboring E186 were not detectable by Western blotting (Fig. 2E). Since NS1 binding to CPFS30 is known to block general gene expression (12), we performed coimmunoprecipitation assays using an equine CPSF30 and NS1 O/03 and revertant proteins expressed in vitro. We conﬁrmed that the presence of E186 deter- mines CPSF30 binding, regardless of the length of NS1 (Fig. 3). It should be noted that FIG 1 Legend (Continued) plasmid encoding the indicated NS1 proteins (or with empty plasmid), together with either a ﬁreﬂy luciferase IFN- promoter reporter construct (pIFN--FF-Luc) (A) or a plasmid expressing ﬁreﬂy luciferase under the control of a promoter containing an IFN-stimulated regulatory element (pISRE-FF-Luc) (B), as well as with a constitutively active Renilla luciferase expression plasmid (pREN-Luc). At 24 hpt, the cells were either infected with SeV () or mock infected () (A) or treated with uIFN () or mock treated () (B) for 18 h. The relative activity of FF-Luc in cells transfected with the pIFN--FF-Luc (A) or pISRE-FF-Luc (B) plasmid was determined as the ratio between FF-Luc and REN-Luc in each corresponding sample. The values were normalized to the empty pCAGGS plasmid plus SeV (A) or the empty pCAGGS plasmid plus uIFN (B) (set to 100%). (C) Human 293T cells were transiently cotransfected individually with the indicated NS1 expression plasmids together with pREN-Luc. Total REN-Luc levels were measured 24 h later, and the values were normalized to empty-pCAGGS- transfected cells. (A to C) The bars represent means of the results from three independent experiments, and the error bars represent SEM. Signiﬁcance was calculated as indicated in Materials and Methods. , P 0.0001 for pCAGGS plus SeV or pCAGGS plus uIFN versus all other conditions. ££, P 0.01; ££, P 0.001; £££, P 0.0001 for the indicated NS1 against all other NS1 proteins. (D) The level of expression of the indicated NS1 was analyzed by Western blotting from parallel transfected lysates using a rabbit polyclonal anti-EIV NS1 antibody. (E) Alignment of NS1 amino acid sequences of the 13 EIVs used in this study. Yellow shading, polymorphism between the 13 NS1 proteins; red shading, NS1 amino acid changes investigated in the study; gray shading, other polymorphisms present at high frequency in the EIV population. jvi.asm.org 5 March 2018 Volume 92 Issue 5 e01875-17 FIG 2 NS1 E186 controls general gene expression. (A) Protein alignment showing the sequence of interest and the introduced mutations in EIV NS1. Below the alignment is a diagram of the three different NS1 proteins that represent evolutionarily distinct NS1 proteins: early NS1, O/03-230, 230 amino acids long and possessing E186; intermediate NS1, O/03-230, 230 amino acids long and possessing K186; and late NS1, 219 amino acids long and possessing K186. FIG 1 Legend (Continued) (B to D) IFN- (B), ISRE (C), and general gene (D) expression reporter assays were done using the indicated NS1 pCAGGS expression constructs as described for Fig. 1. Experiments were performed three times independently, and signiﬁcance was calculated as described in Materials and Methods. , P 0.0001 for pCAGGS plus SeV or pCAGGS plus uIFN versus all other conditions. ££££, signiﬁcant difference (P 0.0001) between K186E-containing revertant(s), O/03, and O/03-230 revertant; €€€€, signiﬁcant (P 0.0001) difference between O/03-K186E and all other conditions; $$$$, signiﬁcant difference (P 0.0001) between O/03 and O/03-230 revertant. The error bars represent SEM. (E) Western blot of lysates from parallel samples performed as described for Fig. 1. Chauché et al. Journal of Virology Chauché et al. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from on March 3, 2020 at UNIVERSITY OF EDINBURGH rg/ FIG 2 NS1 E186 controls general gene expression. (A) Protein alignment showing the sequence of interest and the introduced mutations in EIV NS1. Below the alignment is a diagram of the three different NS1 proteins that represent evolutionarily distinct NS1 proteins: early NS1, O/03-230, 230 amino acids long and possessing E186; intermediate NS1, O/03-230, 230 amino acids long and possessing K186; and late NS1, 219 amino acids long and possessing K186. (B to D) IFN- (B), ISRE (C), and general gene (D) expression reporter assays were done using the indicated NS1 pCAGGS expression constructs as described for Fig. 1. Experiments were performed three times independently, and signiﬁcance was calculated as described in Materials and Methods. , P 0.0001 for pCAGGS plus SeV or pCAGGS plus uIFN versus all other conditions. ££££, signiﬁcant difference (P 0.0001) between K186E-containing revertant(s), O/03, and O/03-230 revertant; €€€€, signiﬁcant (P 0.0001) difference between O/03-K186E and all other conditions; $$$$, signiﬁcant difference (P 0.0001) between O/03 and O/03-230 revertant. The error bars represent SEM. (E) Western blot of lysates from parallel samples performed as described for Fig. 1. jvi.asm.org 6 March 2018 Volume 92 Issue 5 e01875-17 Journal of Virology Functional Evolution of the EIV NS1 Gene FIG 3 Amino acid 186 is a determinant of NS1 interaction with CPSF30. NS1 variants were synthesized in vitro. Human 293T cells were transiently transfected with 2,000 ng of a pCAGGS plasmid expressing an HA-tagged version of the equine CPSF30. FIG 1 Legend (Continued) At 30 hpt, the cells were lysed, and cleared cell lysates expressing HA-CPSF30 were incubated overnight at 4°C with the in vitro-synthesized NS1 proteins and 20 l of an anti-HA afﬁnity resin. Precipitated proteins were dissociated from the resin and analyzed by Western blotting (WB). Representative blots showing protein expression levels (Input) and coimmunoprecipitation results (IP anti-HA) are shown. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from FIG 3 Amino acid 186 is a determinant of NS1 interaction with CPSF30. NS1 variants were synthesized in vitro. Human 293T cells were transiently transfected with 2,000 ng of a pCAGGS plasmid expressing an HA-tagged version of the equine CPSF30. At 30 hpt, the cells were lysed, and cleared cell lysates expressing HA-CPSF30 were incubated overnight at 4°C with the in vitro-synthesized NS1 proteins and 20 l of an anti-HA afﬁnity resin. Precipitated proteins were dissociated from the resin and analyzed by Western blotting (WB). Representative blots showing protein expression levels (Input) and coimmunoprecipitation results (IP anti-HA) are shown. binding of NS1 to CPSF30 is consistent with the inhibition of NS1 expression upon transfection (Fig. 1D and 2D). This is because transcription from the pCAGGS plasmid is driven by a polymerase II (Pol II)-dependent promoter through binding to CPSF30. Overall, these results suggested that the NS1 proteins of early EIVs were able to control general gene expression through CPSF30 binding and that the K186E substi- tution, which arose in NS1 approximately 10 years after emergence of the virus, inﬂuenced the control of IFN and ISG induction. Moreover, this substitution seems to have released the block of general gene expression and improved NS1’s ability to control IFN-mediated signal transduction. on March 3, 2020 at UNIVERSITY OF EDINBURGH Mutations in NS1 acquired throughout evolution affect virus replication and cell-to-cell spread. Three individual O/03 revertant viruses were generated and tested for growth in Madin-Darby canine kidney (MDCK) cells: one carrying the K186E substi- tution (O/03-K186E), another carrying the C terminus extension (O/03-230), and another one carrying both changes (O/03-K186E-230). As indicated in Fig. 4A, none of the introduced changes modiﬁed the nuclear export protein (NEP) amino acid sequence. While the O/03-K186E and O/03-230 viruses exhibited growth kinetics similar to those of O/03 (Fig. FIG 1 Legend (Continued) 4B), the double revertant O/03-K186E-230 displayed a higher replication rate until 12 h postinfection (hpi) but never reached a titer as high as those of the three other viruses over 72 h (Fig. 4B). In equine cells, the O/03 virus showed a signiﬁcant advantage over all the revertant viruses after 24 hpi. Indeed, O/03 peaked at a titer 3 to 4 log units higher than those of the revertants between 24 and 72 hpi (Fig. 4B). Interestingly, during the ﬁrst 16 hpi, no difference in growth kinetics was observed between O/03 and natural revertant viruses (O/03-230 and O/03-K186E-230). More importantly, O/03-K186E-230 displayed the highest replication rate during the ﬁrst 12 hpi, reaching a titer 2 log units higher than that of any other virus tested. The O/03-K186E revertant, which expressed an NS1 protein that was never isolated in nature, showed strong attenuation compared to O/03 between 16 and 72 hpi. When we tested the abilities of O/03 and revertant viruses to spread among neighboring cells by examining their plaque phenotypes in MDCK cells, all the revertant viruses displayed signiﬁcantly smaller plaques than O/03 (Fig. 4C), and the O/03-K186E- 230 revertant was the most affected, showing pinpoint plaques. This suggested that NS1 evolution resulted in more efﬁcient cell-to-cell spread. As NS1 proteins harboring E186 bind CPSF30, we wanted to check if during viral infection NS1 would be present in the same intracellular compartment as CPSF30 (i.e., the nucleus). All the proteins exhibited similar nucleocytoplasmic localization patterns (Fig. 4D), which suggests that the introduced mutations did not affect NS1 subcellular localization. NS1 amino acid 186 and the length of the C-terminal tail are key determinants of EIV control of cellular protein synthesis and the establishment of an antiviral state in equine cells. To compare the viruses’ abilities to limit the establishment of an jvi.asm.org 7 March 2018 Volume 92 Issue 5 e01875-17 Journal of Virology Chauché et al. FIG 4 Characterization of EIVs carrying mutations in NS1. (A) Schematic representation of the mutations introduced in the O/03 NS segment at NS1 codon 186 and codon 220 (nucleotides 582 to 584 and 684 to 686 in the O/03 NS segment, respectively) and the resulting amino acid changes in the different NS1 proteins (late, artiﬁcial, intermediate, and early). FIG 1 Legend (Continued) A(n) indicates a poly(A) tail, and an asterisk (Continued on next page) on March 3, 2020 at U http://jvi.asm.org/ Downloaded from on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from on March 3, 2020 at UNIVERSITY OF EDINBURGH rg/ FIG 4 Characterization of EIVs carrying mutations in NS1. (A) Schematic representation of the mutations introduced in the O/03 NS segment at NS1 codon 186 and codon 220 (nucleotides 582 to 584 and 684 to 686 in the O/03 NS segment, respectively) and the resulting amino acid changes in the different NS1 proteins (late, artiﬁcial, intermediate, and early). A(n) indicates a poly(A) tail, and an asterisk (Continued on next page) jvi.asm.org 8 jvi.asm.org 8 March 2018 Volume 92 Issue 5 e01875-17 Functional Evolution of the EIV NS1 Gene Journal of Virology antiviral state, we infected equine cells (multiplicity of infection [MOI], 0.1) with each of the viruses and monitored the expression of two ISGs (ISG15 and MX1) by Western blotting at different times postinfection. As expected, ISG15 and MX1 were detected in lysates of cells infected with both the O/03-230 and O/03-K186E-230 revertant viruses, but not O/03 (Fig. 5A), at 24 hpi. We did not detect the proteins in cells infected with O/03-K186E. To determine the effects of the NS1 revertants on the synthesis of cellular proteins, we performed puromycin assays at various times postinfection in equine cells (21). Puromycin is incorporated at the C termini of all nascent proteins (22). After cell lysis, immunoblotting against puromycin was performed to allow the detection of nascent proteins of various sizes (appearing as a black smear) that were being produced at the moment of cell lysis. The darker and longer the smear was, the more proteins were being produced at the moment of cell lysis. The double-revertant virus (O/03-K186E- 230) induced strong protein shutoff (Fig. 5B), particularly at early times postinfection, and it was associated with high expression levels of NS1 from 8 to 24 hpi (Fig. 5A). As expected, the presence of E186 did not lead to self-inhibition upon infection (in contrast with the self-inhibition of NS1 observed in transfections [Fig. 3]). This is because, during viral infections, the viral polymerase complex drives transcription of NS1, and this process does not require CPSF30. Differences in molecular mechanisms between transfections and infections have also been reported in the literature (18, 19, 23). FIG 4 Legend (Continued) indicates a stop codon (TGA). The introduced changes did not change the NEP amino acids at positions 28 (TTA/G, coding for leucine) and 68 (AAT/C, coding for asparagine). (B) Growth kinetics of O/03, O/03-K186E, O/03-230, and O/03-K186E-230 viruses in MDCK cells (MOI, 0.01) and E-derm cells (MOI, 0.1). The data points represent virus titers in supernatants at different times postinfection, and the error bars represent SEM. (C) Plaque phenotype of O/03, O/03-K186E, O/03-230, and O/03-K186E-230 in MDCK cells at 48 hpi. The average plaque size for each virus was determined by counting over 100 plaques per condition. Signiﬁcance was calculated as described in Materials and Methods. , P 0.001 for O/03-K186E-230 versus O/03. , P 0.0001; , P 0.001; , P 0.01 for O/03 versus the other three viruses. £, P 0.05 for O/03-K186E-230 versus O/03-K186E. (D) Subcellular localization of NS1 in infected equine cells. E-derm cells were infected with O/03, O/03-K186E, O/03-230, and O/03-K186E-230 (MOI, 0.1) for 24 h. NS1 was detected by immunoﬂuorescence as described in Materials and Methods. The experiment was performed three times independently, and representative images of NS1 are shown. Scale bars, 20 m. FIG 1 Legend (Continued) (A) Cells were lysed and immunoblotted for the indicated proteins at the indicated times postinfection (p.i.). (B) To measure protein shutoff upon infection, culture media were replaced with a puromycin-containing suspension and incubated for 1 h. The cells were then lysed and immunoblotted for puromycin at the indicated times, as described in Materials and Methods. Western blot quantiﬁcations for puromycin and -tubulin signals were determined for three independent experiments and expressed relative to mock-infected cells. Signiﬁcance was calculated as indicated in Materials and Methods. , P 0.001 for the indicated virus versus all other conditions at 12 hpi. £, P 0.05 for O/03-K186E-230 versus 230; ££, P 0.01 for O/03-K186E-230 versus O/03-K186E; £££, P 0.0001 for O/03-K186E-230 versus O/03 and mock infection. , P 0.05 for the indicated virus versus mock-infected samples at 24 hpi. Gamma-tubulin was used as a loading control. The error bars indicate SEM. Chauché et al. Journal of Virology Chauché et al. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from on March 3, 2020 at UNIVERSITY OF EDINBURGH FIG 5 NS1 amino acid 186 and C-terminal tail affect EIV control of general protein production, apoptosis, and response to IFN in equine cells. E-derm cells were infected (MOI, 0.1) with O/03, O/03-K186E, O/03-230, and O/03-K186E-230 viruses. (A) Cells were lysed and immunoblotted for the indicated proteins at the indicated times postinfection (p.i.). (B) To measure protein shutoff upon infection, culture media were replaced with a puromycin-containing suspension and incubated for 1 h. The cells were then lysed and immunoblotted for puromycin at the indicated times, as described in Materials and Methods. Western blot quantiﬁcations for puromycin and -tubulin signals were determined for three independent experiments and expressed relative to mock-infected cells. Signiﬁcance was calculated as indicated in Materials and Methods. , P 0.001 for the indicated virus versus all other conditions at 12 hpi. £, P 0.05 for O/03-K186E-230 versus 230; ££, P 0.01 for O/03-K186E-230 versus O/03-K186E; £££, P 0.0001 for O/03-K186E-230 versus O/03 and mock infection. , P 0.05 for the indicated virus versus mock-infected samples at 24 hpi. Gamma-tubulin was used as a loading control. The error bars indicate SEM. was produced upon O/03 infection, peaking at 24 hpi (Fig. 6A). FIG 1 Legend (Continued) While this result is consistent with E186 playing an important role in blocking general gene expression via a CPSF30-dependent mechanism, it also suggests that a complementary role of the NS1 C-terminal tail might be necessary to induce strong protein shutoff, as O/03-K186E did not block protein synthesis at any time postinfection (Fig. 5B). However, we cannot rule out the possibility that this lack of protein shutoff was due to a low infection level or low expression of O/03-K186E NS1 protein in equine cells, likely due to strong attenuation of the virus in equine cells (Fig. 6A). on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from on March 3, 2020 at UNIVERSITY OF EDINBURGH Interestingly, the O/03 virus induced a signiﬁcant increase in protein synthesis at 12 hpi and low but signiﬁcant protein shutoff at 24 hpi (Fig. 5B). As the double-revertant virus (O/03-K186E-230) induced strong protein shutdown and an early antiviral state, we assumed that cellular homeostasis would be compro- mised and the cells would undergo apoptosis prematurely. To test this hypothesis, we compared the expression levels of caspase 3 (cleaved and total) in O/03 and revertant virus-infected E-derm cells (MOI, 0.1) at different times postinfection (Fig. 5A). As expected, cleavage of caspase 3 was already detectable at 24 hpi in cells infected with the O/03-K186E-230 revertant virus. Interestingly, the O/03-230 revertant virus also induced premature apoptosis, suggesting that the tail of NS1 plays a role in controlling programmed cell death. EIV O/03 grows to high levels despite eliciting large amounts of antiviral cytokines and can replicate in IFN-primed equine cells. To assess whether the IFN system was the limiting factor of O/03 revertant virus replication, we measured the antiviral activities of the supernatants of equine cells infected with the viruses (MOI, 0.1) at different times postinfection using a recombinant vesicular stomatitis virus (rVSV)- green ﬂuorescent protein (GFP)-based bioassay (24) and compared them with the growth kinetics of each virus (Fig. 6). Surprisingly, the highest level of antiviral cytokines jvi.asm.org 9 March 2018 Volume 92 Issue 5 e01875-17 was produced upon O/03 infection, peaking at 24 hpi (Fig. 6A). This did not seem to FIG 5 NS1 amino acid 186 and C-terminal tail affect EIV control of general protein production, apoptosis, and response to IFN in equine cells. E-derm cells were infected (MOI, 0.1) with O/03, O/03-K186E, O/03-230, and O/03-K186E-230 viruses. FIG 1 Legend (Continued) This did not seem to affect viral growth, since the virus rapidly reached a very high titer (107 PFU/ml at 24 hpi), as previously shown (Fig. 4B). The patterns of antiviral-cytokine production compared to viral growth were similar between O/03-230 and O/03-K186E-230 rever- tant viruses (Fig. 6C and D, respectively), where low levels of antiviral cytokines were jvi.asm.org 10 March 2018 Volume 92 Issue 5 e01875-17 Functional Evolution of the EIV NS1 Gene Journal of Virology FIG 6 Comparison of growth kinetics and production of antiviral cytokines in equine cells infected with EIV O/03 and NS1 revertant viruses. E-derm cells were infected (MOI, 0.1) for 72 h with O/03 (A), O/03-K186E (B), O/03-230 (C), and O/03-K186E-230 (D). Supernatants were collected, and the cells were ﬁxed at the indicated times postinfection. The total amount of antiviral cytokines produced upon infection was measured using an IFN bioassay, and virus growth kinetics was determined by immunofocus assay. The error bars represent SEM. Signiﬁcance was calculated as indicated in Materials and Methods. *, P 0.0001 for the indicated virus at the indicated time postinfection versus the other three viruses at the same time postinfection. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from on March 3, 2020 at UNIVERSITY OF EDINBURGH FIG 6 Comparison of growth kinetics and production of antiviral cytokines in equine cells infected with EIV O/03 and NS1 revertant viruses. E-derm cells were infected (MOI, 0.1) for 72 h with O/03 (A), O/03-K186E (B), O/03-230 (C), and O/03-K186E-230 (D). Supernatants were collected, and the cells were ﬁxed at the indicated times postinfection. The total amount of antiviral cytokines produced upon infection was measured using an IFN bioassay, and virus growth kinetics was determined by immunofocus assay. The error bars represent SEM. Signiﬁcance was calculated as indicated in Materials and Methods. , P 0.0001 for the indicated virus at the indicated time postinfection versus the other three viruses at the same time postinfection. associated with limited virus growth. In contrast, cells infected with the O/03-K186E revertant virus exhibited a signiﬁcantly high peak of antiviral cytokines at 8 hpi that seemed to control virus growth effectively (Fig. 6B), since we could not detect any viral growth at later times postinfection. Consistent with this, the level of antiviral cytokines decreased progressively and reached a plateau at 16 hpi (Fig. 6B). March 2018 Volume 92 Issue 5 e01875-17 FIG 1 Legend (Continued) E-derm cells were treated for 24 h with uIFN (A) or ruxolitinib (B) prior to infection (MOI, 0.1) with O/03, O/03-K186E, O/03-230, or O/03-K186E-230. Cells and supernatants were collected at different times postinfection. Viral titers were measured and signiﬁcance was calculated as indicated in Materials and Methods. , P 0.001, and , P 0.05 for O/03 at the indicated time postinfection versus the other three viruses at the same time postinfection. The error bars indicate SEM. adaptive mutations on virus-host interactions, we compared the transcriptomes of equine cells infected with the O/03 and revertant viruses. We ﬁrst determined the total number of differentially expressed genes (DEGs) compared to mock-infected samples (Fig. 8) at 8 hpi (the end of the eclipse phase in E-derm cells) in order to avoid any bias due to different replication efﬁciencies between viruses. Cells infected with the O/03 virus exhibited the highest ratio of DEGs to mock-infected cells (n 429), which correlates with increased production of proteins at 12 hpi (Fig. 5B) and likely reﬂects productive infection. Furthermore, the number of DEGs in revertant virus-infected cells compared to mock-infected samples decreased as follows: 241 for O/03-230-infected samples, 193 for O/03-K186E-infected samples, and 158 for O/03-K186E-230-infected samples (Fig. 8A; a detailed list of DEGs, including Gene ID, gene name, and log2-fold change, is shown in Table S1 in the supplemental material). This indicates that these two natural mutations in NS1 have a high impact on the response of equine cells to EIV infection, highlighting their roles in virus adaptation. on March 3, 2020 at UNIVERSITY OF EDINBURGH As the O/03 virus replicated to high titers despite inducing high levels of antiviral cytokines, we compared the number of ISGs and interferon-induced elements that were upregulated in cells infected with the revertant viruses but absent in O/03-infected samples (Fig. 8B). As expected, a large number of ISGs (i.e., ISG15, MX1, and OAS1/2/3) that were maintained at a basal level in cells infected with O/03 were upregulated in cells infected with the revertant viruses (Fig. 8B). We also looked for variations in viral gene expression between O/03 and revertant viruses and did not observe any signiﬁcant differences (data not shown). EIV NS1 evolved to block the induction of ISGs at a pretranscriptional level. Our reporter assays showed that the NS1 proteins that did not bind CPSF30 were able to control ISRE-containing promoters more efﬁciently (Fig. FIG 1 Legend (Continued) As the O/03 virus (but not the NS1 revertants) was able to replicate to high levels in the presence of antiviral cytokines, we wanted to determine if it was able to replicate in cells in an antiviral state induced by exogenous IFN. E-derm cells were pretreated with 500 U of uIFN for 24 h and then inoculated with O/03 and NS1 revertant viruses (MOI, 0.1). While all the viruses exhibited a reduction in virus growth compared to their observed replication abilities in untreated E-derm cells, the O/03 virus reached a titer of 105 PFU/ml at 24 hpi, signiﬁcantly higher than those of the other three viruses (Fig. 7A). These results show that the evolution of NS1 resulted in an increased ability to replicate in cells that had been exposed to uIFN. To conﬁrm the role of IFN in limiting the replication of the NS1 revertant viruses, we treated equine cells 24 h prior to infection (MOI, 0.1) with ruxolitinib (Selleck Chemicals) to block the JAK/STAT pathway and thus impaired the ability of the cells to respond to IFN (25). We compared the growth kinetics of each virus (Fig. 7B) in the presence of ruxolitinib. Although the O/03 virus maintained a signiﬁcantly higher titer than the three other viruses at 24 and 48 hpi, the revertant viruses reached a higher titer than under normal conditions (Fig. 4B), conﬁrming the central role of the IFN response in limiting the replication of O/03 NS1 revertant viruses in equine cells. The length of NS1 and the nature of residue 186 impact EIV-mediated control of ISG transcription and general gene expression. To identify further effects of these jvi.asm.org 11 March 2018 Volume 92 Issue 5 e01875-17 Journal of Virology Chauché et al. FIG 7 JAK1/2 inhibition restores growth kinetics of NS1 revertant viruses. E-derm cells were treated for 24 h with uIFN (A) or ruxolitinib (B) prior to infection (MOI, 0.1) with O/03, O/03-K186E, O/03-230, or O/03-K186E-230. Cells and supernatants were collected at different times postinfection. Viral titers were measured and signiﬁcance was calculated as indicated in Materials and Methods. **, P 0.001, and , P 0.05 for O/03 at the indicated time postinfection versus the other three viruses at the same time postinfection. The error bars indicate SEM. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from FIG 7 JAK1/2 inhibition restores growth kinetics of NS1 revertant viruses. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from on March 3, 2020 at UNIVERSITY OF EDINBURGH org/ FIG 8 NS1 amino acid 186 and the C-terminal tail affect EIV control of gene expression in equine cells. (A) Heat map of DEGs between O/03- and revertant-virus-infected E-derm cells versus mock-infected samples. A log2 FC value of 0.58 and a P value of 0.05 were regarded as indicating statistically differential expression relative to mock infection. The total number of DEGs for each condition is indicated below the heat map. DEGs relative to mock-infected conditions were classiﬁed in 11 groups: 1, DEGs shared between O/03 and mutants; 2, DEGs shared between all viruses but O/03-230; 3, DEGs shared between all viruses but O/03-K186E; 4, DEGs shared between O/03 and O/03-K186E; 5, DEGs shared between O/03 and O/03-230; 6, DEGs shared between O/03 and O/03-K186E-230; 7, DEGs shared between all revertants; 8, DEGs present in O/03 only; 9, DEGs present in O/03-K186E only; 10, DEGs present in O/03-230 only; and 11, DEGs present in O/03-K186E-230 only. The complete list of genes in presented in Table S1 in the supplemental material. (B) DEGs (including some ISGs) present only in revertant virus-infected samples (group 7). The values are log2 FC. A log2 FC value of 0.58 and a P value of 0.05 were regarded as statistically signiﬁcant. FIG 8 NS1 amino acid 186 and the C-terminal tail affect EIV control of gene expression in equine cells. (A) Heat map of DEGs between O/03- and revertant-virus-infected E-derm cells versus mock-infected samples. A log2 FC value of 0.58 and a P value of 0.05 were regarded as indicating statistically differential expression relative to mock infection. The total number of DEGs for each condition is indicated below the heat map. DEGs relative to mock-infected conditions were classiﬁed in 11 groups: 1, DEGs shared between O/03 and mutants; 2, DEGs shared between all viruses but O/03-230; 3, DEGs shared between all viruses but O/03-K186E; 4, DEGs shared between O/03 and O/03-K186E; 5, DEGs shared between O/03 and O/03-230; 6, DEGs shared between O/03 and O/03-K186E-230; 7, DEGs shared between all revertants; 8, DEGs present in O/03 only; 9, DEGs present in O/03-K186E only; 10, DEGs present in O/03-230 only; and 11, DEGs present in O/03-K186E-230 only. The complete list of genes in presented in Table S1 in the supplemental material. (B) DEGs (including some ISGs) present only in revertant virus-infected samples (group 7). The values are log2 FC. FIG 1 Legend (Continued) 1 to 3). The transcriptomics data showed that cells infected with O/03 (but not the revertants) exhibited signiﬁcant control of ISG transcripts (Fig. 8B) despite inducing large amounts of antiviral cytokines (Fig. 6A). Furthermore, experiments using ruxolitinib treatment conﬁrmed the central role of the JAK/STAT pathway in limiting revertant virus growth in equine cells (Fig. 7B). Taken together, these results suggested that NS1 of O/03 virus blocked the induction of ISGs at a pretranscriptional level and that this involved the JAK/STAT pathway, since the JAK/STAT signaling pathway, upon IFN stimulation, leads to the transcription of ISGs and this involves the nuclear translocation of the ISGF3 complex. As a marker of ISGF3 nuclear translocation, we compared the amounts of nuclear STAT1 at 8 and 24 hpi in equine cells infected with O/03 and revertant viruses. As expected, O/03 infection did not result in STAT1 nuclear localization, despite the presence of high levels of antiviral cytokines (Fig. 6A, 24 hpi). In addition, residue 186 played a central role in limiting STAT1 nuclear translocation, as O/03-K186E virus infection resulted in abun- jvi.asm.org 12 March 2018 Volume 92 Issue 5 e01875-17 Functional Evolution of the EIV NS1 Gene Journal of Virology on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from A log2 FC value of 0.58 and a P value of 0.05 were regarded as statistically signiﬁcant. dant nuclear STAT1 (Fig. 9A). STAT1 also remained in the cytoplasm of cells infected with O/03-230 or O/03-K186E-230 revertant virus, probably due to the low level of antiviral cytokines produced (Fig. 6C and D). However, we cannot rule out a possible contribution of the NS1 C-terminal tail to the control of STAT1 nuclear translocation. jvi.asm.org 13 March 2018 Volume 92 Issue 5 e01875-17 FIG 9 STAT1 nuclear localization in equine cells infected with EIV O/03 and NS1 revertant viruses. E-derm cells were infected (MOI, 0.1) with O/03, O/03-K186E O/03-230 and O/03-K186E-230 viruses or mock infected for 8 or 24 h (A) (Top) Confocal images showing coimmunostaining of viral NP and Chauché et al. Journal of Virology on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from Journal of Virology Chauché et al. FIG 9 STAT1 nuclear localization in equine cells infected with EIV O/03 and NS1 revertant viruses. E-derm cells were infected (MOI, 0.1) with O/03, O/03-K186E, O/03-230, and O/03-K186E-230 viruses or mock infected for 8 or 24 h. (A) (Top) Confocal images showing coimmunostaining of viral NP and STAT1. The experiment was performed three times independently, and representative images of STAT1 and NP are shown. Scale bars, 20 m. (Bottom) Graphical representation of nuclear localization of STAT1 (nSTAT1). nSTAT1 was quantiﬁed as described in Materials and Methods and expressed as a percentage of tSTAT1. Statistical signiﬁcance was calculated as described in Materials and Methods. , P 0.01 for O/03-K186E versus other conditions at 8 hpi. No other signiﬁcant differences were detected. The error bars indicate SEM. (B) Infected cells were lysed, and total STAT1 expression was Chauché et al. Journal of Virology on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from Chauché et al. Journal of Virology on March 3, 2020 at U http://jvi.asm.org/ Downloaded from on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from on March 3, 2020 at /jvi.asm.org/ on March 3, 2020 at UNIVERSITY OF EDINBURGH FIG 9 STAT1 nuclear localization in equine cells infected with EIV O/03 and NS1 revertant viruses. E-derm cells were infected (MOI, 0.1) with O/03, O/03-K186E, O/03-230, and O/03-K186E-230 viruses or mock infected for 8 or 24 h. (A) (Top) Confocal images showing coimmunostaining of viral NP and STAT1. DISCUSSION We investigated the role of NS1 evolution in the posttransfer adaptation of an avian-origin IAV to a mammalian host, focusing on the roles of two mutations that appeared sequentially during EIV evolution. The E186K mutation appeared early after EIV emergence, while the 11-amino-acid C-terminal truncation appeared 20 years later. Our results showed that isogenic viruses carrying the evolved version of NS1 exhibit signiﬁcantly higher ﬁtness in equine cells. The O/03 virus from 2003 was able to replicate to high titers despite high levels of cytokines being produced by infected cells (Fig. 6A). This was achieved by blocking the induction of ISGs at a pretranscriptional level (Fig. 8B), which is consistent with the lack of STAT1 nuclear localization in infected cells (Fig. 9A). The ability to replicate in the presence of IFN is a signiﬁcant ﬁtness trait, as it not only renders an important arm of the host antiviral response ineffective, but also acts as a barrier to other respiratory viruses that might compete for the same ecological niche (i.e., the epithelium of the respiratory tract). Coinfection experiments will shed light on this issue. In contrast, an isogenic virus carrying a revertant version of NS1 (O/03-K186E-230) that mimics the early, more avian-like NS1 proteins of EIV replicated to signiﬁcantly lower titers over a 72-hour period in equine cells (Fig. 4B). This virus induced cellular- protein shutdown (Fig. 5B) and high levels of apoptosis (Fig. 5A), and this was associated with inefﬁcient cell-to-cell spread (Fig. 4C). However, O/03-K186E-230 efﬁ- ciently produced infectious particles at 12 hpi (Fig. 4B and 6D). This suggests that the emerging, nonadapted virus was highly susceptible to the host IFN response and therefore relied on blocking general gene expression nonspeciﬁcally to transiently control the innate immune response. However, this would likely affect cellular homeo- stasis and lead to premature apoptosis, depriving the virus of the cellular resources it needs to replicate. Thus, high replication efﬁciency during this early period (Fig. 4B) would enhance the chances of onward transmission. on March 3, 2020 at UNIVERSITY OF EDINBURGH Early EIV NS1 proteins blocked general gene expression by binding to CPSF30, a host protein that plays a central role in pre-mRNA processing (12). A group of residues centered around residue 186 (184 to 188) (23, 26, 27), as well as other residues (103, 106, 108, and 125) (18, 19, 28), have been previously shown to be involved in this function. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from The experiment was performed three times independently, and representative images of STAT1 and NP are shown. Scale bars, 20 m. (Bottom) Graphical representation of nuclear localization of STAT1 (nSTAT1). nSTAT1 was quantiﬁed as described in Materials and Methods and expressed as a percentage of tSTAT1. Statistical signiﬁcance was calculated as described in Materials and Methods. , P 0.01 for O/03-K186E versus other conditions at 8 hpi. No other signiﬁcant differences were detected. The error bars indicate SEM. (B) Infected cells were lysed, and total STAT1 expression was determined by Western blotting. Gamma tubulin was used as a loading control. 14 March 2018 Volume 92 Issue 5 e01875-17 jvi.asm.org 14 Journal of Virology Functional Evolution of the EIV NS1 Gene Furthermore, the observed differences were not due to different levels of expression of STAT1, as no signiﬁcant differences in total STAT1 expression in cell lysates were detected by Western blotting (Fig. 9B). Finally, the percentages of infected E-derm cells at 8 hpi were comparable between O/03 and revertant viruses (data not shown), and a higher percentage of cells infected with O/03 was found at 24 hpi, further conﬁrming the signiﬁcant advantage of O/03 over all the revertant viruses after 24 hpi. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from March 2018 Volume 92 Issue 5 e01875-17 DISCUSSION The red branches represent NS1 proteins containing E186 and 230 amino acids (early NS1), the orange branches represent NS1 proteins containing K186 and 230 amino acids (inter- mediate NS1), and the blue branches represent NS1 proteins containing K186 and 219 amino acids (late NS1). FIG 10 K186 and C-terminal truncation were ﬁxed at the virus population level during EIV NS1 evolutionary history. Shown is a phylogenetic tree highlighting the ﬁxation of E186K substitution and C-terminal truncation. The red branches represent NS1 proteins containing E186 and 230 amino acids (early NS1), the orange branches represent NS1 proteins containing K186 and 230 amino acids (inter- mediate NS1), and the blue branches represent NS1 proteins containing K186 and 219 amino acids (late NS1). on March 3, 2020 at UNIVERSITY OF EDINBURGH apoptosis in infected cells (Fig. 5A). The last could be caused by the presence of a putative PDZ-binding domain in the tail of NS1 that has been previously associated with apoptosis (30, 31). Truncation of NS1 would therefore be beneﬁcial for EIV, as it would provide better control of the IFN response and extend the life span of infected cells. It is important to note that E186 is present in 98.2% of NS1 unique sequences derived from avian, human, swine, canine, and equine viruses (n 12,775) (data not shown). However, K186 is more common among equine and canine IAVs. With regard to NS1 length, the majority (63.7%) of NS1 proteins are longer than 220 amino acids, and such “long” NS1 proteins are highly prevalent among IAVs derived from birds, humans, dogs, and horses, but not from pigs, as 77% of unique swine NS1 sequences are shorter than 220 residues. Further experimental work is required to unveil the roles of residue 186 and the C-terminal length of NS1 in the adaptation of IAVs to other species. In summary, we have shown that mutations in the NS1 gene that became ﬁxed during the continuous circulation of EIV in horses led to at least two temporally distinct changes in NS1 function that resulted in better control of the mammalian host innate immune response and likely contributed to the adaptation of an avian-origin virus to its new host, the horse. Comparisons of other mammalian IAV lineages of avian origin will show whether such dynamic strategies are common features of inﬂuenza A viruses while adapting to mammals. DISCUSSION Interestingly, for EIV, that binding ability was lost with the introduction of K186, a substitution that must have been selectively advantageous for the virus. This is supported by the fact that it became quickly ﬁxed at the virus population scale (Fig. 10) and because E186 in a contemporary EIV NS1 resulted in severe restriction of viral replication (Fig. 4B and 6B) and cell-to-cell spread (Fig. 4C). That single mutation impaired the ability of EIV to block the induction of the IFN response (Fig. 9A), resulting in the release of cellular cytokines (Fig. 6B) and subsequent upregulation of ISGs (Fig. 8B). These results are consistent with work we recently published on the evolution of the NS1 protein of H3N8 canine inﬂuenza virus, a virus that originated from the H3N8 EIV lineage in the early 2000s (29). The second evolutionary change in NS1 that became rapidly ﬁxed at the population level was a C-terminal truncation of the protein that appeared in the mid-1990s (Fig. 10). While determining the molecular mechanism underpinning the effect of the C-terminal tail was beyond the scope of this study, our experimental infections sug- gested that this truncation also increased viral ﬁtness. Indeed, extending the tail of NS1 reduced virus replication (Fig. 4B and 6C) and plaque size (Fig. 4C), increased viral susceptibility to IFN (Fig. 7A and B), reduced viral control of ISG transcription (Fig. 8B) and expression of ISG15 and MX1 (Fig. 5A), and also resulted in early induction of jvi.asm.org 15 March 2018 Volume 92 Issue 5 e01875-17 Journal of Virology Chauché et al. FIG 10 K186 and C-terminal truncation were ﬁxed at the virus population level during EIV NS1 evolutionary history. Shown is a phylogenetic tree highlighting the ﬁxation of E186K substitution and C-terminal truncation. The red branches represent NS1 proteins containing E186 and 230 amino acids (early NS1), the orange branches represent NS1 proteins containing K186 and 230 amino acids (inter- mediate NS1), and the blue branches represent NS1 proteins containing K186 and 219 amino acids (late NS1). on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from FIG 10 K186 and C-terminal truncation were ﬁxed at the virus population level during EIV NS1 evolutionary history. Shown is a phylogenetic tree highlighting the ﬁxation of E186K substitution and C-terminal truncation. jvi.asm.org 16 MATERIALS AND METHODS The rVSV-GFP virus stock used in the bioassay was generated by transiently transfecting HEK293T cells in Opti-MEM with an expression plasmid encoding the VSV surface glycoprotein (pVSV-G) (24) using TransIT-LT1 transfection reagent (Cambridge Biosciences) at 37°C and 5% CO2 for 36 h (100-mm sterile-dish format; 5 106 cells). At the end of the transfection period, the cells were infected with a previous stock of rVSV-GFP (2.5 102 50% tissue culture infective doses [TCID50]) for 4 h at 37°C and 5% CO2 in growth medium and then washed with phosphate-buffered saline (PBS) and maintained at 37°C and 5% CO2 in growth medium for a further 16 h. The supernatant was then collected and ﬁltered through a 0.45-m ﬁlter (Fisher Scientiﬁc) before being aliquoted and stored at 80°C. Cloning of NS1. Mammalian expression constructs for untagged NS1 used in the reporter assay were generated as previously described (18, 19). pcDNA3 plasmids encoding NS1 were also generated by subcloning from pCAGGS (18). The sequence of each pCAGGS- or pcDNA3-NS1 insert was conﬁrmed by Sanger sequencing and compared to those available in the NCBI Inﬂuenza Database. Viral RNA (vRNA) was extracted from the corresponding viral stocks using the QIAmp viral RNA kit (Qiagen), the RNA quality was assessed with a NanoDrop 2000 spectrophotometer (ThermoFisher Scientiﬁc), and the RNA was stored at 80°C. Reverse transcription (RT) was done using Uni12 primer (33), 500 ng of RNA, and SuperScript III reverse transcriptase (Invitrogen) following the manufacturer’s protocol. PCR was done with 100 ng of cDNA and PfuUltra II fusion HS DNA polymerase (Agilent), following the manufacturer’s protocol, and a speciﬁc set of primers was used for each NS1. The cycling parameters and primers are available upon request. In order to prevent expression of NEP in the pCAGGS-NS1 plasmids, a silent mutation in the splice acceptor site of NS1 (splice acceptor mutation [SAM]) was introduced for each construct as previously described (18, 19). on March 3, 2020 at UNIVERSITY OF EDINBURGH To engineer the three NS1 revertants, lysine (K)-to-glutamic acid (E) (codon 186, AAA changed to GAA) and stop codon-to-arginine (R) (codon 220, TGA replaced by CGA) mutations were introduced into the pCAGGS-splice acceptor revertant O/03 NS1 constructs and the ambisense pDP2002 plasmid carrying the O/03 NS gene using PFUTurbo DNA polymerase (Agilent) and speciﬁc sets of primers. The presence of introduced mutations was conﬁrmed by Sanger sequencing. The cycling parameters and primers are available upon request. MATERIALS AND METHODS Cells. MDCK (ATCC CCL-34) and human embryonic kidney (293T; ATCC CRL-11268) cells were grown at 37°C and 5% CO2 in Dulbecco’s modiﬁed Eagle’s medium (DMEM) high glucose, GlutaMax, and pyruvate (ThermoFisher Scientiﬁc) supplemented with 10% fetal calf serum (Gibco Life Technologies) and 1% PS (100 units/ml penicillin, 100 g/ml streptomycin; Gibco Life Technologies). E-derm cells (ATCC CCL-57) were grown at 37°C and 5% CO2 in DMEM high glucose, GlutaMax, and pyruvate supplemented jvi.asm.org 16 March 2018 Volume 92 Issue 5 e01875-17 Journal of Virology Functional Evolution of the EIV NS1 Gene with 15% fetal calf serum (Gibco Life Technologies), 1% nonessential amino acids (Gibco, Life Technol- ogies), 1% PS (Gibco Life Technologies). with 15% fetal calf serum (Gibco Life Technologies), 1% nonessential amino acids (Gibco, Life Technol- ogies), 1% PS (Gibco Life Technologies). with 15% fetal calf serum (Gibco Life Technologies), 1% nonessential amino acids (Gibco, Life Technol- ogies), 1% PS (Gibco Life Technologies). Viruses. Viral stocks (accession number; abbreviation) of A/equine/Uruguay/1/1963 (ACD85423; U/63), A/equine/Miami/1/1963 (ABY81497; M/63), A/equine/SaoPaulo/1/1969 (ACD85390; SP/69), A/equine/Fontainebleau/1/1979 (ACD85401; F/79), A/equine/Sussex/1/1989 (ACD97430; S/89), A/equine/ Kentucky/1/1991 (ACA24672; K/91), A/equine/LaPlata/1995 (MF182460; LP/95), A/equine/Kentucky/1995 (MF182451; K/95), A/equine/Kentucky/1999 (MF182443; K/99), A/equine/Kentucky/5/2002 (ABA42429; K/02), A/equine/Newmarket/5/2003 (ACI48802; N/03), A/equine/Ohio/1/2003 (ABA42431; O/03), and A/equine/Mongolia/3/2013 (MF182459; M/13) were grown at passage 2 in MDCK cells (MOI, 0.1) and then aliquoted and stored at 80°C. Viruses. Viral stocks (accession number; abbreviation) of A/equine/Uruguay/1/1963 (ACD85423; U/63), A/equine/Miami/1/1963 (ABY81497; M/63), A/equine/SaoPaulo/1/1969 (ACD85390; SP/69), A/equine/Fontainebleau/1/1979 (ACD85401; F/79), A/equine/Sussex/1/1989 (ACD97430; S/89), A/equine/ Kentucky/1/1991 (ACA24672; K/91), A/equine/LaPlata/1995 (MF182460; LP/95), A/equine/Kentucky/1995 (MF182451; K/95), A/equine/Kentucky/1999 (MF182443; K/99), A/equine/Kentucky/5/2002 (ABA42429; K/02), A/equine/Newmarket/5/2003 (ACI48802; N/03), A/equine/Ohio/1/2003 (ABA42431; O/03), and A/equine/Mongolia/3/2013 (MF182459; M/13) were grown at passage 2 in MDCK cells (MOI, 0.1) and then aliquoted and stored at 80°C. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from Reverse genetic inﬂuenza A/equine/Ohio/1/03 (O/03), H3N8 O/03, and NS1 (O/03-K186E, O/03-230, and O/03-K186E-230) revertant viruses were grown in MDCK cells at 37°C and 5% CO2. For infections, virus stocks were diluted in infection medium (DMEM, 0.3% bovine serum albumin [BSA], 1% PS, and 1 g/ml of tosylsulfonyl phenylalanyl chloromethyl ketone [TPCK]-treated trypsin [Sigma]) (32). SeV strain Cantell was purchased from Charles River Laboratories and stored at 80°C. MATERIALS AND METHODS Reporter assay. For analysis of IFN- and ISRE promoter activation and general gene expression, 293T cells (12-well plate format; 2.5 105 cells/well) were transiently cotransfected using TransIT-LT1 transfection reagent (Cambridge Biosciences), with either 50 ng of pIFN--FF-Luc or 50 ng of pISRE-FF- Luc (reporter plasmids encoding FF-Luc under the control of the IFN- promoter or the ISRE promoter, respectively) and 50 ng of a plasmid constitutively expressing Renilla luciferase (pREN-Luc) under the simian virus 40 (SV40) promoter (24) (kindly provided by Benjamin G. Hale), as well as 1,000 ng of the indicated pCAGGS-SAM NS1-expressing plasmids (or empty pCAGGS; 1,000 ng). After 24 h of transfec- tion, the cells were infected with 50 hemagglutinating units (HAU) of SeV for 18 h and then lysed with 250 l of passive lysis buffer (Promega). IFN-–FF-Luc, ISRE-FF-Luc, and REN-Luc activities were measured using the dual-luciferase reporter assay system (Promega), as directed by the manufacturer’s protocol. All transfections were carried out in triplicate, and experiments were repeated independently three times. Coimmunoprecipitation of NS1 with CPSF30. Equine CPSF30 was cloned as previously described for human CPSF30 (18). Brieﬂy, the equine CPSF30 gene was ampliﬁed by RT-PCR from equine cells using oligo(dT) and PCR with speciﬁc primers. The RT-PCR product was then cloned into the pCAGGS HA-COOH plasmid as described previously (34). NS1 proteins were synthesized in vitro using pcDNA3 plasmids and the TNT7 transcription/translation kit (Promega), following the manufacturer’s recommendations. Human 293T cells (1.5 106 cells/well; 6-well format; triplicates) were transiently transfected with 2 g/well of an HA-equine CPSF30-expressing pCAGGS plasmid. Forty-eight hours posttransfection, the cells were lysed in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5 mM EDTA, 5% glycerol, 1% Triton X-100 supplemented with a complete mini-protease inhibitor cocktail (Pierce). Cleared cell lysates were incubated overnight at 4°C with the in vitro-synthesized O/03 and revertant NS1 proteins and 20 l of an anti-HA afﬁnity resin (Sigma). After extensive washing, precipitated proteins were dissociated from the resin using Laemmli buffer plus -mercaptoethanol. Total proteins from cell lysates were then analyzed by SDS-PAGE and Western blotting as described below, using a primary speciﬁc rabbit polyclonal antibody against NS1 (GenScript) or an HA tag for CPSF30 (Sigma). Rescue of recombinant H3N8 EIVs. Viruses were rescued as previously described (35). Brieﬂy, cocultures (2:3) of 293T and MDCK cells (6-well plate format; 2 106 cells/well) were seeded 24 h prior to viral rescue. MATERIALS AND METHODS Ruxolitinib was prepared as 10 mM stocks in dimethyl sulfoxide (DMSO) and used at a concentration of 4 M (25). Treatment was started 24 h prior to infection and maintained at the same concentration for the whole experiment. For plaque phenotype, a conﬂuent monolayer of MDCK cells (6-well plate format; triplicates; 6.4 104 cells/well) were infected with serial dilutions (1:2) of the viral stock of interest and placed for incubation at 37°C and 5% CO2 in a humidiﬁed atmosphere. The plates were gently rocked every 10 min. After 1 h of incubation, the cells were gently washed with PBS, and the infection medium was replaced with a 50:50 2.4% Avicell-2 minimal essential medium (MEM) overlay for exactly 48 h. At 48 hpi, the overlay was discarded, and the cells were gently washed 3 times with PBS and ﬁxed with 80% ice-cold acetone solution (Sigma-Aldrich) for 10 min at room temperature. The plates were then allowed to dry overnight at room temperature before being treated with 1% Triton X-100 (Sigma-Aldrich)-PBS solution for 10 min at room temperature, followed by 1 h of incubation with 10% normal goat serum (NGS)-PBS solution at room temperature. This was followed by overnight immunoblotting at 4°C in 10% normal goat serum plus PBS with a monoclonal anti-inﬂuenza A virus NP antibody (clone HB65; European Veterinary Laboratory; 1/500 dilution) under gentle agitation. After a 3-step washing with PBS, a horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody (AbD Serotec, United Kingdom; 1/1,000 dilution) was used in PBS solution for a further 4 h at room temperature under gentle agitation. A color development method was used to reveal the immunofocus using the TrueBlue peroxidase substrate (Insight Biotechnology); 2 ml of substrate was used per well for exactly 10 min before being stopped with tap water. on March 3, 2020 at UNIVERSITY OF EDINBURGH Viral and cellular protein staining for FACS and confocal microscopy. Cells were permeabilized with 1% Triton X-100 for 10 min and blocked in PBS-10% normal goat serum (Gibco, Life Technologies) for 1 h. The cells were then incubated with anti-inﬂuenza A virus NP antibody, rabbit polyclonal anti-NS1 protein antibody (GenScript), or rabbit STAT1 polyclonal antibody (Santa Cruz) overnight at 4°C. MATERIALS AND METHODS The cells were transiently cotransfected using TransIT-LT1 (Cambridge Bioscience) with 2.5 jvi.asm.org 17 March 2018 Volume 92 Issue 5 e01875-17 Journal of Virology Chauché et al. g of seven ambisense O/03 plasmids (pDP2002-PB2, -PB1, -PA, -HA, -NP, -NA, and -M) plus ambisense O/03 NS plasmid (O/03) or the NS revertant constructs (O/03-K186E, O/03-230, and O/03-K186E-230). At 24 hpt, the medium was replaced by infection medium. Virus-containing tissue culture supernatants were collected 2 or 3 days posttransfection, clariﬁed, and used to infect fresh MDCK cells (P1 stock) for 2 to 3 days postinfection. Viral titers of P1 stocks were determined by immunofocus assay (focus forming units [FFU] per milliliter) in MDCK cells, using mouse monoclonal anti-NP antibody (clone HB-65; European Veterinary Laboratory), horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG anti- body (AbD Serotec), and TrueBlue peroxidase substrate (Insight Biotechnology), as previously described (35). These P1 stocks were then used to grow P2 viral stocks at an MOI of 0.01 in MDCK cells for further use in experiments. For experimental infections, a minimum of two viral stocks for each virus were rescued and grown independently. The NS segment of each virus was sequenced by the Sanger method to conﬁrm the sequence of the O/03 or revertant viruses. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from q Viral infection. Conﬂuent monolayers of MDCK cells (12-well plate format; triplicates; 5 105 cells/well) or E-derm cells (12-well plate format; triplicates; 2.5 105 cells/well) were infected (MOI, 0.01 and 0.1, respectively) with the indicated viruses and placed at 37°C and 5% CO2. The cells were grown on coverslips for confocal microscopy. After 1 h of incubation, the cells were washed with PBS, and the infection medium was replaced with 500 l of fresh growth medium. Tissue culture supernatants were collected at various times postinfection and stored at 80°C, and the cells were ﬁxed in 0.1% buffered formalin at 4°C for 16 h and kept for confocal microscopy or ﬂuorescence-activated cell sorting (FACS) analysis. Each experiment was repeated three times independently. Viral titers were determined by immunofocus assay in MDCK cells. Titrations were repeated three times independently, and the mean value and standard error of the mean (SEM) were calculated using GraphPad Prism 7. To measure virus growth kinetics in the presence of an inhibitor of the IFN response, E-derm cells were treated with ruxolitinib. MATERIALS AND METHODS The cells were then washed twice with PBS and incubated for 4 h with rabbit anti-mouse IgG Alexa Fluor 488 (Cell Signaling) or donkey anti-rabbit IgG Alexa Fluor 555 (Cell Signaling) before FACS analysis (Guava ﬂow cytometer; Merck) or ﬁxed in Vectashield antifade mounting medium with DAPI (4=,6-diamidino-2- phenylindole) (Vector Laboratories) and analyzed by confocal microscopy. p y y y py For confocal microscopy, images were taken with a 63 oil objective on a Zeiss LSM 880 confocal microscope with gallium arsenide phosphide (GaAsP) detector. All images were taken with cross talk minimized using best signal calculations in Zen software; 3 by 3 tilescans were collected in three different positions for each sample. The experiment was repeated three times independently. The images were imported into ImageJ, and STAT1 nuclear localization was analyzed in NP-positive cells, with a minimum of 1,000 NP-expressing cells analyzed for each sample. The nuclear localization of STAT1 was analyzed using the AND function in the image calculator and was accomplished by detecting colocalization signals between STAT1 and DAPI, using the colocalization threshold function in ImageJ. The quantity of nuclear STAT1 (nSTAT1) was expressed as a percentage of total STAT1 (tSTAT1) in infected cells. For the representative pictures of nSTAT1, the signal intensity of nSTAT1 was normalized to O/03-K186E at 8 hpi. SDS-PAGE and Western blotting. Cells were lysed in Laemmli buffer plus -mercaptoethanol and stored immediately at 80°C. Samples were boiled for 15 min at 95°C prior to polypeptide separation by SDS-PAGE on NuPAGE Novex 4 to 12% Bis-Tris protein gels (ThermoFisher Scientiﬁc). Proteins were detected by Western blotting following transfer to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in 5% milk–Tris-buffered saline with 0.1% Tween 20 (TBST) and immunoblotted overnight at 4°C in 5% milk-TBST (0.1% Tween 20) with the following antibodies: mouse monoclonal anti-Mx1 (clone M143; provided by Georg Kochs, University of Freiburg, Freiburg, Germany), rabbit polyclonal anti-ISG15 (Proteintech), rabbit polyclonal anti-NS1 (GenScript), rabbit monoclonal anti-cleaved caspase-3 and rabbit monoclonal anti-caspase-3 (Cell Signaling), rabbit monoclonal anti-- For confocal microscopy, images were taken with a 63 oil objective on a Zeiss LSM 880 confocal microscope with gallium arsenide phosphide (GaAsP) detector. All images were taken with cross talk minimized using best signal calculations in Zen software; 3 by 3 tilescans were collected in three different positions for each sample. The experiment was repeated three times independently. March 2018 Volume 92 Issue 5 e01875-17 jvi.asm.org 18 MATERIALS AND METHODS The images were imported into ImageJ, and STAT1 nuclear localization was analyzed in NP-positive cells, with a minimum of 1,000 NP-expressing cells analyzed for each sample. The nuclear localization of STAT1 was analyzed using the AND function in the image calculator and was accomplished by detecting colocalization signals between STAT1 and DAPI, using the colocalization threshold function in ImageJ. The quantity of nuclear STAT1 (nSTAT1) was expressed as a percentage of total STAT1 (tSTAT1) in infected cells. For the representative pictures of nSTAT1, the signal intensity of nSTAT1 was normalized to O/03-K186E at 8 hpi. SDS-PAGE and Western blotting. Cells were lysed in Laemmli buffer plus -mercaptoethanol and stored immediately at 80°C. Samples were boiled for 15 min at 95°C prior to polypeptide separation by SDS-PAGE on NuPAGE Novex 4 to 12% Bis-Tris protein gels (ThermoFisher Scientiﬁc). Proteins were detected by Western blotting following transfer to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in 5% milk–Tris-buffered saline with 0.1% Tween 20 (TBST) and immunoblotted overnight at 4°C in 5% milk-TBST (0.1% Tween 20) with the following antibodies: mouse monoclonal anti-Mx1 (clone M143; provided by Georg Kochs, University of Freiburg, Freiburg, Germany), rabbit polyclonal anti-ISG15 (Proteintech), rabbit polyclonal anti-NS1 (GenScript), rabbit monoclonal anti-cleaved caspase-3 and rabbit monoclonal anti-caspase-3 (Cell Signaling), rabbit monoclonal anti-- jvi.asm.org 18 March 2018 Volume 92 Issue 5 e01875-17 Functional Evolution of the EIV NS1 Gene Journal of Virology tubulin (Sigma), mouse monoclonal anti-puromycin (clone 12D10; Millipore), HRP-conjugated anti-mouse IgG (Dako), and HRP-conjugated anti-rabbit IgG (Dako). The chemiluminescent signal was detected using Amersham ECL Prime Western blotting detection reagent (GE Healthcare) and captured with a ChemiDoc XRS system (Bio-Rad). Antiviral cytokine production and general protein shutdown. E-derm cells (12-well plate format; triplicates; 2.5 105 cells/well) were infected (MOI, 0.1) with the indicated viruses for a total of 72 h. At the indicated times postinfection, the supernatants were collected and stored at 80°C for further analysis by bioassay, while the cells were treated for 1 h at 37°C and 5% CO2 with puromycin (20 g/ml in DMEM, 15% FBS) prior to lysis in Laemmli buffer plus -mercaptoethanol and stored at 80°C for further analysis by Western blotting. Puromycin is a well-known antibiotic that competes against aminoacyl tRNA on the ribosome A site (36). MATERIALS AND METHODS As such, puromycin enables examination of total protein production without requiring transfection, radiolabeling, or the prior choice of a candidate gene (37). If cells are incubated with puromycin, lysed, and immunoblotted using an anti-puromycin antibody, all the proteins produced will be immunostained, as puromycin is incorporated at the C termini of all the nascent proteins. For the bioassay, supernatants were UV inactivated for 5 min at room temperature and used to treat fresh E-derm cells (48-well plate format; 6 104 cells/well; triplicates) for 24 h. The cells were then infected with rVSV-GFP (2.5 102 TCID50) for 8 h and then trypsinized and ﬁxed in 0.1% buffered formalin for 16 h at 4°C. The percentage of GFP-expressing cells was then analyzed by FACS. For controls, E-derm cells were mock treated or treated with 500 units of uIFN. The GFP expression of mock-treated cells infected with rVSV-GFP was considered 100%, and the GFP expression of uIFN-treated cells infected with rVSV-GFP was considered 0%. Mean values and standard errors of the mean were calculated with GraphPad Prism 7. on March 3, 2020 at UNIVERSITY OF EDINBURGH http://jvi.asm.org/ Downloaded from Transcriptome sequencing (RNA-seq). Conﬂuent monolayers of E-derm cells (12-well plates; trip- licates; 2.5 105 cells/well) were infected with O/03 and revertant viruses (MOI, 1) or mock infected at least three times independently. At 8 hpi, the cells were washed with PBS and immunostained with anti-NP antibody, and the proportion of infected cells was determined by FACS. The samples containing similar proportions of infected cells were selected for transcriptomic analysis, and the cells were lysed with 500 l of TRIzol (ThermoFisher Scientiﬁc) for further RNA extraction. Total RNA was extracted using the TRIzol method and further puriﬁed using RNeasy mini-spin columns (Qiagen), including an on-column DNase I digestion step (Qiagen) according to the manufac- turer’s protocol. The RNA concentration was measured with Qubit and the Qubit RNA HS assay kit (ThermoFisher Scientiﬁc) following the manufacturer’s protocol. The rRNA integrity number was mea- sured using an Agilent 2100 BioAnalyzer (Agilent Technologies). on March 3, 2020 at UNIVERSITY OF EDINBURGH Total RNA (4.5 g) was enriched by selectively depleting rRNA using the RiboMinus Eukaryote kit v2 (Ambion, Life Technologies). The sequence reads (BioProject accession number PRJEB21264) were processed according to the Tuxedo pipeline (38). Read quality was assessed using FastQC, and TopHat2 and Bowtie2 were used to map short reads against the Equus caballus 2 genome (GCA_000002305.1). MATERIALS AND METHODS A list of differentially expressed genes compared to mock-infected samples was generated using CuffDiff2 (genes with Benjamini Hochberg P values of 0.05 were considered signiﬁcant) (39). g j g g Phylogenetic analysis of EIV NS1 sequences. We collected 170 EIV H3N8 NS sequences from the NCBI Inﬂuenza Virus Resource database (the sequences are available upon request). We also included four EIV H3N8 NS sequences that we sequenced for this study (accession numbers MF182460, MF182451, MF182443, and MF182459) (40). SeaView version 4.6.1 (41) was used to align the NS1 coding regions, and the ﬁnal alignment was edited manually. NEP coding regions were removed from the alignment. We used BEAST version 1.8.4 (42) to infer maximum clade credibility (MCC) trees. We used a strict molecular clock and an HKY85G model of nucleotide substitution. Each codon position was estimated with separate substitution rates and nucleotide frequencies. Two individual chains were run until convergence was achieved. Statistical analysis. Unless otherwise indicated, signiﬁcance was calculated by two-way analysis of variance (ANOVA), followed by Bonferroni’s multiple-comparison test. Accession number(s). Data determined in this study may be found in the NCBI BioProject database under accession number PRJEB21264 and in GenBank under accession numbers MF182460, MF182451, MF182443, and MF182459. SUPPLEMENTAL FILE 1, PDF ﬁle, 0.2 MB. SUPPLEMENTAL FILE 1, PDF ﬁle, 0.2 MB. SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/JVI .01875-17. REFERENCES Lipsitch M, Barclay W, Raman R, Russell CJ, Belser JA, Cobey S, Kasson PM, Lloyd-Smith JO, Maurer-Stroh S, Riley S, Beauchemin CA, Bedford T, Friedrich TC, Handel A, Herfst S, Murcia PR, Roche B, Wilke CO, Russell CA. 2016. Viral factors in inﬂuenza pandemic risk assessment. Elife 5:e18491. https://doi.org/10.7554/eLife.18491. 24. Martinez-Sobrido L, Zuniga EI, Rosario D, Garcia-Sastre A, de la Torre JC. 2006. Inhibition of the type I interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus. 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https://openalex.org/W4239940540	https://www.qeios.com/read/JMY2AJ/pdf	English	null	Hypopharyngeal Cancer pT1 TNM Finding v7	Definitions	2,020	cc-by	82	Open Peer Review on Qeios Open Peer Review on Qeios Open Peer Review on Qeios Hypopharyngeal Cancer pT1 TNM Finding v7 National Cancer Institute National Cancer Institute Qeios ID: JMY2AJ · https://doi.org/10.32388/JMY2AJ Qeios · Definition, February 8, 2020 Source National Cancer Institute. Hypopharyngeal Cancer pT1 TNM Finding v7. NCI Thesaurus. Code C89032. Code C89032. Hypopharyngeal cancer with tumor limited to one subsite of hypopharynx and/or 2 cm or less in greatest dimension. (from AJCC 7th Ed.) Qeios ID: JMY2AJ · https://doi.org/10.32388/JMY2AJ 1/1
https://openalex.org/W3116890422	https://www.researchprotocols.org/2021/3/e23492/PDF	English	null	Impact of Intersecting Systems of Oppression on Diabetic Retinopathy Screening Among Those Who Identify as Women of Low Socioeconomic Status: Protocol for a Convergent Mixed Methods Study	JMIR research protocols	2,021	cc-by	8,042	Corresponding Author: Corresponding Author: Valeria Rac, MD, PhD Ted Rogers Centre for Heart Research at Peter Munk Cardiac Centre Toronto General Hospital Research Institute University Health Network 200 Elizabeth Street Toronto, ON Canada Phone: 1 416 634 8799 Email: valeria.rac@theta.utoronto.ca Corresponding Author: Valeria Rac, MD, PhD Ted Rogers Centre for Heart Research at Peter Munk Cardiac Centre Toronto General Hospital Research Institute University Health Network 200 Elizabeth Street Toronto, ON Canada Phone: 1 416 634 8799 Email: valeria.rac@theta.utoronto.ca Corresponding Author: Valeria Rac, MD, PhD Ted Rogers Centre for Heart Research at Peter Munk Cardiac Centre Toronto General Hospital Research Institute University Health Network 200 Elizabeth Street Toronto, ON Canada Phone: 1 416 634 8799 Email: valeria.rac@theta.utoronto.ca JMIR RESEARCH PROTOCOLS JMIR RESEARCH PROTOCOLS Stanimirovic et al 1Program for Health System and Technology Evaluation, Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada 2Ted Rogers Centre for Heart Research at Peter Munk Cardiac Centre, Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada https://www.researchprotocols.org/2021/3/e23492 JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 1 (page number not for citation purposes) Impact of Intersecting Systems of Oppression on Diabetic Retinopathy Screening Among Those Who Identify as Women of Low Socioeconomic Status: Protocol for a Convergent Mixed Methods Study Impact of Intersecting Systems of Oppression on Diabetic Retinopathy Screening Among Those Who Identify as Women of Low Socioeconomic Status: Protocol for a Convergent Mixed Methods Study Aleksandra Stanimirovic1,2,3,4,5, BSc, MA, PhD; Troy Francis1,2,3,5, BSc, MSc; Anna Cooper Reed3, MSW; Sonia Meerai1,2,5,6,7, MSW; Olivera Sutakovic4,8, MD; Rebecca Merritt9, BA; Michael Brent4,8, MD; Valeria Rac1,2,3,4,5, MD, PhD Aleksandra Stanimirovic1,2,3,4,5, BSc, MA, PhD; Troy Francis1,2,3,5, BSc, MSc; Anna Cooper Reed3, MSW; Sonia Meerai1,2,5,6,7, MSW; Olivera Sutakovic4,8, MD; Rebecca Merritt9, BA; Michael Brent4,8, MD; Valeria Rac1,2,3,4,5, MD, PhD Aleksandra Stanimirovic , BSc, MA, PhD; Troy Francis , BSc, MSc; Anna Cooper Reed , MSW; Sonia Meerai1,2,5,6,7, MSW; Olivera Sutakovic4,8, MD; Rebecca Merritt9, BA; Michael Brent4,8, MD; Valeria Rac1,2,3,4,5, MD, PhD 1Program for Health System and Technology Evaluation, Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada 2Ted Rogers Centre for Heart Research at Peter Munk Cardiac Centre, Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada 3Institute of Health Policy, Management and Evaluation, University of Toronto, Toronto, ON, Canada 4Diabetes Action Canada, CIHR SPOR Network, Toronto, ON, Canada 5Toronto Health Economics and Technology Assessment Collaborative, Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada 6Gender, Feminist & Women Studies, Faculty of Graduate Studies, York University, Toronto, ON, Canada 7Lyle S Hallman Faculty of Social Work, Wilfrid Laurier University, Brantford, ON, Canada 8Department of Ophthalmology, Toronto Western Hospital, Toronto, ON, Canada 9South Riverdale Community Health Centre, Toronto, ON, Canada 1Program for Health System and Technology Evaluation, Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada 2Ted Rogers Centre for Heart Research at Peter Munk Cardiac Centre, Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada ON, Canada 3Institute of Health Policy, Management and Evaluation, University of Toronto, Toronto, ON, Canada 4Diabetes Action Canada, CIHR SPOR Network, Toronto, ON, Canada 5Toronto Health Economics and Technology Assessment Collaborative, Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada 6Gender, Feminist & Women Studies, Faculty of Graduate Studies, York University, Toronto, ON, Canada 7Lyle S Hallman Faculty of Social Work, Wilfrid Laurier University, Brantford, ON, Canada 8Department of Ophthalmology, Toronto Western Hospital, Toronto, ON, Canada 9South Riverdale Community Health Centre, Toronto, ON, Canada Aleksandra Stanimirovic1,2,3,4,5, BSc, MA, PhD; Troy Francis1,2,3,5, BSc, MSc; Anna Cooper Reed3, MSW; Sonia Meerai1,2,5,6,7, MSW; Olivera Sutakovic4,8, MD; Rebecca Merritt9, BA; Michael Brent4,8, MD; Valeria Rac1,2,3,4,5, MD, PhD Health Disparities in Diabetes and Its Complications and Comorbidities Health disparities in diabetes and its complications exist globally [12]. In Canada, ethnic minorities and Indigenous populations have a higher prevalence of diabetes than nonminority populations [13]. Diabetes appears to be more common among men than women. Socioeconomic status (SES) is inversely related to the prevalence of diabetes, but income-related disparities are greater among women [13]. In comparison to men with diabetes, women were more likely to be in the lowest income quintiles than the highest [14]. The odds ratio of developing diabetes doubles in men and almost triples in women in the lowest income category compared with those in the highest income category [15], and among Aboriginal Canadians, two-thirds (66.6%) of diagnosed individuals are women [16]. The Project for an Ontario Women’s Health Evidence–based Report (POWER) study found that women of lower SES with diabetes had worse health and functional statuses than men and stressed the importance of addressing gender differences, which may interfere with diabetes self-care among the general diabetic population [14]. In 2016, 1.8 million Canadian males and 2.2 million females aged 45 to 85 years experienced vision loss. Prevalence increased from 8.7% to 16.9% between 2011 and 2016 [17]. Prevalence proportions increased with age but decreased exponentially with the severity of impairment, and vision loss remains more common among females [17]. One-third of adult diabetic patients did not receive an eye examination for diabetic retinopathy within 2 years [5], and more specifically, 25.3% of people with diabetes over the age of 60 years had not seen an eye care provider in the last year [7]. The prevalence of vision loss in Canada is expected to increase nearly 30% in the next decade [8]. The financial implication of vision loss in Canada in 2007 was estimated to be CAD $15.8 billion per annum, with CAD $8.6 billion (54.6%) associated with direct health system expenditure; CAD $4.4 billion (28.0%) associated with productivity loss resulting from lower employment, higher absenteeism, and premature death of Canadians with vision loss; CAD $1.8 billion (11.1%) associated with costs to society created by market inefficiency from transfers including welfare payments and taxation forgone; CAD $0.7 billion (4.4%) associated with the value for the care of people with vision loss; and CAD $305 million (1.9%) associated with other indirect costs such as aids, home modifications, and funeral costs [9]. Abstract Background: By 2025, 5 million Canadians will be diagnosed with diabetes, and women from lower socioeconomic groups will likely account for most new diagnoses. Diabetic retinopathy is a primary vision complication of diabetes and a leading cause of blindness among adults, with 26% prevalence among women. Tele-retina is a branch of telemedicine that delivers eye care remotely. Screening for diabetic retinopathy has great potential to reduce the incidence of blindness, yet there is an adverse association among screening, income, and gender. Objective: We aim to explore gender disparity in the provision of tele-retina program services for diabetic retinopathy screening in a cohort of women of low socioeconomic status (SES) receiving services in South Riverdale Community Health Centre (SRCHC) between 2014 and 2019. Methods: Using a convergent mixed methods design, we want to understand patients’, providers’, administrators’, and decision makers’ perceptions of the facilitators and barriers associated with the implementation and adoption of tele-retina. Multivariate logistic regression will be utilized to assess the association among client characteristics, referral source, and diabetic retinopathy screening. Guided by a grounded theory approach, systematic coding of data and thematic analysis will be utilized to identify key facilitators and barriers to the implementation and adoption of tele-retina. Results: For the quantitative component, we anticipate a cohort of 2500 patients, and we expect to collect data on the overall patterns of tele-retina program use, including descriptions of program utilization rates (such as data on received and completed diabetic retinopathy screening referrals) along the landscape of patient populations receiving these services. For the qualitative XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS Stanimirovic et al component, we plan to interview up to 21 patients and 14 providers, administrators, and decision makers, and to conduct up to 14 hours of observations alongside review of relevant documents. The interview guide is being developed in collaboration with our patient partners. Through the use of mixed methods research, the inquiry will be approached from different perspectives. Mixed methods will guide us in combining the rich subjective insights on complex realities from qualitative inquiry with the standard generalizable data that will be generated through quantitative research. The study is under review by the University Health Network Research Ethics Board (19-5628). We expect to begin recruitment in winter 2021. Conclusions: In Ontario, the screening rate for diabetic retinopathy among low income groups remains below 65%. Social and Economic Impacts of Diabetes and Its Complications Diabetes is a significant public health burden, affecting 382 million people worldwide [1]. In Canada, the prevalence was estimated at 3.4 million (9.3%) in 2015 and is expected to increase to 5 million (12.1%) by 2025 [2]. Diabetic retinopathy is the primary vision complication caused by diabetes [3] and is the leading cause of new cases of blindness in adults aged 20 to 65 years [4]. The prevalence of diabetic retinopathy in Canada ranges from 20% to 30% [4]. Over a million people from Ontario were affected by diabetic retinopathy in 2016 [5]. Among Canadian adults, 5.7% have visual impairments with a variation in the provincial prevalence of visual impairment from 2.4% in Manitoba to a staggering 10.9% in Newfoundland and Labrador [6]. Lower income and type 2 diabetes have been shown to be associated with increased odds of visual impairment [6]. KEYWORDS to a financial cost of CAD $19,370 per person with vision loss per annum, and considering the value of lost well-being, the cost is CAD $33,704 per person per annum [9]. The Canadian National Institute for the Blind (CNIB) estimated costs of associated complications of vision loss are as follows: falls, CAD $25.8 million; depression, CAD $175.2 million; hip fractures, CAD $101.7 million; and nursing home admission, CAD $713.6 million [10]. The National Coalition for Vision Health noted that health care costs for vision loss in Canada have been projected to increase to CAD $30.3 billion per year by 2032 [11]. Introduction Social and Economic Impacts of Diabetes and Its Complications Abstract Understanding the facilitators and barriers to diabetic retinopathy screening may be a prerequisite in the development of a successful screening program. This study is the first Ontario study to focus on diabetic retinopathy screening practices in women of low SES, with the aim to improve their health outcomes and revolutionize access to quality care. ternational Registered Report Identifier (IRRID): PRR1-10.2196/23492 International Registered Report Identifier (IRRID): PRR1-10.2196/23492 (JMIR Res Protoc 2021;10(3):e23492) doi: 10.2196/23492 KEYWORDS gender; screening; diabetes; diabetic retinopathy; blindness; technology; tele-retina screening; health equity; intersectionalit theory JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 2 (page number not for citation purposes) https://www.researchprotocols.org/2021/3/e23492 Social Determinants of Health Social determinants of health are conceptualized in a manner that takes into account how environmental and material conditions further increase the risk for marginalized populations at the intersection of identities such as race, age, gender, and income [22-24]. Social determinants of health in the context of mobile health screening among marginalized populations take into consideration environmental stressors that an individual may be exposed to, including but not limited to toxins, dwelling living conditions, access to education, food, employment, the role of a globalized economy and its effects, and current social exclusionary measures exacerbated through limited access to basic needs [22-24]. Tele-retina is one of the diabetic retinopathy screening modalities. It is focused on reducing eye care disparities that lead to avoidable vision loss. Tele-retina is a branch of telemedicine that delivers eye care remotely. Retinal images and data are collected and transferred via telecommunication technology to eye specialists [19]. In many developing countries, tele-retina has been utilized to provide quality eye care to the underserved urban population and the unserved remote rural population [19]. Alternatives to in-person examinations, such as tele-retina, can triage patients to proper levels of care and reduce barriers to specialized eye care [19]. Screening and detection of diabetic retinopathy are important to reduce the incidence of blindness, as they can detect early sight-threatening lesions, which can be treated effectively. Factors contributing to patients’ missed opportunities in access to timely treatment can include limited number of specialists and challenges related to time and travel. Integrating a socioeconomic approach provides an integrative synergistic framework in further understanding the material effects and experiences of diabetic retinopathy screening among women with low SES. Embedded within the framework is a social, economic, and political system approach for uncovering health inequities that are heavily influenced by these systems [23-25]. In this context, social determinants of health are elevated to include systems that perpetuate oppressions in relation to these material effects, such as poverty and inaccessibility to housing, food, and employment, which further compounds the overall health and well-being of women with low SES [23-26]. The eye care pathway does not end with diabetic retinopathy screening. Individuals who are screened and those who remain unscreened and develop severe vision loss have access to comprehensive vision rehabilitation services. Social Determinants of Health In Canada, comprehensive vision rehabilitation represents a multidisciplinary pathway that encompasses the full spectrum of a patient’s rehabilitation journey after vision loss, from initial assessment through intensive rehabilitation therapy [19]. Health Disparities in Diabetes and Its Complications and Comorbidities The value of the lost well-being (inclusive of disability and premature death) was estimated at CAD $11.7 billion. In per capita terms, this adds An adverse association between screening and income has been found previously. A published report indicated that women of https://www.researchprotocols.org/2021/3/e23492 XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS Stanimirovic et al October each year [20,21]. The CNIB Eye Van was cancelled in 2020 owing to the COVID-19 pandemic, leaving patients without care. A tele-retina program is a viable option that could address the needs of these patients. lower SES may not be screened for breast, lung, and colorectal cancers [3]. In fact, they may not have symptoms recognized early or receive the most effective treatment. Similar to cancer screening, diabetic retinopathy screening is essential for the early detection and treatment of diabetes-related visual impairments and blindness [18]. Yet, it is commonly underutilized among women of lower SES [18]. Because diabetic morbidity and mortality are associated with low SES, the need to address socioeconomic barriers for women must take precedence over simply ensuring the provision of diabetes medical management. This is the first Ontario study to focus on diabetic retinopathy screening practices in women of low SES, with great potential to improve their health outcomes and access to quality care. Tele-Retina Screening for Diabetic Retinopathy There are disparities in eye care utilization among community-dwelling Canadians, where eye care utilization is defined as the self-report of a visit to an optometrist or ophthalmologist in the past 12 months [7]. Of concern, 25.3% of people with diabetes above the age of 60 years had not seen an eye care provider in the last year. Men in comparison to women and people with lower income (linear trend P<.05) were less likely to use eye care [7]. Conceptual Frameworks The Conceptual Social Determinants of Health framework adapted from the World Health Organization, Danaher framework, and Multi-Construct Intersectionality framework (Synergies of Oppression) will provide theoretical insights into the potential facilitators and barriers of the implementation and adoption of tele-retina screening for diabetic retinopathy. The frameworks will be used as analytical lenses to assess the system-, organizational-, and patient/provider-level causal factors of the tele-retina program. This approach will provide in-depth insights into the experiences, impacts, and outcomes of tele-retina screening for diabetic retinopathy among women in low socioeconomic groups. https://www.researchprotocols.org/2021/3/e23492 JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 3 (page number not for citation purposes) Health Equity Health equity outcomes are directly correlated with the distribution of resources within any particular context. Women of low SES with multiple health conditions and limited access to resources have decreased access to achieving health equity. When deconstructing health equity, the construct of power relations within social, economic, and political systems is taken into consideration, as it determines the success rates for health- and well-being–specific interventions, including but not limited to promotion of health, interventions, and evaluation of the effectiveness of health-specific programs [22]. In considering the Social Determinants of Health framework, health equity, specifically the concept of power relations with a distribution of resources, is integral to understanding how successful health Of note, in this study, tele-retina was utilized in an urban setting. It is also being utilized in rural settings such as First Nations reserves. It has the potential to scale to many rural communities that are underserviced with respect to diabetic retinopathy screening and could be used as a strategy in conjunction with the Medical Mobile Care Unit (known as the CNIB Eye Van). The CNIB Eye Van is a fully equipped medical mobile eye care clinic that travels (with an ophthalmologist on board) to patients in Northern Ontario, from March through XSL•FO RenderX JMIR RESEARCH PROTOCOLS Stanimirovic et al [22], and Figure 2 illustrates the Danaher framework (2011), which integrates the role of community in health policy advocacy for addressing health disparities [27]. [22], and Figure 2 illustrates the Danaher framework (2011), which integrates the role of community in health policy advocacy for addressing health disparities [27]. promotion and intervention will be sustainable for marginalized populations [22]. Figure 1 illustrates the synergy of adapting the Social Determinants of Health framework with health equity promotion and intervention will be sustainable for marginalized populations [22]. Figure 1 illustrates the synergy of adapting the Social Determinants of Health framework with health equity [22], and Figure 2 illustrates the Danaher framework (2 which integrates the role of community in health p advocacy for addressing health disparities [27]. Figure 1. Conceptual Social Determinants of Health framework (adapted from the World Health Organization). Figure 2. Danaher framework. SDoH: Social Determinants of Health. Figure 1. Conceptual Social Determinants of Health framework (adapted from the World Health Organization). Figure 1. Conceptual Social Determinants of Health framework (adapted from the World Health Organization). Figure 1. Health Equity Conceptual Social Determinants of Health framework (adapted from the World Health Organization). Figure 2. Danaher framework. SDoH: Social Determinants of Health. igure 2. Danaher framework. SDoH: Social Determinants of Health. JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 4 (page number not for citation purposes) JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 4 (page number not for citation purposes) https://www.researchprotocols.org/2021/3/e23492 https://www.researchprotocols.org/2021/3/e23492 XSL•FO RenderX JMIR RESEARCH PROTOCOLS Stanimirovic et al environmental factors are integrated [23-26]. Figure 3 illustrates these categories/framings within the context of the synergy of oppressions, specifically the outcomes of increased marginalization through oppressions and intersections of social determinants of health. This synergy is also compounded with access to health equity [26,28]. https://www.researchprotocols.org/2021/3/e23492 Intersectionality Intersectionality is a theoretical and pragmatic tool within health service and system research to further nuance the complexities of the impact of health promotion and intervention among marginalized populations. Originally developed by Black feminist scholar Kimberle Crenshaw (1989), it has been adapted and interrogated to be applied in many other contexts within the sciences, social sciences, and humanities [23]. Marginality in relation to an intersectional approach takes into consideration that identity markers, such as age, race, gender, disability, and SES, are not viewed as separate and are rather interwoven with the outcomes involving how power relations within social, economic, and political systems further create health inequities through inaccessibility to resources for health promotion and intervention [23-26]. Intersectionality is taken up in this conceptual framing of an intersectional categorical axis where social determinants of health, systems of oppressions, and For the purpose of this study, social determinants of health, health equity, and an intersectional approach to analysis are integral for understanding the impacts and material effects of diabetic retinopathy screening among women of low SES. This adapted framework provides an in-depth approach to understand how power relations within social, economic, and political systems impact community-based health screening programs for marginalized populations. Further, it integrates the individual standpoint based on intersectional identities and the environment. Figure 3. Multiconstruct intersectionality framework (Synergies of Oppression) for addressing social determinants of health (SDH) inequities (adapted from McGibbon & McPherson [28]). Figure 3. Multiconstruct intersectionality framework (Synergies of Oppression) for addressing social determinants of health (SDH) inequities (adapted from McGibbon & McPherson [28]). Multiconstruct intersectionality framework (Synergies of Oppression) for addressing social determinants of health (SDH) inequities (adapted Gibbon & McPherson [28]). The tele-retina program is offered to patients at no cost in partnership with primary care organizations with a population focus (Anishnawbe Health Toronto) or in low-income communities with high prevalence of diabetes and low diabetic retinopathy screening rates (Parkdale, Flemingdon Park Community Health Centre, Scarborough Academic Family Health Team, LAMP Community Health Centre, Unison Health, Community Services-Lawrence Heights Site, and Unison Health and Community Services sublocations [Bathurst-Finch, Jane-Trethewey, and Keele-Rogers]) [29,30]. JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 5 (page number not for citation purposes) Program Description Program Description In 2014, South Riverdale Community Health Centre (SRCHC), in partnership with Dr Michael H Brent, Chief of Retina Services at the University of Toronto, received funding from Toronto Central Local Health Integration Network to develop a mobile screening program to assess the retinal health of individuals diagnosed with diabetes (Figure 4) [29]. This strategy is driven by the recognition that access to optometrists and ophthalmologists is difficult for individuals with diabetes who live in certain neighborhoods in Toronto. As of December 2016, the Toronto Health Economics and Technology Assessment Collaborative has created a very strong https://www.researchprotocols.org/2021/3/e23492 JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 5 (page number not for citation purposes) XSL•FO RenderX JMIR RESEARCH PROTOCOLS Stanimirovic et al collaboration with SRCHC where Toronto Health Economics and Technology Assessment assessed the cost-effectiveness of the screening program [31]. In 2019, findings from a cost-effectiveness study suggested that tele-retina is a more cost-effective means of screening for diabetic retinopathy than the standard of care screening in urban and rural underscreened communities, and this study represents a natural progression of the previous collaborative work. Figure 4. South Riverdale Community Health Centre (SRCHC) tele-retina project process map [30]. EMR: electronic medical records; OTN: Ontario Telemedicine Network; RPN: Registered Practical Nurse. elemedicine Network; RPN: Registered Practical Nurse. Guidelines The population of interest includes individuals of low SES (income less than CAD $25,000) attending diabetic retinopathy screening services at various sites (listed in Table 1) from 2014 to 2019, who have a diagnosis of diabetes, are at high risk for remaining unscreened for diabetic retinopathy, and have limited access to eye care. This protocol has been developed in accordance with SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) guidelines [32]. Qualitative Component The population of interest includes those who identify as women of low SES who attend or decline to attend tele-retina screening for diabetic retinopathy within one of the identified sites. The study population also includes health care providers, administrators, and decision makers who are involved in the tele-retina screening program. Aims We aim to explore the gender disparity in the provision of tele-retina program services for diabetic retinopathy screening in a cohort of women of low SES receiving services in SRCHC between 2014 and 2019, including but not limited to the evaluation of the overall patterns of tele-retina program use, and to conduct a qualitative study of patients, providers, administrators, and decision makers in order to understand their perceptions regarding the facilitators and barriers associated with the implementation and adoption of the tele-retina program. SRCHC annually serves a population of 10,000 individuals, of which about 13% are diagnosed with diabetes and should undergo diabetic retinopathy screening. Annually, close to 500 individuals diagnosed with diabetes and receiving services from SRCHC are screened for diabetic retinopathy. As the program has been in place since 2014, we anticipate the study cohort will consist of 2500 individuals (500 individuals screened for diabetic retinopathy per year × 5 years [from 2014 to 2019]). Table 1. Breakdown of recruitment (qualitative component) based on participating sites. Number of hours of observation Number of health care provider, administrator, and decision maker interviews Number of patient interviews Site 1-2 1-2 2-3 Anishnawabe Health Toronto 1-2 1-2 2-3 Flemingdon Health Centre 1-2 1-2 2-3 LAMP Community Health Centre 1-2 1-2 2-3 Parkdale Community Health Centre 1-2 1-2 2-3 Scarborough Academic Family Health Team 1-2 1-2 2-3 South Riverdale Community Health Centre 1-2 1-2 2-3 Unison Health and Community Services Table 1. Breakdown of recruitment (qualitative component) based on participating sites. JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 6 https://www.researchprotocols.org/2021/3/e23492 (page number not for citation purposes) FO https://www.researchprotocols.org/2021/3/e23492 (page number not for citation purposes) •FO derX JMIR RESEARCH PROTOCOLS Stanimirovic et al Quantitative Component In the retrospective cohort study, we will collect data on the overall patterns of tele-retina program use, including descriptions of program utilization rates (such as data on received and completed diabetic retinopathy screening referrals) along the landscape of patient populations receiving these services. Within the qualitative component, in addition to interviewing providers, administrators, and decision makers, we will purposely select a subset of the population from the quantitative component, more specifically those identifying as women of low SES who attend or decline to attend tele-retina screening for diabetic retinopathy within one of the identified sites. Inclusion Criteria We anticipate a total of 7 to 14 hours of nonparticipatory observations (1-2 hours of observation per site) to observe how providers carry out their work on a daily basis, how administrators interact with providers, how patients and providers interact, and how patients interact with their environments. Field work provides excellent opportunities to identify and engage respondents for interviews and to collect grey literature. For all such field work, we will document activities, impressions, and interactions through field notes. Interviews lasting approximately 60 minutes will be guided by open-ended semistructured interview guides, and recorded and transcribed verbatim [33]. Interview guides will ensure that interactions among the researcher and participants remain focused and will be modified as new themes or issues arise, in line with the qualitative approach [34]. Textbox 1 provides a detailed description of ethnographic field work and semistructured interviews. Patients who are aged 18 years or above; identify as women of low SES (income less than CAD $25,000); are able and willing to provide verbal and/or written informed consent; and attend (complete or decline) tele-retina screening for diabetic retinopathy are considered for inclusion. Please note that there are no language requirements, as we will use a translation services for non-English speaking clients. Health care providers who currently provide care and are part of the screening process within the tele-retina program are considered for inclusion. Administrators who coordinate the tele-retina screening for diabetic retinopathy and decision makers who inform and/or are part of the decision-making process for tele-medicine–specific programming within community health centers in Ontario are considered for inclusion. Qualitative Component The qualitative component will entail collection of primary data sources to ensure rigor and data quality, including (1) nonparticipatory observations (ethnographic observations), (2) semistructured interviews, and (3) document review. JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 7 (page number not for citation purposes) Quantitative Data Analysis Continuous variables will be described using measures of central tendency and dispersion, such as mean/median and standard deviation/interquartile range, with appropriate statistical methods, and compared using analysis of variance (ANOVA) or the Kruskal-Wallis test as appropriate. Categorical variables will be described using contingency tables and compared using the chi-square test. Multivariate logistic regression will be utilized to assess the association between client characteristics, screening referral sources, and diabetic retinopathy screening. Analyses will be conducted using SAS v 9.4 (SAS Institute). Convergent Analysis and Interpretation We will utilize a convergent mixed methods design, which will combine and contrast the data collected in the quantitative and qualitative components in order to triangulate similarities and differences in the results of both research methods [43]. The findings will summarize and interpret to what extent the results from the two components converge, diverge, and produce a more complete understanding of the use of tele-retina services among women of low SES receiving care at SRCHC [43]. Exclusion Criteria Ethnographic fieldwork • Process and interaction between the patient and health care provider during the tele-retina screening process for diabetic retinopathy (7-14 hours in total) Semistructured interviews per identified site Patients • 2-3 one-on-one semistructured interviews per site • Patients who identify as women of low socioeconomic status • Impact, experience, and outcomes of participating in the screening process Health care providers • 7-14 semistructured interviews • Nurses, physicians, and/or ophthalmologists involved in the screening process • Impact and experience of coordination, and delivery of the program Administrators/decision makers • Up to 14 semistructured interviews • Health care administrators/decision makers • Values and beliefs that inform the screening program • Description of the coordination, delivery, funding, and policies that inform the screening program Textbox 1. Description of ethnographic field work and semistructured interviews per identified site. Ethnographic fieldwork • Process and interaction between the patient and health care provider during the tele-retina screening process for diabetic retinopathy (7-14 hours in total) Semistructured interviews per identified site Patients • 2-3 one-on-one semistructured interviews per site • Patients who identify as women of low socioeconomic status • Impact, experience, and outcomes of participating in the screening process Health care providers • 7-14 semistructured interviews • Nurses, physicians, and/or ophthalmologists involved in the screening process • Impact and experience of coordination, and delivery of the program Administrators/decision makers • Up to 14 semistructured interviews • Health care administrators/decision makers • Values and beliefs that inform the screening program • Description of the coordination, delivery, funding, and policies that inform the screening program • Description of the coordination, delivery, funding, and policies that inform the screening program • Description of the coordination, delivery, funding, and policies that inform the screening program will facilitate self-reflection on how the analysis evolved to allow the qualitative lead to fully interrogate potential assumptions or biases reflected in the interpretation of the data [37]. Reliability of the findings will be strengthened by maintaining a chain of evidence throughout the study to ensure that the evolution of qualitative results can be followed by an external observer in order to ensure credibility of the data collection and analytical process. Data will be stored and managed electronically using the qualitative research software NVivo 11 (QSR International). Qualitative Data Analysis Data analysis will be an iterative and inductive process using a grounded theory approach [37]. This will involve systematic coding of data and theme abstraction to identify key facilitators and barriers to tele-retina intervention across the sites to ensure that our findings will inform recommendations appropriate to their context. Thematic analysis of the interview transcripts, interview notes, and observation notes will occur in the following three stages: open coding (data reduction), axial coding (data display), and selective coding (conclusion drawing). Comparisons within and across the interview data will be conducted (constant comparison technique) [38]. Multiple readings will be used, and alternative explanations of the data will be explored [39,40] to develop the most plausible and robust interpretation of the findings in order to obtain a comprehensive understanding of the facilitators and barriers to diabetic retinopathy screening [41,42]. All final themes will be informed by continuous dialogue among the research team. This dialogue Exclusion Criteria We will collect and review relevant documentary sources on the operation of the tele-retina program, as processes within organizations are frequently text based and may serve as a substitute for records of activity [35]. Documents (eg, scientific papers, conference reports, organizational histories, press releases, and news stories) may provide access to an accepted body of knowledge about the role, policy, and procedures of an organization [36]. Documents will be collected by searching for publicly available documents (eg, through organizational websites, citations, and database searches). Patients who are aged less than 18 years; do not identify as women of low SES (income less than CAD $25,000); and are unable or unwilling to provide verbal and/or written informed consent are excluded. Health care providers who are not practicing health care providers in the tele-retina screening for diabetic retinopathy program and are unable or unwilling to provide verbal informed consent are excluded. Administrators and/or decision makers who are unable or unwilling to provide verbal informed consent are excluded. JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 7 (page number not for citation purposes) https://www.researchprotocols.org/2021/3/e23492 https://www.researchprotocols.org/2021/3/e23492 JMIR RESEARCH PROTOCOLS Stanimirovic et al Textbox 1. Description of ethnographic field work and semistructured interviews per identified site. Ethnographic fieldwork • Process and interaction between the patient and health care provider during the tele-retina screening process for diabetic retinopathy (7-14 hours in total) Semistructured interviews per identified site Patients • 2-3 one-on-one semistructured interviews per site • Patients who identify as women of low socioeconomic status • Impact, experience, and outcomes of participating in the screening process Health care providers • 7-14 semistructured interviews • Nurses, physicians, and/or ophthalmologists involved in the screening process • Impact and experience of coordination, and delivery of the program Administrators/decision makers • Up to 14 semistructured interviews • Health care administrators/decision makers • Values and beliefs that inform the screening program • Description of the coordination, delivery, funding, and policies that inform the screening program Textbox 1. Description of ethnographic field work and semistructured interviews per identified site. https://www.researchprotocols.org/2021/3/e23492 JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 8 (page number not for citation purposes) Results For the quantitative study, we anticipate a cohort of 2500 patients, which will provide descriptive information on patterns of use of the tele-retina program. In total, we plan to interview 14 to 21 patients and 7 to 14 providers, administrators, and decision makers, and to conduct 7 to 14 hours of observations in order to gain an understanding of the facilitators and barriers to diabetic retinopathy screening. Please note that the interview guide is being developed in collaboration with our patient partners. The study is under review by the University Health Network Research Ethics Board (19-5628). We responded to JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 8 (page number not for citation purposes) JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 8 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS Stanimirovic et al aim to improve access to care, but may not be well adapted to vulnerable populations [47]. Understanding the facilitators and barriers of screening in this population will address the knowledge gap and assist in developing, implementing, and adopting effective, yet culturally sensitive, diabetic retinopathy screening interventions and thus carries promise in reducing the burden of blindness resulting from diabetic retinopathy. The findings should generate a deeper understanding of the ways in which system-level organizational interventions may improve access to screening for vulnerable populations and new knowledge with regard to improvements in the delivery of diabetic retinopathy screening interventions. Considering the widespread burden of diabetic retinopathy across the globe, the findings will be disseminated to ensure that strategies for the prevention and treatment of diabetic retinopathy are sensitive to vulnerable populations and can be implemented and adopted at the global level. the first set of Research Ethics Board comments and are anticipating Research Ethics Board response. We expect to begin recruitment in winter 2021, as the tele-retina program has recently resumed at SRCHC. This work is supported by Patient-Oriented Research Intercentre Trainee Internship in Diabetes and its Complications through Diabetes Action Canada–Canadian Institutes of Health Research funds and an unrestricted educational grant from Novartis Canada. The funding sources have no involvement in the study design; the collection, analysis, or interpretation of data; the writing of the manuscript; or the decision to submit the article for publication. Discussion This protocol outlines a study designed to understand the critical facilitators and barriers of the delivery of diabetic retinopathy screening among vulnerable communities. With the increasing incidence and prevalence of diabetes worldwide, morbidity, mortality, and associated costs due to diabetes-related complications remain a growing public health concern [44]. Diabetic retinopathy represents a global epidemic, as 191 million individuals worldwide will be diagnosed by 2030 [44], and the disease burden remains concentrated among low-income groups [45,46]. Emerging evidence illustrates that often interventions Considering that previous work has found tele-retina screening to be a more cost-effective alternative to standard care and that there is an increasing global burden of diabetic retinopathy, there is a need for improved access to care for vulnerable populations. National and international scaling and adoption of the tele-retina program to assist vulnerable populations may contribute to system-level cost saving. Conflicts of Interest None declared. https://www.researchprotocols.org/2021/3/e23492 References 1. Vujosevic S, Midena E. Diabetic Retinopathy in Italy: Epidemiology Data and Telemedicine Screening Programs. J Diabetes Res 2016;2016:3627465-3627466 [FREE Full text] [doi: 10.1155/2016/3627465] [Medline: 27990441] 1. 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Milbank Q 2011 Jun;89(2):256-288 [FREE Full text] [doi: 10.1111/j.1468-0009.2011.00628.x] [Medline: 21676023] Abbreviations CNIB: Canadian National Institute for the Blind SES: socioeconomic status SRCHC: South Riverdale Community Health Centre ©Aleksandra Stanimirovic, Troy Francis, Anna Cooper Reed, Sonia Meerai, Olivera Sutakovic, Rebecca Merritt, Michael Brent, Valeria Rac. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 05.03.2021. 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 JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on http://www.researchprotocols.org, as well as this copyright and license information must be included. ©Aleksandra Stanimirovic, Troy Francis, Anna Cooper Reed, Sonia Meerai, Olivera Sutakovic, Rebecca Merritt, Michael Brent, Valeria Rac. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 05.03.2021. 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 JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on http://www.researchprotocols.org, as well as this copyright and license information must be included. https://www.researchprotocols.org/2021/3/e23492 JMIR Res Protoc 2021 \| vol. 10 \| iss. 3 \| e23492 \| p. 11 (page number not for citation purposes) https://www.researchprotocols.org/2021/3/e23492
https://openalex.org/W4322720196	https://www.repository.cam.ac.uk/bitstream/1810/346975/2/41467_2023_36496_MOESM1_ESM.pdf	English	null	Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis	Nature communications	2,023	cc-by	5,775	Supplementary figure 1: Characterization of BS4 uptake. (a) Quenching of extracellular dylight-650 BS4 antibody by TCEP. Labelled BS4 was bound to SkBr3 cell surface on ice and after fixation stained with anti-human Alexa-488. Cells were imaged  TCEP in the mounting medium. (b) Endocytosis of pHrodo-labelled monotopic antibodies Tz and 39S and biparatopic BS4 in the absence and presence of serum (FCS) was quantified using an Incucyte live cell imager. Data are presented as means SEM, n= 2 independent experiments (c) Gating strategy to quantify transferrin and BS4 uptake in transfected SkBr3 cells. Cell were transfected with dominant negative AP180ct N-terminally GFP tagged expression plasmid were incubated with dylight650 labelled BS4 and AlexaFluor546-transferrin for 15 min and analysed by flow-cytometry as follows: Initial gating was area vs height to eliminate doublets and gating on forward and side scatter to exclude debris. Cells selected by this strategy were further gated on GFP if transfected, providing intrinsic un-transfected controls for each cell population. Transferrin and BS4 uptake was measured in the red and far-red channel, respectively both in un-transfected (GFP-) and transfected (GFP+) cells. (d) SkBr3 cells transfected with control (GFP only) plasmid were incubated with dylight650 labelled BS4 and AlexaFluor546-transferrin for indicated times over a period of 4 h and analysed by flow- cytometry. Endocytosis of transferrin and BS4 in cells from the same well expressing or not GFP (GFP+, GFP-), (means  SD, n=4 independent experiments). (e) SkBr3 cells transfected with control (GFP only) and dominant negative AP180ct and DynaminS45N N-terminally GFP tagged expression constructs were incubated with dylight650 labelled BS4 and AlexaFluor546-transferrin for 30 min and analysed by confocal microscopy. Transfected cells (GFP channel) are outlined in the images displaying BS4 and transferrin. (f) Endocytosis of Dylight488 labelled BS4in SkBr3 cells in the presence of 80 µM dynasore, 100 µM 5‐(N‐ethyl‐ N‐isopropyl)amiloride (EIPA), 100 µM LY294002, 10 µM ML141 and 0.5 µM CytochalasinD Supplementary figure 1: Characterization of BS4 uptake. (a) Quenching of extracellular dylight-650 BS4 antibody by TCEP. Labelled BS4 was bound to SkBr3 cell surface on ice and after fixation stained with anti-human Alexa-488. Cells were imaged  TCEP in the mounting medium. (b) Endocytosis of pHrodo-labelled monotopic antibodies Tz and 39S and biparatopic BS4 in the absence and presence of serum (FCS) was quantified using an Incucyte live cell imager. Data are presented as means SEM, n= 2 independent experiments (c) Gating strategy to quantify transferrin and BS4 uptake in transfected SkBr3 cells. Cell were transfected with dominant negative AP180ct N-terminally GFP tagged expression plasmid were incubated with dylight650 labelled BS4 and AlexaFluor546-transferrin for 15 min and analysed by flow-cytometry as follows: Initial gating was area vs height to eliminate doublets and gating on forward and side scatter to exclude debris. Cells selected by this strategy were further gated on GFP if transfected, providing intrinsic un-transfected controls for each cell population. Transferrin and BS4 uptake was measured in the red and far-red channel, respectively both in un-transfected (GFP-) and transfected (GFP+) cells. (d) SkBr3 cells transfected with control (GFP only) plasmid were incubated with dylight650 labelled BS4 and AlexaFluor546-transferrin for indicated times over a period of 4 h and analysed by flow- cytometry. Endocytosis of transferrin and BS4 in cells from the same well expressing or not GFP (GFP+, GFP-), (means  SD, n=4 independent experiments). (e) SkBr3 cells transfected with control (GFP only) and dominant negative AP180ct and DynaminS45N N-terminally GFP tagged expression constructs were incubated with dylight650 labelled BS4 and AlexaFluor546-transferrin for 30 min and analysed by confocal microscopy. Transfected cells (GFP channel) are outlined in the images displaying BS4 and transferrin. (f) Endocytosis of (CytoD) for 1h. After fixation surface BS4 was counterstained with anti-human Alexa568 antibody and imaged by confocal microscopy. Representative images for each condition are shown. Scale bars: 10 µm. Source data are provided as a Source Data file. Supplementary figure 2: Inhibition of BS4 uptake by chemical inhibitors. Endocytosis of BS4 (a) and transferrin (b) in presence of increasing concentrations of 5‐(N‐ethyl‐N‐ isopropyl)amiloride (EIPA). After 30 min of uptake SkBr3 cells were processed and analysed by flow-cytometry (means  SD, n=3 independent experiments, ns (non-significant) p>0.05, * p<0.05,** p<0.0001, one-way ANOVA with Dunnett’s multiple comparison test). (c) Confocal microscopy images of BS4 (red) and transferrin (green) uptake into Hela cells ectopically expressing full-length HER2 treated with increasing concentrations of EIPA. After fixation surface bound BS4-dylight 650 was counterstained using Alexa568 conjugated anti- human IgG. Surface subtracted images highlighting endocytosed BS4 are shown on the right of each corresponding image. Results of BS4 and Tf uptake in presence of increasing concentrations of EIPA are quantified in (d). Snapshots of BS4-dylight650 endocytosis in SkBr3 cells at indicated times after antibody addition (see also movie 1). Arrowheads indicate plasma membrane proximal vesicles containing BS4. Dashed boxes show vesicles at higher magnification. (b) BS4 preferentially endocytoses in regions exhibiting high plasma membrane motility. GFP-GPI expressing cells were imaged during BS4 uptake. Maximum intensity time projections of the GFP-GPI channel over the imaging period are displayed, with time frames color-coded as indicated. Arrowheads highlight highly motile regions of the plasma membrane, which coincide with areas of BS4 endocytosis. (c) Accumulation of Rac1 at regions of the plasma membrane harbouring BS4 aggregates (see Fig. 2e). SKBR3 cells before and after 2.5 min incubation with BS4 were immune-stained for endogenous Rac1 protein and imaged by confocal microscopy. (d) Plasma membrane lamellipodia movement is blocked by Rac1 inhibition. GFP-GPI expressing cells were imaged prior to and after incubation with Rac1 inhibitor EHT1864 (see also movie 5). Maximum intensity time projections over the imaging period are displayed, with time frames color- coded as indicated. (e) Rac1 inhibition blocks BS4 endocytosis. Dylight labelled BS4 was added to cells treated with EHT1864 and uptake was allowed for 20 min. Surface bound antibody was quenched. (f) Quantification of the effect of EHT1864 on BS4 uptake (means  SD, n=10 cells from 3 independent experiments; p<0.0001; two-tailed unpaired Student’s t-test). (g) 3D views of the BS4 channel only corresponding to frames from Figure 2 c are displayed. Dashed circles indicate the position of the macropinocytic cup. Scale bars 5 µm (a,f) insets in (a) 1 µm, 10 µm (b,c, d,e). Source data are provided as a Source Data file. Supplementary figure 3: Rac1-dependent plasma membrane rearrangements during BS4 uptake resemble macro-endocytosis. (a) BS4 marks large endocytic vesicles. Snapshots of BS4-dylight650 endocytosis in SkBr3 cells at indicated times after antibody addition (see also movie 1). Arrowheads indicate plasma membrane proximal vesicles containing BS4. Dashed boxes show vesicles at higher magnification. (b) BS4 preferentially endocytoses in regions exhibiting high plasma membrane motility. GFP-GPI expressing cells were imaged during BS4 uptake. Maximum intensity time projections of the GFP-GPI channel over the imaging period are displayed, with time frames color-coded as indicated. Arrowheads highlight highly motile regions of the plasma membrane, which coincide with areas of BS4 endocytosis. (c) Accumulation of Rac1 at regions of the plasma membrane harbouring BS4 aggregates (see Fig. 2e). Each dot represents the measurement from an individual cell, red lines indicate medians (n>50, ns (non-significant),** p<0.0001; two-way ANOVA with Dunnett’s multiple comparison test). Supplementary figure 2: Inhibition of BS4 uptake by chemical inhibitors. Endocytosis of BS4 (a) and transferrin (b) in presence of increasing concentrations of 5‐(N‐ethyl‐N‐ isopropyl)amiloride (EIPA). After 30 min of uptake SkBr3 cells were processed and analysed by flow-cytometry (means  SD, n=3 independent experiments, ns (non-significant) p>0.05, * p<0.05,** p<0.0001, one-way ANOVA with Dunnett’s multiple comparison test). (c) Confocal microscopy images of BS4 (red) and transferrin (green) uptake into Hela cells ectopically expressing full-length HER2 treated with increasing concentrations of EIPA. After fixation surface bound BS4-dylight 650 was counterstained using Alexa568 conjugated anti- human IgG. Surface subtracted images highlighting endocytosed BS4 are shown on the right of each corresponding image. Results of BS4 and Tf uptake in presence of increasing concentrations of EIPA are quantified in (d). Each dot represents the measurement from an individual cell, red lines indicate medians (n>50, ns (non-significant), p<0.0001; two-way ANOVA with Dunnett’s multiple comparison test). Endocytosis of BS4 (e) and transferrin (f) in SkBr3 cells expressing the dominant-negative dynamin 1 S45N mutant or treated with 10 µM CytoD. After indicated times cells were processed and analysed by flow-cytometry (means  SD, n=3 independent experiments). (g) Confocal microscopy images of BS4 and transferrin uptake into cells treated with 10 µM CytoD. After fixation surface bound BS4-dylight 650 was quenched and counterstained using Alexa488 conjugated anti-human IgG. (h) Cells treated with increasing concentrations of CytochalasinD (CytoD) were incubated with pHrodo-labelled BS4 and uptake was quantified using an Incucyte live cell imager (means  SD, n=3 independent experiments). (i) Uptake of BS4 in SkBr3 cells treated with 10 µM CytoD or 300 µM CK666. After 30 min of uptake SkBr3 cells were processed and analysed by flow-cytometry (means  SD, n=3 independent experiments , p<0.0001, one-way ANOVA with Dunnett’s multiple comparison test). (j) Cells endocytosing pHrodo-labelled BS4 were treated with 300 µM CK666, which was subsequently washed out as indicated, (means  SEM, n=2 independent experiments). Scale bars: 10 µm. Source data are provided as a Source Data file. Scale bars: 10 µm. Source data are provided as a Source Data file. Supplementary figure 3: Rac1-dependent plasma membrane rearrangements during BS4 uptake resemble macro-endocytosis. (a) BS4 marks large endocytic vesicles. SKBR3 cells before and after 2.5 min incubation with BS4 were immune-stained for endogenous Rac1 protein and imaged by confocal microscopy. (d) Plasma membrane lamellipodia movement is blocked by Rac1 inhibition. GFP-GPI expressing cells were imaged prior to and after incubation with Rac1 inhibitor EHT1864 (see also movie 5). Maximum intensity time projections over the imaging period are displayed, with time frames color- coded as indicated. (e) Rac1 inhibition blocks BS4 endocytosis. Dylight labelled BS4 was added to cells treated with EHT1864 and uptake was allowed for 20 min. Surface bound antibody was quenched. (f) Quantification of the effect of EHT1864 on BS4 uptake (means  SD, n=10 cells from 3 independent experiments; p<0.0001; two-tailed unpaired Student’s t-test). (g) 3D views of the BS4 channel only corresponding to frames from Figure 2 c are displayed. Dashed circles indicate the position of the macropinocytic cup. Scale bars 5 µm (a,f) insets in (a) 1 µm, 10 µm (b,c, d,e). Source data are provided as a Source Scale bars 5 µm (a,f) insets in (a) 1 µm, 10 µm (b,c, d,e). Source data are provided as a Source Data file. Supplementary figure 4: BS4-induced receptor aggregation triggers HER2 endocytosis (a) Time course of HER2 receptors aggregation by BS4. SkBr3 cells were incubated with BS4 for indicated times and samples lysed in low detergent buffer. Samples were spun to separate soluble (Sup) from insoluble/aggregated proteins (Pelletand assayed by immunoblot for the HER2 protein. (b) Time dependence of BS4-triggered aggregation and concomitant loss in the soluble fraction of HER2 receptor is quantified (means  SD, n=3 independent experiments). (c) Surface binding and internalisation of dylight650-labelled antibodies Trastuzumab (Tz), 39S and BS4 in HER2 expressing CHO cells analysed by microscopy. CHO cells expressing either wt or TZ binding mutant (Tzmut) of full-length (FL) HER2 were incubated with dylighyt650-BS4 for 30 min on ice or at 37 °C. Cells were fixed, surface bound antibody stained with anti-human Alexa488 and imaged by confocal microscopy. (d) Cells were incubated with equal amounts of indicated antibodies, with or without a cross-linking anti-human Alexa488 antibody (2nd) for 1h. After fixation surface bound, dylight650 labelled antibody was quenched and cell sections analysed by confocal microscopy. All control conditions related to Fig 3e are shown. (e) Cross-linking of both monotopic antibodies phenocopies the effect of BS4 on HER2 aggregation and endocytosis. Cells were incubated for 10 min with equal amounts of indicated antibodies, with or without a cross- linking anti-human antibody (2nd) and  CytochalasinD (CytoD). After cell surface biotinylation a fraction of sample was spun to pellet insoluble/aggregated proteins (Pellet). Protein in the remaining sample was solubilised (see materials and methods for protocol) and surface remaining biotinylated proteins concentrated on Streptavidin beads (surface). Samples were analysed by immunoblot for HER2. (f) Abundance of HER2 protein is a prerequisite for BS4-mediated clustering and endocytosis. Indicated cell lines were surface materials and methods section. A 100 fold longer exposure of HER2 blots for MCF7 cells is shown on the right. Scale bars: 10 µm. Source data are provided as a Source Data file. Scale bars: 10 µm. Source data are provided as a Source Data file. Supplementary figure 5: Aggregation-dependent endocytosis re-routes transferrin receptor. (a) Aggregation of transferrin receptor ecto-domain (TfR) by antibody-mediated cross-linking in vitro. TfR was mixed with anti-transferrin receptor antibody 289  secondary antibody (2nd). Samples were spun to separate soluble (S) from insoluble/aggregated protein (P). Total protein coomassie stain is shown, the arrow indicates the MW of the TfR band that moved to the pellet. (b, c) Endocytosis of cross-linked transferrin receptor is independent of clathrin and dynamin, but transferrin remains dependent. SkBr3 cells transfected with control (GFP only) and dominant negative AP180ct and DynaminS45N N-terminally GFP tagged expression constructs were incubated with a panel of anti-TfR antibodies  secondary antibody (2nd) and AlexaFluor546-transferrin for 30 min and analysed by flow-cytometry. Results for transferrin (b) and anti-TfR antibodies (c) endocytosis (means  SD, n=3 independent experiments, ns non-specific p>0.05, , p=0.0014,** p<0.0001 two-way ANOVA with Tukey’s multiple comparison test.). (d) Endocytosis of cross-linked transferrin receptor is independent of clathrin and dynamin. SkBr3 cells transfected with control (GFP only) and dominant negative AP180ct N-terminally GFP tagged expression constructs were incubated with dylight650 labelled anti-TfR antibody  secondary antibody (2nd) for 30 min and analysed confocal microscopy. (e) Cross-linked transferrin-receptor endocytosis is inhibited by 5‐(N‐ethyl‐N‐isopropyl)amiloride (EIPA). Hela cells were treated with 50 µM EIPA for 30 min, followed by incubation with dylight650 labelled anti-transferrin receptor antibody  secondary antibody (2nd) for 30 min. After fixation surface bound antibody was counterstained and samples analysed by confocal microscopy. Subtraction of surface from total antibody signal yielded the endocytosed pool, which is shown in the bottom panels. (f) Cross-linked transferrin-receptor endocytosis requires Rac1. Hela cells were transfected with a vector expressing a dominant-negative mutant (T17N) of Rac1 for 16h incubated with antibodies and analysed by confocal microscopy as described in (e). Transfected cells in the bottom panels showing the endocytosed anti-TfR antibody pool are outlined. Scale bars: 10 µm. Source data are provided as a Source Data file. Scale bars: 10 µm. Source data are provided as a Source Data file. Supplementary figure 6: Aggregation-dependent endocytosis is induced by cross-linking molecules. (a)Uptake dynamics of wheat-germ-agglutinin (WGA) resemble BS4 endocytosis. Dylight650 labelled WGA or biparatopic antibody BS4 was added onto SkBr3 cells, and imaging started immediately after (see also movie 8). Maximum intensity time projections over the imaging period are displayed, with time frames color-coded as indicated. (b) WGA specifically aggregates glycosylated proteins in vitro. WGA was mixed with recombinant glycosylated and non-glycosylated (control) protein as indicated. Samples were spun to separate soluble (S) from insoluble/aggregated protein (P). Total protein coomassie stain is shown. (c) WGA uptake is mediated by both clathrin-dependent and actin-dependent endocytic pathways. Cells transfected with dominant negative AP180ct N-terminally GFP tagged expression construct were incubated  CytochalasinD (CytoD) with dylight650 labelled WGA and analysed by confocal microscopy. (d) Endocytosis of TfR by cross-linked transferrin is independent of clathrin and dynamin. SkBr3 cells transfected with control (GFP only) and dominant negative AP180ct and DynaminS45N N-terminally GFP tagged expression constructs were incubated with dylight650 labelled, biotinylated transferrin  streptavidin (strep) for 30 min and analysed confocal microscopy Supplementary figure 6: Aggregation-dependent endocytosis is induced by cross-linking molecules. (a)Uptake dynamics of wheat-germ-agglutinin (WGA) resemble BS4 endocytosis. Dylight650 labelled WGA or biparatopic antibody BS4 was added onto SkBr3 cells, and imaging started immediately after (see also movie 8). Maximum intensity time projections over the imaging period are displayed, with time frames color-coded as indicated. (b) WGA specifically aggregates glycosylated proteins in vitro. WGA was mixed with recombinant glycosylated and non-glycosylated (control) protein as indicated. Samples were spun to separate soluble (S) from insoluble/aggregated protein (P). Total protein coomassie stain is shown. (c) WGA uptake is mediated by both clathrin-dependent and actin-dependent endocytic pathways. Cells transfected with dominant negative AP180ct N-terminally GFP tagged expression construct were incubated  CytochalasinD (CytoD) with dylight650 labelled WGA and analysed by confocal microscopy. (d) Endocytosis of TfR by cross-linked transferrin is independent of clathrin and dynamin. SkBr3 cells transfected with control (GFP only) and dominant negative AP180ct and DynaminS45N N-terminally GFP tagged expression constructs were incubated with dylight650 labelled, biotinylated transferrin  streptavidin (strep) for 30 min and analysed confocal microscopy Scale bars: 10 µm. Source data are provided as a Source Data file. -=:I' V'I C0 C E 0 .... HER2 HER3 EGFR Na/K-ATPase TfR Sup Figure 7 Supplementary figure 7: BS4 clusters HER2 but no other receptors in the plasma membrane resulting in HER2-specific endocytosis. SkBr3 cells were incubated with HER2-specific, dylight650 labelled biparatopic antibody BS4 (red) for indicated times. After fixation, total receptor tyrosine-protein kinase ErbB-2 (HER2), receptor tyrosine-protein kinase ErbB-3 (HER3), epidermal growth factor receptor (EGFR), sodium/potassium-transporting ATPase Alpha1 (Na/K-ATPase) and Transferrin receptor 1 (TfR) in cell sections were stained (green) and samples were analysed by confocal microscopy. Scale bars: 10 µm. Supplementary figure 8: BS4-triggered ADE of HER2 requires autophosphorylation in the C- terminal domain. BS4 induces Her2 auto-phosphorylation (a,b). SkBr3 cells  dual EGFR/HER2 kinase inhibitor lapatinib (HER1/2i) were incubated with BS4 for 2.5 min as indicated. Samples were analysed by immunoblot for phospho-tyrosine residues (p-Y) and phospho-HER2 (pHER2 (a) related to Fig. 6a. SkBr3 cells  dual EGFR/HER2 kinase inhibitor lapatinib (HER1/2i) were incubated with BS4-dylight650 for 2.5 min, after fixation stained for phosphorylated tyrosine residues (p-Y) and analysed by confocal microscopy (b) related to Fig. 6b. BS4 uptake in cells expressing full-length vs. kinase deficient HER2 (c-f). U2OS cells ectopically expressing full- length (FL) HER2 or lacking the C-terminal cytoplasmic domain (HER2 CT) were incubated with BS4-dylight650 for indicated times. After fixation surface bound BS4 was counterstained and samples analysed by confocal microscopy. Subtraction of surface from total antibody signal represented the endocytosed pool (c). Results are quantified in (d). Dots represent measurements from individual cells, red lines indicate the median; n≥50 cells from 3 independent experiments. (e) U2OS cells ectopically expressing full-length (FL) HER2 or lacking the C-terminal cytoplasmic domain (HER2 CT) were incubated with TZ-or BS4- dylight650 (red) for 30 min. After fixation surface bound antibodies were counterstained (green) and samples analysed by confocal microscopy. (f) U2OS cells ectopically expressing full-length (FL) HER2 or lacking the C-terminal cytoplasmic domain (HER2 CT) were incubated with CytoD and TZ-or BS4-dylight650 for 30 min as indicated. Samples were processed and anlaysed as described in (c). A quantification of antibody uptake is shown in (f). Dots represent measurements from individual cells, red lines indicate the median; n≥50 cells from 3 independent experiments. Supplementary figure 8: BS4-triggered ADE of HER2 requires autophosphorylation in the C- terminal domain. BS4 induces Her2 auto-phosphorylation (a,b). SkBr3 cells  dual EGFR/HER2 kinase inhibitor lapatinib (HER1/2i) were incubated with BS4 for 2.5 min as indicated. Samples were analysed by immunoblot for phospho-tyrosine residues (p-Y) and phospho-HER2 (pHER2 (a) related to Fig. 6a. SkBr3 cells  dual EGFR/HER2 kinase inhibitor lapatinib (HER1/2i) were incubated with BS4-dylight650 for 2.5 min, after fixation stained for phosphorylated tyrosine residues (p-Y) and analysed by confocal microscopy (b) related to Fig. 6b. BS4 uptake in cells expressing full-length vs. kinase deficient HER2 (c-f). U2OS cells ectopically expressing full- length (FL) HER2 or lacking the C-terminal cytoplasmic domain (HER2 CT) were incubated with BS4-dylight650 for indicated times. Scale bars: 10 µm. Source data are provided as a Source Data file. After fixation surface bound BS4 was counterstained and samples analysed by confocal microscopy. Subtraction of surface from total antibody signal represented the endocytosed pool (c). Results are quantified in (d). Dots represent measurements from individual cells, red lines indicate the median; n≥50 cells from 3 independent experiments. (e) U2OS cells ectopically expressing full-length (FL) HER2 or lacking the C-terminal cytoplasmic domain (HER2 CT) were incubated with TZ-or BS4- dylight650 (red) for 30 min. After fixation surface bound antibodies were counterstained (green) and samples analysed by confocal microscopy. (f) U2OS cells ectopically expressing full-length (FL) HER2 or lacking the C-terminal cytoplasmic domain (HER2 CT) were incubated with CytoD and TZ-or BS4-dylight650 for 30 min as indicated. Samples were processed and anlaysed as described in (c). A quantification of antibody uptake is shown in (f). Dots represent measurements from individual cells, red lines indicate the median; n≥50 cells from 3 independent experiments. S l b 10 S d t id d S D t fil Scale bars: 10 µm. Source data are provided as a Source Data file. Scale bars: 10 µm. Source data are provided as a Source Data file. Supplementary figure 9: PI3K and VAV dependence of BS4 uptake and Ras-induced macropinocytosis. Localisation of PIP3 to BS4-induced HER2 aggregates (a) related to Fig. 7c. SkBr3 cells were transfected with a vector expressing the PIP3 sensor PH-AKT-GFP (green) for 16h treated or not with the PI3K inhibitor LY294002 (PI3Ki) followed by incubation with BS4- dylight650 (red) for 2.5 min. After fixation samples were analysed by confocal microscopy. PI3K and Rac dependence of Ras-induced dextran uptake (b). SkBr3 cells stably expressing constitutively active mutant Ras (G12V) were transfected with Rac1 dominant negative mutant (T17N) or dominant-negative VAV2 comprising only the interaction domains (amino acids 546-878) C-terminally GFP tagged expression vectors for 16 h or treated with LY294002 (PI3Ki) as indicated and incubated with dextran(70kDa)-TMR for 30 min. After fixation samples were analysed by confocal microscopy and dextran uptake was quantified. Dots represent measurements from individual cells, red lines indicate the median; n≥25 cells from 3 independent experiments; ns (non-significant) p>0.05, p<0.0001; one-way ANOVA with Sidak’s multiple comparison test. Expression of VAV1-3 in SkBr3 cells (c, d). Normalized transcript expression values (nTPM) retrieved from ProteinAtlas.org for VAV1-3 in SKBR3 cells are displayed (c). SkBr3 cells were immunoblotted for VAV1-3 and HER2 and actin as controls (d). BS4-induced interaction of HER2 with VAV2 is phospho-tyrosine dependent (e). SkBr3 cells were transfected with VAV2 C-terminally GFP tagged expression vector (green) for 16h treated with lapatinib (HER1/2i) and incubated with BS4-dylight650 (red) for 2.5 min as indicated. VAV2 wt and SH2 (lacking residues 665-772) steady-state protein levels (f) related to Fig. 8b. SkBr3 cells were transfected with VAV2 wt or a lacking residues 665-772 (SH2) C- terminally GFP tagged expression vector for 16h followed by immunoblot analysis for GFP and actin as a loading control. Scale bars: 10 µm. Source data are provided as a Source Data file. Supplementary figure 10: BS4-induced ADE of HER2 requires VAV proteins. Characterisation of VAV1-3 KO cells (a-d). Wt as well as VAV1-3 KO SkBr3 (a) and U2OS_HER2 (c) cells were immunoblotted for VAV2, HER2 and actin. VAV1-3 KO cell clones in SkBr3 (b) and U2OS_HER2 (d) backgrounds were analysed by sequencing of genomic loci targeted by gRNAs. Sanger sequencing files were analyses using the Inference of CRISPR Edits (ICE) tool from ice.synthego.com. VAV proteins are required for BS4 uptake in U2OS cells ectopically expressing HER2 (e, f). Scale bars: 10 µm. Source data are provided as a Source Data file. U2OS_HER2 wt or VAV1-3 knock-out (KO) cells were incubated with BS4-dylight650 (red) for 30 min. After fixation, surface bound BS4 antibody was counterstained (green) and samples were analysed by confocal microscopy (e). Subtraction of surface from total BS4 signal shows endocytosed pool (BS4 uptake). Results are quantified in (f). Dots represent measurements from individual cells, red lines indicate the median; n≥50 cells from 3 independent experiments, p<0.0001; one-way ANOVA with Dunnett’s multiple comparison test. VAV 1-3 steady-state protein levels (g) related to Fig. 6n. SkBr3 wt or VAV1-3KO cells were transfected with C-terminally HA-tagged VAV1-3 expression vectors for 16 h followed by immunoblot analysis for HA, VAV2 and actin as a loading control. VAV2 wt and SH2 (lacking residues 665-772) steady-state protein levels (h) related to Fig. 6o. SkBr3 wt and VAV1-3KO cells were transfected with VAV2 wt or a lacking residues 665-772 (SH2) C-terminally GFP tagged expression vectors for 16h followed by immunoblot analysis for VAV2 and actin as a loading control. Scale bars: 10 µm. Source data are provided as a Source Data file. Supplementary figure 10: BS4-induced ADE of HER2 requires VAV proteins. Characterisation of VAV1-3 KO cells (a-d). Wt as well as VAV1-3 KO SkBr3 (a) and U2OS_HER2 (c) cells were immunoblotted for VAV2, HER2 and actin. VAV1-3 KO cell clones in SkBr3 (b) and U2OS_HER2 (d) backgrounds were analysed by sequencing of genomic loci targeted by gRNAs. Sanger sequencing files were analyses using the Inference of CRISPR Edits (ICE) tool from ice.synthego.com. VAV proteins are required for BS4 uptake in U2OS cells ectopically expressing HER2 (e, f). U2OS_HER2 wt or VAV1-3 knock-out (KO) cells were incubated with BS4-dylight650 (red) for 30 min. After fixation, surface bound BS4 antibody was counterstained (green) and samples were analysed by confocal microscopy (e). Subtraction of surface from total BS4 signal shows endocytosed pool (BS4 uptake). Results are quantified in (f). Dots represent measurements from individual cells, red lines indicate the median; n≥50 cells from 3 independent experiments, p<0.0001; one-way ANOVA with Dunnett’s multiple comparison test. VAV 1-3 steady-state protein levels (g) related to Fig. 6n. SkBr3 wt or VAV1-3KO cells were transfected with C-terminally HA-tagged VAV1-3 expression vectors for 16 h followed by immunoblot analysis for HA, VAV2 and actin as a loading control. VAV2 wt and SH2 (lacking residues 665-772) steady-state protein levels (h) related to Fig. 6o. SkBr3 wt and VAV1-3KO cells were transfected with VAV2 wt or a lacking residues 665-772 (SH2) C-terminally GFP tagged expression vectors for 16h followed by immunoblot analysis for VAV2 and actin as a loading control. Scale bars: 10 µm. Source data are provided as a Source Data file. Scale bars: 10 µm. Source data are provided as a Source Data file. Supplementary figure 11: Stress-induced cell surface receptor aggregates are endocytosed by ADE. (a) Titration of pH dependence of HER2 aggregation and endocytosis. After surface biotinylation, SkBr3 cells  CytochalasinD (CytoD) were washed in buffers of different pH. After incubation of all samples at 37°C for 30 min, surface remaining biotin was removed and samples lysed in low detergent buffer. A fraction was spun to precipitate insoluble/aggregated proteins (Pellet). Protein in the remaining sample was solubilised (see materials and methods for protocol) and endocytosed biotinylated proteins concentrated on Streptavidin beads (uptake). Samples were assayed by immunoblot for HER2. A representative western blot is shown along with the quantification of band intensities, means SD are displayed, n= 3 independent experiments . (b) Temperature dependence of HER2 aggregation and endocytosis. After surface biotinylation, SkBr3 cells  CytochalasinD (CytoD) were incubated for 5 min at different temperatures. After incubation of all samples at 37°C for 30 min, samples were prepared and analysed as descried in (a). (c) Stress-induced aggregation-dependent endocytosis occurs independent of dynamin but requires actin polymerisation. SkBr3 cells transfected with RFP control, or dominant negative AP180ct N- terminally RFP tagged expression vector (RFP channel displayed in white) were heat shocked for 5 min (at 50 °C)  CytochalasinD (CytoD) or washed in an acidic buffer (pH3.5) followed by 30 min incubation at 37 °C. After fixation surface HER2 was stained (false colour-coded in red) before permeabilization and staining for total HER2 (green channel). Cell sections were analysed by confocal microscopy and post-processed by subtraction of surface from total HER2 staining, which allowed for specific visualisation of intracellular HER2. (d) Co-localisation of HER2 and TfR in endocytic carriers after stress-induced ADE. SkBr3 cells were treated as described in (c) and stained for TfR (red) and HER2 (green). Right hand panels show intracellular signal after subtraction of cell surface signal (white) from green and red channels. Supplementary figure 11: Stress-induced cell surface receptor aggregates are endocytosed by ADE. (a) Titration of pH dependence of HER2 aggregation and endocytosis. After surface biotinylation, SkBr3 cells  CytochalasinD (CytoD) were washed in buffers of different pH. After incubation of all samples at 37°C for 30 min, surface remaining biotin was removed and samples lysed in low detergent buffer. A fraction was spun to precipitate insoluble/aggregated proteins (Pellet). Protein in the remaining sample was solubilised (see materials and methods for protocol) and endocytosed biotinylated proteins concentrated on Streptavidin beads (uptake). Samples were assayed by immunoblot for HER2. A representative western blot is shown along with the quantification of band intensities, means SD are displayed, n= 3 independent experiments . (b) Temperature dependence of HER2 aggregation and endocytosis. After surface biotinylation, SkBr3 cells  CytochalasinD (CytoD) were incubated for 5 min at different temperatures. After incubation of all samples at 37°C for 30 min, samples were prepared and analysed as descried in (a). (c) Stress-induced aggregation-dependent endocytosis occurs independent of dynamin but requires actin polymerisation. SkBr3 cells transfected with RFP control, or dominant negative AP180ct N- terminally RFP tagged expression vector (RFP channel displayed in white) were heat shocked for 5 min (at 50 °C)  CytochalasinD (CytoD) or washed in an acidic buffer (pH3.5) followed by 30 min incubation at 37 °C. After fixation surface HER2 was stained (false colour-coded in red) before permeabilization and staining for total HER2 (green channel). Cell sections were analysed by confocal microscopy and post-processed by subtraction of surface from total HER2 staining, which allowed for specific visualisation of intracellular HER2. (d) Co-localisation of HER2 and TfR in endocytic carriers after stress-induced ADE. SkBr3 cells were treated as (e) Physical and chemical stress triggers ADE of multiple receptors. Samples were prepared as described in (a) and immunoblotted for HER2, EGFR and TfR. Actin blots are shown as loading controls. Scale bars: 10 µm. Source data are provided as a Source Data file. Scale bars: 10 µm. Source data are provided as a Source Data file. Supplementary figure 12: Dependence of stress-induced ADE on PI3K and VAV proteins. Heat stress-induced fluid-phase uptake partially depends on VAV proteins, HER1/2 tyrosine- phosphorylation and PI3K (a-d). Wt or VAV1-3 KO SkBr3 cells (a,c) or Hela cells (b,d)  CytochalasinD (CytoD) or LY294002 (PI3Ki) or Lapatinib (HER1/2i) were incubated with dextran(70kDa)-fluorescein for 5 min at 50 °C followed by 30 min at 37°C. After fixation samples were analysed by confocal microscopy (a,b). Quantification of heat-stress induced dextran uptake is shown in (c,d). Dots represent measurements from individual cells, red lines indicate the median; n≥50 cells from 3 independent experiments, * p=0.0003, ** p<0.0001; one-way ANOVA with Sidak’s multiple comparison test. Antibody-mediated aggregation-dependent endocytosis of TfR depends on PI3K/Ras but is independent of VAV proteins (e-g). Hela cells  LY294002 (PI3Ki) were incubated with dylight650 labelled anti- transferrin receptor antibody 289 (red)  secondary antibody (2nd) for 30 min. After fixation surface bound antibody was counterstained (green) and samples analysed by confocal microscopy. Subtraction of surface from total antibody signal yielded the endocytosed pool (e). Hela cells were transfected with a plasmid expressing a dominant-negative mutant (S17N) of Ras or with a dominant-negative mutant of VAV2 comprising only the interaction domains (amino acids 546-878) for 16h followed by incubation with dylight650 labelled anti-transferrin receptor antibody 289 (red) (f). Samples were treated and analysed as described in (e). Quantification of anti-TfR antibody  secondary antibody (2nd) uptake is shown in (g). Dots represent measurements from individual cells, red lines indicate the median; n>50 from 3 Supplementary figure 12: Dependence of stress-induced ADE on PI3K and VAV proteins. Heat stress-induced fluid-phase uptake partially depends on VAV proteins, HER1/2 tyrosine- phosphorylation and PI3K (a-d). Wt or VAV1-3 KO SkBr3 cells (a,c) or Hela cells (b,d)  CytochalasinD (CytoD) or LY294002 (PI3Ki) or Lapatinib (HER1/2i) were incubated with dextran(70kDa)-fluorescein for 5 min at 50 °C followed by 30 min at 37°C. After fixation samples were analysed by confocal microscopy (a,b). Quantification of heat-stress induced dextran uptake is shown in (c,d). Dots represent measurements from individual cells, red lines indicate the median; n≥50 cells from 3 independent experiments, * p=0.0003, ** ( ) y samples were analysed by confocal microscopy (a,b). Quantification of heat-stress induced dextran uptake is shown in (c,d). Dots represent measurements from individual cells, red lines indicate the median; n≥50 cells from 3 independent experiments, * p=0.0003, ** p<0.0001; one-way ANOVA with Sidak’s multiple comparison test. Antibody-mediated aggregation-dependent endocytosis of TfR depends on PI3K/Ras but is independent of VAV proteins (e-g). Hela cells  LY294002 (PI3Ki) were incubated with dylight650 labelled anti- transferrin receptor antibody 289 (red)  secondary antibody (2nd) for 30 min. After fixation surface bound antibody was counterstained (green) and samples analysed by confocal microscopy. Subtraction of surface from total antibody signal yielded the endocytosed pool (e). Hela cells were transfected with a plasmid expressing a dominant-negative mutant (S17N) Scale bars: 10 µm. Source data are provided as a Source Data file. Supplementary figure 13: Lysosomal degradation of HER2 receptor aggregates after BS4- and stress triggered ADE. (a) Quantitation of western blot analysis (see Fig 10a) of steady state HER2 protein levels after exposure to monotopic antibodies Tz and 39S and biparatopic BS4 for increasing length of time (means  SD, n=3 independent experiments). (b) HER2 proteolysis is acid-dependent. SkBr3 cell lysates were incubated for 5 h under indicated conditions and blotted for HER2. (c) BS4-induced degradation is HER2-specific and blocked by inhibition of lysosomal acidification. SkBr3 cells were incubated with BS4 and  bafilomycinA1 (Baf) for indicated times and blotted for HER2, the biparatopic Ab heavy chain (BS4) and other cell surface receptors as controls. (d,e) BS4-triggered ADE uptake is not inhibited by bafilomycinA1. SkBr3 cells treated with CytoD or Baf as indicated were incubated with dylight650 labelled BS4 antibody (red) for 30 min and after fixation and surface staining with anti-human 488 (green) analysed by confocal microscopy and post-processed by subtraction of surface from total BS4 staining, which allowed for specific visualisation of intracellular BS4 (d), or flow-cytometry (e) means  SD, n=8 independent experiments, ns (non-significant) p>0.05, ** p<0.0001, one-way ANOVA with Dunnett’s multiple comparison test. (f) SkBr3 cells endocytosing BS4 were fixed after indicated times and stained for lysosomes using a LAMP-1 specific antibody. (g) Quantification of BS4 localisation to lysosomes (non-linear fitted curve, n=8 randomly chosen fields of view with a total of at least 40 cells per condition). (h) Co-localisation of stress-induced endocytic vesicles with LAMP-1. SkBr3 cells treated with Baf were heat shocked for 5 min (at 50 °C) or washed in an acidic buffer (pH3.5) followed by 0.5 or 16 h incubation at 37 °C. Scale bars: 10 µm. Source data are provided as a Source Data file. After fixation surface HER2 was stained (false colour-coded in white) before permeabilization and staining for total HER2 (green) and LAMP-1 (red). Cell
https://openalex.org/W4220725883	https://zenodo.org/record/6535183/files/A_Hidden_Chaotic_Attractor_with_an_Indep.pdf	English	null	A Hidden Chaotic Attractor with an Independent Amplitude-Frequency Controller	Complexity	2,022	cc-by	8,358	Hindawi Complexity Volume 2022, Article ID 3086747, 11 pages https://doi.org/10.1155/2022/3086747 Hindawi Complexity Volume 2022, Article ID 3086747, 11 pages https://doi.org/10.1155/2022/3086747 Research Article A Hidden Chaotic Attractor with an Independent Amplitude-Frequency Controller Yousuf Islam,1,2 Chunbiao Li ,1,2 Yicheng Jiang,1,2 Xu Ma,1,2 and Akif Akgul 3 1School of Electronic Information Engineering, Nanjing University of Information Science & Technology, Nanjing 210044, China 2Jiangsu Collaborative Innovation Center of Atmospheric Environment and Equipment Technology (CICAEET), Nanjing University of Information Science & Technology, Nanjing 210044, China 3Department of Computer Engineering, Faculty of Engineering, Hitit University, Corum 19030, Turkey Correspondence should be addressed to Akif Akgul; akifakgul@hitit.edu.tr Received 10 January 2022; Accepted 11 February 2022; Published 6 March 2022 Academic Editor: M. De Aguiar Copyright © 2022 Yousuf Islam et al. Tis 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. In this paper, a three-dimensional chaotic system with a line equilibrium is studied, in which a single nonbifurcation parameter is used to control the amplitude and frequency. A variety of chaotic signals can be modiﬁed using the amplitude-frequency control switch. Te realization of circuit simulation based on multisim further veriﬁes the theoretical analysis. Finally, the method for encrypting color images is tested, and the process performance is valued. It shows that the novel chaotic oscillation has a promising application prospect in image encryption. information encryption, chaotic sequence signals have a signiﬁcant application value. Chaos has been extensively applied for many aspects due to the intricate relationship between chaos and cryptography, and signiﬁcant ad- vancement has been performed in this area. Because of the irregularities and unpredictability of chaotic signals, chaos and corresponding fundamental systems have received a lot of attention [18–24]. A high-dimensional system’s chaotic dynamics seem to be more complex, and sometimes, they show hyperchaos for more than two positive Lyapunov exponents [25, 26]. Terefore, it has been proven that chaotic signals can contribute to enhancing the security of chaos-based communication as well as digital encryption. Furthermore, a hidden chaotic attractor is an important phenomenon found ten years ago. Te hidden oscillation has received a lot of attention because of its potential threat and possible applications. Finding hidden chaotic attrac- tors in nonlinear dynamical systems has become a major issue in nonlinear dynamical system research [27–29]. Many hidden attractors have been found in memristive systems [30] and hyperchaotic systems [31]. However, there is not much attention on hidden attractors with amplitude-frequency control. Recently, Wang et al. [32] 1. Introduction Since chaos was ﬁrst modeled by Edward Norton Lorenz in 1963 [1], great attention has been attracted for the reason that in such deterministic systems, chaos refers to the presence of seemingly random irregular motion [2]. In fact, chaos is a universal phenomenon in nonlinear systems and an inherent property of nonlinear dynamic systems. Chaos is a fundamental concept in nonlinear systems, and it is frequently used to characterize phenomena including bi- furcation and periodic motion [3, 4]. It is found that a chaotic system will display bifurcation under speciﬁc pa- rameters and that periodic and aperiodic motion can be- come entangled. Many famous 3-D chaotic systems have been proposed, including Arneodo systems, Sprott systems, Chen systems, Lv-Chen systems, Cai systems, and T sys- tems [5–8]. A chaotic circuit is a signiﬁcant research topic and an important representation of chaos. Chua proposed the ﬁrst chaotic circuit in 1984 [9–12], trying to bridge the gap between chaos theory and chaotic circuits. People have performed signiﬁcant research on chaos theory and pro- duced numerous novel breakthroughs in recent years as a result of the wide application of chaos [13–17]. For 2 Complexity studied the amplitude control and encryption application of chaotic signal, and they found that modular circuit cells with systematically conﬁgured parameters are useful for implementing multipiecewise Chua’s diode. Wang et al. [33, 34] also studied the hidden oscillations in Chua’s circuit and modiﬁed Sprott-A systems, where all the basins of attraction are not intersected with any equilibrium point indicating hidden attractor. representing a derivative of time t. When a 0.9, b 1, c 1, and IC (2, −2, 2), the system (1) exhibits chaos in the region with negative y, as shown in Figure 1. Tere are six terms in this new system, with two nonlinear items. Te Lyapunov exponents are L1 0.1314, L2 0, and L3 −0.8453. 3. Dynamical Analysis g In this paper, a chaotic system with a line of equilibrium points is focused on, where the attractor stands in the region with negative y, and thus, the basin of attraction does not intersect with all equilibrium points indicating hidden attractor. Furthermore, the amplitude and frequency of hidden oscillation can be controlled by a single knob. Circuit implementation shows the convenience of amplitude-fre- quency control of the chaotic signal. Image encryption proves the merits of this system. Te remaining of this paper is organized as follows. In Section 2, the system model is elaborated. In Section 3, the dynamics of the system are analyzed. In Section 4, the system is implemented in a simulated circuit. Finally, a chaotic system is applied to image encryption. Te conclusions are presented in the last section. 3.1. Equilibrium Points. Let _x _y _z 0. Te equilibrium points of the system can be calculated as cy + y2 −ayz 0, −z2 + byz 0, xy 0, ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ (2) (2) EO x, 0, 0, E1 0, −c, 0, E2 0, c ab −1, bc ab −1, ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ (3) (3) where a 0.9, b 1, and c 1. Solving this equation, the dynamical system (1) has three nontrivial equilibrium points in (2) which is independent of the value of the parameters a, b, and c. where a 0.9, b 1, and c 1. Solving this equation, the dynamical system (1) has three nontrivial equilibrium points in (2) which is independent of the value of the parameters a, b, and c. 2. A Novel Hidden Attractor In reference [35], the 3-dimensional chaotic system is ar- ticulated as J 0 c + 2y −az −ay 0 bz by −2z y x 0 􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌 􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌􏼌 . (4) _x cy + y2 −ayz, _y −z2 + byz, _z xy. ⎧ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎩ (1) (4) (1) As \|J −λI\| 0, the characteristic equation is As \|J −λI\| 0, the characteristic equation is Te system state variables are x, y, and z, while the constants a, b, and c are the real coeﬃcients, with dots det(J −λI) −aλy2 + 2ayz2 + bcy2 + bλ2z + bλxy + 2by3 −2cyz −λ3 −2λxz −4y2 det(J −λI) −aλy2 + 2ayz2 + bcy2 + bλ2z + bλxy + 2by3 −2cyz −λ3 −2λxz −4y2z 0. (5) (5) In the Jacobian matrix of system (1), the equilibrium point E is deﬁned as Te eigenvalues of the matrix at the equilibrium point can be determined as follows: Te eigenvalues of the matrix at the equilibrium point can be determined as follows: J EO 􏼁 J E1 􏼁 J E2 􏼁 0 c + 2y −az −ay 0 bz by −2z y x 0 ⎛ ⎜ ⎜ ⎜ ⎜ ⎜ ⎜ ⎜ ⎜ ⎜ ⎝ ⎞⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠. (6) 0, EO: λ1 0; λ2 0; λ3 0, E1: λ1 −0.7113; λ2 0.3556 + 1.1311i; λ3 0.3556 −1.1311i, E2: λ1 0.9911; λ2 −5.4956 + 8.4079i, λ3 −5.4956 −8.4079i. ⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ (7) EO: λ1 0; λ2 0; λ3 0, E1: λ1 −0.7113; λ2 0.3556 + 1.1311i; λ3 0.3556 −1.1311i, E2: λ1 0.9911; λ2 −5.4956 + 8.4079i, λ3 −5.4956 −8.4079i ⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ (7) Te eigenvalues of the system are obtained while a 0.9, b 1, and c 1. Te obtained all eigenvalues are given in Equation (7). 2. A Novel Hidden Attractor Te equilibrium E1(0, −c, 0) is a saddle-focus point of index-2; therefore, this equilibrium point E1 is unstable, and we can see in E2 that λ1 is a positive real number, and λ2 and λ3 are a pair of complex conjugate 3 Complexity 3 -1 -1 -2 -3 -4 -5 0 1 2 X 3 Y (a) -1 0 1 2 X 3 0 -2 -4 Z (b) -5 0 -2 -4 -4 -3 -2 Y Z -1 (c) Y X 0 -2 -4 0 -2 -4 0 2 -2 Z (d) Figure 1: Chaotic attractor of system (1) with a 0.9, b 1, and c 1 under initial condition IC (2, −2, 2): (a) (x)-(y), (b) (x)-(z), (c) (y)-(z), and (d) (x)-(y)-(z). -1 0 1 2 X 3 0 -2 -4 Z -1 -1 -2 -3 -4 -5 0 1 2 X 3 Y (a) -5 0 -2 -4 -4 -3 -2 Y Z -1 (c) (b) (b) Y X 0 -2 -4 0 -2 -4 0 2 -2 Z (d) (d) (c) igure 1: Chaotic attractor of system (1) with a 0.9, b 1, and c 1 under initial condition IC (2, −2, 2): (a) (x)-(y), (b) (x)-(z), (c) (y)-(z), nd (d) (x)-(y)-(z). Figure 1: Chaotic attractor of system (1) with a 0.9, b 1, and c 1 under initial condition IC (2, −2, 2): (a) (x)-(y), (b) (x)-(z), (c) (y)-(z), and (d) (x)-(y)-(z). Terefore, we can obtain V(t) ecV(0); if ∇× F < 0, then system (1) is dissipative and the state of the system is bounded by the state of the system (when a 0.9, b 1, and c 1). eigenvalues with a negative real number. Te equilibrium E2(0, c/ab −1, bc/ab −1) is a saddle-focus point of index-1; therefore, this equilibrium point E2 is also unstable. 3.2. Dissipativity Analysis. Inference µ is a region in the horizontal surface A3, and V(t) is set to be the volume of µ(t). 3.3. Amplitude and Frequent Control. In system (1), pa- rameter c is a single knob for amplitude and frequency control. 2. A Novel Hidden Attractor Let x ⟶mx, y ⟶my, z ⟶mz, t ⟶t/m (m > 0); then, system (1) turns to be Here, we obtain Here, we obtain _x c m y + y2 −ayz, _y −z2 + byz, _z xy, ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ (11) _V(t) 􏽚 ∞ μ(t)(∇× F)dxdydz. (8) (8) (11) Terefore, the dissipativity of the proposed chaotic system is ∇× F z _x x + z _y y + z _z z , z cy + y2 −ayz 􏼐 􏼑 x + z −z2 + byz 􏼐 􏼑 y + z(xy) z (9) ∇× F z _x x + z _y y + z _z z , ∇× F z _x x + z _y y + z _z z , indicating that the parameter c can control the amplitude and frequency of all variables x, y, and z, as shown in Figure 2. In Figure 3(a), when the linear coeﬃcient c is increased, system (1) keeps the chaotic state of all the time and the chaotic area continue to increase. Te rescaled amplitude and frequency can also be proved by Lyapunov exponents, as shown in Figure 3(b). z cy + y2 −ayz 􏼐 􏼑 x + z −z2 + byz 􏼐 􏼑 y + z(xy) z (9) (9) 0 + bz + 0 z c, where F is the 3-dimensional chaotic system, and b and z are the real parameters. Te above equation is rewritten as where F is the 3-dimensional chaotic system, and b and z are the real parameters. Te above equation is rewritten as 4. Circuit Implementation Te state variables x, y, and z in the system (1) correspond to the state voltages of the capacitors C1, C1, and C3 in the simpliﬁed circuit, and the corresponding circuit components can be selected as follows: V1 V2 15V, C1 C2 C3 10nF, correctness. Meanwhile, the key issue in the circuit is how to implement a circuit expression for a 3D chaotic system by converting and transforming it into a realistic circuit using electronic devices such as capacitors and resistors. To au- thenticate the eﬃciency of our proposed 3-dimensional chaotic system, the circuit implementation is designed and simulated with the NI multisim circuit simulation software, and the simulation results are detailed in this section. 1 2 3 R1 R2 R4 R5 R6 R7 R8 100kΩ, R3 106kΩ; For this circuit construction, it is transformed to be LM741CN is selected as an operational ampliﬁer; there is a general time scale 1000 for better displaying in the oscilloscope. _x 1 R1C1 y + 1 R2C1 y2 − 1 R3C1 yz, _y −1 R4C2 z2 + 1 R5C2 yz, _z 1 R8C3 xy. ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ (12) Figure 5 shows the phase trajectory of the system (1) in the analog oscilloscope. Te area of the phase track will change with the organization of the resistance R1, as shown in Figure 5(d). (12) 4. Circuit Implementation _V(t) 􏽚 ∞ μ(t) cdxdydz cV(t). (10) (10) Circuit veriﬁcation is also an essential step in the imple- mentation of the proposed chaotic system to ensure its Complexity 4 -5 0 5 10 X Y 0 -5 -10 -15 -20 c = 4 c = 2 c = 1 (a) x (t) 15 10 5 0 -5 0 100 200 300 400 500 Time/Sec c = 4 c = 2 c = 1 (b) Figure 2: Rescaled attractor of system (1) with a 0.9, b 1, and initial values (2, −2, 2) under the parameter c: (a) (x)-(y) and (b) signal x (t). x (t) 15 10 5 0 -5 0 100 200 300 400 500 Time/Sec c = 4 c = 2 c = 1 (b) -5 0 5 10 X Y 0 -5 -10 -15 -20 c = 4 c = 2 c = 1 (a) (b) Figure 2: Rescaled attractor of system (1) with a 0.9, b 1, and initial values (2, −2, 2) under the parameter c: (a) (x)-(y) and (b) signal x (t). Figure 2: Rescaled attractor of system (1) with a 0.9, b 1, and initial values (2, −2, 2) under the parameter c: (a) 1 2 3 4 5 c xmax 10 5 0 (a) 1 2 3 4 5 c LEs 1 0 -1 -2 -3 -4 -5 LE1 LE2 LE3 (b) Figure 3: Dynamical evolution of system (1) with a 0.9, b 1, and initial values (2, −2, 2) when c is in [1, 5]: (a) bifurcation diagram and (b) Lyapunov exponents. 1 2 3 4 5 c LEs 1 0 -1 -2 -3 -4 -5 LE1 LE2 LE3 (b) 1 2 3 4 5 c xmax 10 5 0 LE (b) (a) Figure 3: Dynamical evolution of system (1) with a 0.9, b 1, and initial values (2, −2, 2) when c is in [1, 5]: (a) bifurcation diagram and (b) Lyapunov exponents. expression of this system is obtained utilizing a summation, an integrator, and a transformer. Te relevant circuit implementation is depicted in Figure 4 based on the above explanations. 5. Application in Image Encryption Te control parameters and initial conditions of the system are (Table 1) μ 3.9999 and x0 0.6209, respectively, and a standard color image is chosen for testing, as illustrated in Figure 6. Te new chaotic system parameters are a 0.9, b 1, and c 1. Te initial conditions are set as X0 0.7361, Y0 0.4663, Z0 0.1501, and U0 0.7653. Table 2 describes the selected keys that were chosen. M1 and N1 are the zeroing parameters used throughout encryption; k1 is the average gray level of G in the original image, and the original image k2 is the average gray level of B. Figure 6(b) shows the encrypted image after simulation. Te image after encryption is chaotic and fundamentally diﬀerent from the image before processing, as it can be observed. Te properly decrypted image, which can be seen in Figure 6(c), is identical to the original image (Table 2). entropyH(m) −􏽘 H i1 p xi 􏼁log2 p xi 􏼁, (13) (13) where p(xi) represents the probability of the occurrence of the gray value xi and H indicates the gray level of the image. Teoretically, for a completely random digital image with a grayscale of 256 has an evenly distributed pixel value in [0, 255], then p(xi) 1/256 (i ∈∈[0, 255]), and the estimated information entropy is 8 bits. If the image is encrypted, the closer the image’s information entropy is near 8, the better the encryption features. 5.2. Security Analysis. Secure analysis is the most essential and fundamental requirement for an encrypted system. In general, a chaotic image encryption algorithm requires a substantial keyspace, reversible encryption and decryption, strong antiattack characteristics, and the other perfor- mances, and then the performance of chaos-based en- cryption is the next to determine by using the following eligibility requirements: keyspace analysis, histogram anal- ysis, information entropy analysis, and correlation analysis. 5.2. Security Analysis. Secure analysis is the most essential and fundamental requirement for an encrypted system. In general, a chaotic image encryption algorithm requires a substantial keyspace, reversible encryption and decryption, strong antiattack characteristics, and the other perfor- mances, and then the performance of chaos-based en- cryption is the next to determine by using the following eligibility requirements: keyspace analysis, histogram anal- ysis, information entropy analysis, and correlation analysis. 5.6. Correlation Statistical Analysis. 5. Application in Image Encryption On the one hand, the correlation level of adjacent pixels is larger when the cor- relation coeﬃcient degree of adjacent pixels is higher. On the other hand, the lower the correlation, the smaller the co- eﬃcient. As a result, calculating the correlation coeﬃcient can be justiﬁed the algorithm’s security. Te lower the correlation and the advanced security, the smaller the co- eﬃcient. Te correlation coeﬃcients were calculated from the three channels (R, G, and B) with three directions: horizontal, vertical, and diagonal, to measure the correlation between the original image and adjacent pixels of the ci- phertext image; N pairs of adjacent pixels were selected from the image, and the correlation coeﬃcients were calculated from the three channels (R, G, B) with three directions: horizontal, vertical, and diagonal. To equivalence the au- tocorrelation of an unadorned and encrypted image, we have calculated the correlation coeﬃcient r of each pair of pixels by using the following formula: 5.3. Keyspace Analysis. Te keyspace can approach (1016)10 10160, satisfying the security level of the keyspace (greater than 2128) using a 64-bit processor, ﬂoating-point precision up to 10−16. Te modiﬁed system can be observed to be more resistant to the attacker’s comprehensive attack. Te decrypted image cannot be acquired accurately when the algorithm key is changed slightly, as shown in Figure 7. As a result, the new system has a high level of key sensitivity. Te images used throughout the investigations are 256 × 256 conventional images with a 23 8-bit grayscale. 5.4. Histogram Analysis. Te distribution of pixel intensity values within an image is represented by a histogram. It can be utilized to defend against statistical attacks. Te frequency distribution of each gray level pixel can be visually displayed using a gray histogram, which is a statistical analytical method. Te histogram of the image becomes smooth even after encryption, compared to the ﬂuctuation before en- cryption, thus preventing the attacker from accessing the original image information through statistical analysis, resulting in information leakage, and ensuring information. Figures 8(a)–8(c) are the histograms of the original image, and Figures 8(d)–8(f) are the histograms of the encrypted images. It is clear that the new system can withstand a more powerful onslaught. 5. Application in Image Encryption Te chaotic system with a linear equilibrium in this section has a higher level of unpredictability, a larger keyspace, and a higher level of complexity, all of which make the encryption stronger secure in concept. A chaotic system is deﬁned as a system that has several causes and multiplication, addition, and diﬀerentiation, and diﬀerentiation exists in the system equations, and a realistic 5 Complexity -y R1 R2 R3 R4 R7 R8 R5 R6 1V/V 0V 1V/V 0V 1V/V 0V 1V/V 0V A5 A1 A2 A3 A4 C3 C1 U1 U2 U3 U4 V2 V1 C2 -y y y z z y -y Vn Vn Vn z x x -y Vp Vp Vp Vn Vp Vp Vn z -y z Figure 4: Circuit realization of the system (1). (a) (b) (c) (d) gure 5: Chaotic attractor of system (1): (a) (x)-(y), (b) (x)-(z), (c) (y)-(z), and (d) the attractor changes due to the diﬀerence of sistance. -y R1 R2 R3 R4 R7 R8 R5 R6 1V/V 0V 1V/V 0V 1V/V 0V 1V/V 0V A5 A1 A2 A3 A4 C3 C1 U1 U2 U3 U4 V2 V1 C2 -y y y z z y -y Vn Vn Vn z x x -y Vp Vp Vp Vn Vp Vp Vn z -y z Figure 4: Circuit realization of the system (1). Figure 4: Circuit realization of the system (1). (a) (b) (c) (d) Figure 5: Chaotic attractor of system (1): (a) (x)-(y), (b) (x)-(z), (c) (y)-(z), and (d) the attractor changes due to the diﬀerence of R1 resistance. or of system (1): (a) (x)-(y), (b) (x)-(z), (c) (y)-(z), and (d) the attractor changes due to the diﬀerence of R1 Figure 5: Chaotic attractor of system (1): (a) (x)-(y), (b) (x)-(z), (c) (y)-(z), and (d) the attractor changes due to the diﬀerence of R1 resistance. 6 Complexity entropy, the more consistent the image’s gray distribu- tion. In a grayscale image, each pixel is coded in 8 bits. As a result, an image’s maximum entropy value is 8. Te information entropy of the encrypted image should be near 8, which indicates the best amount of uncertainty, owing to a decent encryption process. To compute the information entropy, many users use the method as follows: 5.1. Encryption Application with a Chaotic System. 5. Application in Image Encryption rXY cov(X, Y) D(X)D(Y) 􏽰 , E(X) 1 N 􏽘 N i1 xi 􏼁, D(X) 1 N 􏽘 N i1 xi −E(X) 􏼁, cov(X, Y) 1 N 􏽘 N i1 xi −E(X) 􏼁yi −E(Y) 􏼁, (14) (14) cov(X, Y) 1 N 􏽘 N i1 xi −E(X) 􏼁yi −E(Y) 􏼁, where cov(X, Y) represents the correlation and autocorre- lation function, X and Y are the grayscale values of two adjacent pixels in the image, and N denotes the sample. E is the expected value operator, and D(X) represents the variance of the variable x. Te values of rXY lie in the range 5.5. Information Analysis for Entropy. Te image’s infor- mation entropy can be used to evaluate the level of uncertainty and randomness in the image distribution of its pixel gray value. Typically, the higher the image’s Table 1: Algorithm key. Key μ x0 X0 Y0 Z0 U0 M1 N1 k1 k2 Value 3.9999 0.6209 0.7361 0.4663 0.1501 0.7653 0 0 0.4761 0.1255. Figure 6: Encryption images: (a) original pepper image; (b) encryption pepper image; (c) decryption pepper image. Complexity 7 Table 1: Algorithm key. Key μ x0 X0 Y0 Z0 U0 M1 N1 k1 k2 Value 3.9999 0.6209 0.7361 0.4663 0.1501 0.7653 0 0 0.4761 0.1255. Figure 6: Encryption images: (a) original pepper image; (b) encryption pepper image; (c) decryption pepper image. Complexity 7 7 Complexity Table 1: Algorithm key. gure 6: Encryption images: (a) original pepper image; (b) encryption pepper image; (c) decryption pepper im Figure 6: Encryption images: (a) original pepper image; (b) encryption pepper image; (c) decryption pepper image. Table 2: Te information entropy of the three channels in the original and encrypted on the pepper image. Image R channel entropy G channel entropy B channel entropy Original picture 5.6871 7.6856 4.686 Encrypted image 7.9997 7.9997 7.9998 e information entropy of the three channels in the original and encrypted on the pepper image. Figure 7: Initial condition perturbation ciphertext. Figure 7: Initial condition perturbation ciphertext. 0 50 150 15000 10000 5000 0 100 250 200 Original image R channel histogram (a) 8000 7000 6000 5000 4000 3000 2000 1000 0 0 50 150 100 Original picture G channel histogram 250 200 (b) 15000 10000 5000 0 Original picture B channel histogram 0 50 150 100 250 200 (c) Figure 8: Continued. 5. Application in Image Encryption Image Channel Horizontal Vertical Diagonal Original image Red 0.97489 0.98660 0.96227 Green 0.97532 0.98731 0.96377 Blue 0.95167 0.97112 0.92931 Encrypted image Red 0.00070 0.01175 0.01539 Green 0.00855 −0.01537 −0.01660 Blue 0.00122 0.00135 0.01235 0 50 150 100 250 200 150 100 50 0 250 200 B channel random point pixel gray value Te gray value of horizontal adjacent point B channel horizontal adjacent autocorrelation point 250 200 150 100 50 0 Te gray value of vertical adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel vertical adjacent autocorrelation point 250 200 150 100 50 0 Te gray value of diagonal adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel diagonal adjacent correlation point 250 200 150 100 50 0 Te gray value of vertical adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel vertical adjacent correlation point 0 50 150 100 250 200 B channel random point pixel gray value 250 200 150 100 50 0 Te gray value of horizontal adjacent point B channel horizontal adjacent correlation point 250 200 150 100 50 0 Te gray value of diagonal adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel diagonal adjacent autocorrelation point Figure 9: Correlation and autocorrelation of R channel adjacent pixels of the pepper image and its ciphered image: (a) horizontal direction of the pepper image; (b) vertical direction of the pepper image; (c) diagonal direction of the pepper image; (d) horizontal direction of the pepper ciphered image; (e) vertical direction of the pepper ciphered image; (f) diagonal direction of the pepper ciphered image. 5. Application in Image Encryption Image Channel Horizontal Vertical Diagonal Original image Red 0.97489 0.98660 0.96227 Green 0.97532 0.98731 0.96377 Blue 0.95167 0.97112 0.92931 Encrypted image Red 0.00070 0.01175 0.01539 Green 0.00855 −0.01537 −0.01660 Blue 0.00122 0.00135 0.01235 0 50 150 100 250 200 150 100 50 0 250 200 B channel random point pixel gray value Te gray value of horizontal adjacent point B channel horizontal adjacent autocorrelation point 250 200 150 100 50 0 Te gray value of vertical adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel vertical adjacent autocorrelation point 250 200 150 100 50 0 Te gray value of diagonal adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel diagonal adjacent correlation point 250 200 150 100 50 0 Te gray value of vertical adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel vertical adjacent correlation point 0 50 150 100 250 200 B channel random point pixel gray value 250 200 150 100 50 0 Te gray value of horizontal adjacent point B channel horizontal adjacent correlation point 250 200 150 100 50 0 Te gray value of diagonal adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel diagonal adjacent autocorrelation point Figure 9: Correlation and autocorrelation of R channel adjacent pixels of the pepper image and its ciphered image: (a) horizontal direction of the pepper image; (b) vertical direction of the pepper image; (c) diagonal direction of the pepper image; (d) horizontal direction of the pepper ciphered image; (e) vertical direction of the pepper ciphered image; (f) diagonal direction of the pepper ciphered image. Table 3: A correlation coeﬃcient of two adjacent pixels in the original and encrypted on the pepper image. Image Channel Horizontal Vertical Diagonal Original image B 0.98645 0.98782 0.97851 G 0.99529 0.99282 0.98965 R 0.99428 0.99168 0.98792 Encrypted image B 0.00494 −0.02531 0.00540 G −0.01262 −0.01606 0.00444 R −0.00282 −0.01656 0.00919 able 3: A correlation coeﬃcient of two adjacent pixels in the original and encrypted on the pepper image. Image Channel Horizontal Vertical Diagonal Original image B 0.98645 0.98782 0.97851 G 0.99529 0.99282 0.98965 R 0.99428 0.99168 0.98792 Encrypted image B 0.00494 −0.02531 0.00540 G −0.01262 −0.01606 0.00444 R −0.00282 −0.01656 0.00919 Table 4: Correlation coeﬃcient test result [36]. 5. Application in Image Encryption 0 50 150 15000 10000 5000 0 100 250 200 Original image R channel histogram (a) 8000 7000 6000 5000 4000 3000 2000 1000 0 0 50 150 100 Original picture G channel histogram 250 200 (b) 15000 10000 5000 0 Original picture B channel histogram 0 50 150 100 250 200 (c) Figure 8: Continued. 15000 10000 5000 0 Original picture B channel histogram 0 50 150 100 250 200 (c) (c) (a) (b) Figure 8: Continued. Figure 8: Continued. 8 Complexity 0 50 150 100 250 200 5000 4000 3000 2000 1000 0 R channel histogram afer encryption (d) 0 50 150 100 250 200 5000 4000 3000 2000 1000 0 G channel histogram afer encryption (e) 0 50 150 100 250 200 5000 4000 3000 2000 1000 0 B channel histogram afer encryption (f) Figure 8: Histogram experiment images. (a–c) Histogram of the original image. (d–f) Histogram of the encrypted image. 0 50 150 100 250 200 5000 4000 3000 2000 1000 0 B channel histogram afer encryption (f) 0 50 150 100 250 200 5000 4000 3000 2000 1000 0 G channel histogram afer encryption (e) 0 50 150 100 250 200 5000 4000 3000 2000 1000 0 R channel histogram afer encryption (d) (f) (d) (e) Figure 8: Histogram experiment images. (a–c) Histogram of the original image. (d–f) Histogram of the encrypted image. Figure 8: Histogram experiment images. (a–c) Histogram of the original image. (d–f) Histogram of the en Table 3: A correlation coeﬃcient of two adjacent pixels in the original and encrypted on the pepper image. Image Channel Horizontal Vertical Diagonal Original image B 0.98645 0.98782 0.97851 G 0.99529 0.99282 0.98965 R 0.99428 0.99168 0.98792 Encrypted image B 0.00494 −0.02531 0.00540 G −0.01262 −0.01606 0.00444 R −0.00282 −0.01656 0.00919 Table 4: Correlation coeﬃcient test result [36]. 5. Application in Image Encryption 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of vertical adjacent point R channel vertical adjacent autocorrelation point 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of horizontal adjacent point R channel horizontal adjacent autocorrelation point 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of diagonal adjacent point R channel diagonal adjacent autocorrelation point 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of vertical adjacent point R channel vertical adjacent correlation point 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of diagonal adjacent point R channel diagonal adjacent correlation point R channel vertical adjacent correlation point R channel diagonal adjacent correlation point 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of horizontal adjacent point R channel horizontal adjacent correlation point Figure 10: Correlation and autocorrelation of G channel adjacent pixels of the pepper image and its ciphered image: (a) horizontal direction of the pepper image; (b) vertical direction of the pepper image; (c) diagonal direction of the pepper image; (d) horizontal direction of the pepper ciphered image; (e) vertical direction of the pepper ciphered image; (f) diagonal direction of the pepper ciphered image. 5. Application in Image Encryption 250 200 150 100 50 0 Te gray value of vertical adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel vertical adjacent autocorrelation point 250 200 150 100 50 0 Te gray value of diagonal adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel diagonal adjacent autocorrelation point 0 50 150 100 250 200 150 100 50 0 250 200 B channel random point pixel gray value Te gray value of horizontal adjacent point B channel horizontal adjacent autocorrelation point 250 200 150 100 50 0 Te gray value of vertical adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel vertical adjacent autocorrelation point 250 200 150 100 50 0 Te gray value of diagonal adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel diagonal adjacent autocorrelation point 0 50 150 100 250 200 150 100 50 0 250 200 B channel random point pixel gray value Te gray value of horizontal adjacent point B channel horizontal adjacent autocorrelation point 250 200 150 100 50 0 Te gray value of diagonal adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel diagonal adjacent correlation point 250 200 150 100 50 0 Te gray value of vertical adjacent point 0 50 150 100 250 200 B channel random point pixel gray value B channel vertical adjacent correlation point 0 50 150 100 250 200 B channel random point pixel gray value 250 200 150 100 50 0 Te gray value of horizontal adjacent point B channel horizontal adjacent correlation point B channel random point pixel gray value Figure 9: Correlation and autocorrelation of R channel adjacent pixels of the pepper image and its ciphered image: (a) horizontal direction of the pepper image; (b) vertical direction of the pepper image; (c) diagonal direction of the pepper image; (d) horizontal direction of the pepper ciphered image; (e) vertical direction of the pepper ciphered image; (f) diagonal direction of the pepper ciphered image. 5. Application in Image Encryption 9 Complexity 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of vertical adjacent point R channel vertical adjacent correlation point 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of diagonal adjacent point R channel diagonal adjacent correlation point 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of horizontal adjacent point R channel horizontal adjacent autocorrelation point 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of vertical adjacent point R channel vertical adjacent autocorrelation point 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of diagonal adjacent point R channel diagonal adjacent autocorrelation point 0 50 150 100 250 200 R channel random point pixel gray value 250 200 150 100 50 0 Te gray value of horizontal adjacent point R channel horizontal adjacent correlation point Figure 10: Correlation and autocorrelation of G channel adjacent pixels of the pepper image and its ciphered image: (a) horizontal direction of the pepper image; (b) vertical direction of the pepper image; (c) diagonal direction of the pepper image; (d) horizontal direction of the pepper ciphered image; (e) vertical direction of the pepper ciphered image; (f) diagonal direction of the pepper ciphered image. 5. Application in Image Encryption 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of vertical adjacent point G channel vertical adjacent autocorrelation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of diagonal adjacent point G channel diagonal adjacent autocorrelation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of horizontal adjacent point G channel horizontal adjacent autocorrelation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of vertical adjacent point G channel vertical adjacent autocorrelation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of diagonal adjacent point G channel diagonal adjacent autocorrelation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of horizontal adjacent point G channel horizontal adjacent autocorrelation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of diagonal adjacent point G channel diagonal adjacent autocorrelation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of diagonal adjacent point G channel diagonal adjacent correlation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of vertical adjacent point G channel vertical adjacent correlation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of horizontal adjacent point G channel horizontal adjacent correlation point 50 150 100 200 G channel random point pixel gray value G channel random point pixel gray value G channel random point pixel gray value Figure 11: Correlation and autocorrelation of B channel adjacent pixels of the pepper image and its ciphered image: (a) horizontal direction of the pepper image; (b) vertical direction of the pepper image; (c) diagonal direction of the pepper image; (d) horizontal direction of the pepper ciphered image; (e) vertical direction of the pepper ciphered image; (f) diagonal direction of the pepper ciphered image. 5. Application in Image Encryption 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of horizontal adjacent point G channel horizontal adjacent autocorrelation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of vertical adjacent point G channel vertical adjacent autocorrelation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of diagonal adjacent point G channel diagonal adjacent autocorrelation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of horizontal adjacent point G channel horizontal adjacent correlation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of vertical adjacent point G channel vertical adjacent correlation point 0 50 150 100 250 200 G channel random point pixel gray value 250 200 150 100 50 0 The gray value of diagonal adjacent point G channel diagonal adjacent correlation point Figure 11: Correlation and autocorrelation of B channel adjacent pixels of the pepper image and its ciphered image: (a) horizontal direction of the pepper image; (b) vertical direction of the pepper image; (c) diagonal direction of the pepper image; (d) horizontal direction of the pepper ciphered image; (e) vertical direction of the pepper ciphered image; (f) diagonal direction of the pepper ciphered image. Data Availability Te data used to support the ﬁndings of this study are available from the corresponding author upon reasonable request. [15] J. P. Singh and B. K. Roy, “Te simplest 4-D chaotic system with line of equilibria, chaotic 2-torus and 3-torus behaviour,” Nonlinear Dynamics, vol. 89, no. 3, pp. 1845–1862, 2017. 5. Application in Image Encryption [−1, 1], with 1 indicating perfect correlation, −1 indicating anticorrelation, and 0 representing no correlation. very close to 0, indicating that the encrypted image’s pixel point distribution is very highly discrete. p g Here, we can see from Table 3 that the correlation co- eﬃcients of the original image are all very close to 1, whereas the correlation coeﬃcients of the encrypted image are all y g y If we compare Tables 3 and 4 with each other, we can see some diﬀerent points. In Table 3, the original correlation coeﬃcient values are average horizontal 99.2%, vertical 10 Complexity 99.07%, and diagonal 98.54%, and in Table 4, the original correlation coeﬃcient values are average horizontal 96.72%, vertical 98.17%, and diagonal 95.18%, that is, the original points in Table 3 are closer to 1 and the encrypted points are also closer to 0 than Table 4. So, Table 3 indicates that the encrypted points of distribution are highly discrete. [8] Q. Yang, G. Chen, and T. Zhou, “A uniﬁed Lorenz-Type system and its canonical form,” International Journal of Bi- furcation and Chaos, vol. 16, no. 10, pp. 2855–2871, 2006. [9] L. O. Chua, T. Matsumoto, and M. Komuro, “Te double scroll family. 2. rigorous analysis of bifurcation phenomena,” IEEE Transactions on Circuits and Systems, vol. 33, no. 11, pp. 1097–1118, 1986. Te correlation and autocorrelation plots for each image are shown in Figures 9–11. In Table 3, horizontal, vertical, and diagonal directions provide their correlation and au- tocorrelation coeﬃcients. Table 3 also includes signiﬁcant data from references for comparison. [10] Z. Wei, P. Yu, W. Zhang, and M. Yao, “Study of hidden attractors, multiple limit cycles from Hopf bifurcation and boundedness of motion in the generalized hyperchaotic Rabinovich system,” Nonlinear Dynamics, vol. 82, no. 1-2, pp. 131–141, 2015. [11] A. Akgul, S. Hussain, and I. Pehlivan, “A new three-di- mensional chaotic system, its dynamical analysis and elec- tronic circuit applications,” Optik, vol. 127, no. 18, pp. 7062–7071, 2016. 6. Conclusion Te single nonbifurcation parameter of the system can ef- fectively modify the amplitude and frequency of the dem- onstrated three-dimensional chaotic system. Numerical simulation and circuit experiment based on multisim agree to each other by proving the phenomenon. As a typical application, the property of image encryption is exhaustively analyzed. With the chaotic signal from the new system, a color image is well encrypted and decrypted in the keyspace. Histogram and correlation of adjacent pixels are used for showing the high encryption performance. [12] Z. Wei, V.-T. Pham, T. Kapitaniak, and Z. Wang, “Bifurcation analysis and circuit realization for multiple-delayed Wang- Chen system with hidden chaotic attractors,” Nonlinear Dynamics, vol. 85, no. 3, pp. 1635–1650, 2016. [13] S. M. Tabatabaie Nezhad, M. Nazari, and E. A. Gharavol, “A novel dos and ddos attacks detection algorithm using arima time series model and chaotic system in computer networks,” IEEE Communications Letters, vol. 20, no. 4, pp. 700–703, 2016. [14] Z. Wei, I. Moroz, J. C. Sprott, Z. Wang, and W. Zhang, “Detecting hidden chaotic regions and complex dynamics in the self-exciting homopolar disc dynamo,” International Journal of Bifurcation and Chaos, vol. 27, no. 2, Article ID 1730008, 2017. Conflicts of Interest y [16] Z. Wang, A. Akgul, V. T. Pham, and S. Jafari, “Chaos-based application of a novel no-equilibrium chaotic system with coexisting attractors,” Nonlinear Dynamics, vol. 89, no. 46, pp. 1877–1887, 2017. Te authors declare that there are no conﬂicts of interest regarding the publication of this paper. [17] J. P. Singh and B. K. Roy, “Second order adaptive time varying sliding mode control for synchronization of hidden chaotic orbits in a new uncertain 4-D conservative chaotic system,” Transactions of the Institute of Measurement and Control, vol. 40, no. 13, pp. 3573–3586, 2018. References 28, no. 7, Article ID 1850085, 2018. [23] A. Akg¨ul, S. Kaçar, B. Aricıo˘glu, and I. Pehlivan, “Text en- cryption by using one-dimensional chaos generators and nonlinear equations,” in Proceedings of the 2013 8th Inter- national Conference on Electrical and Electronics Engineering (ELECO), pp. 320–323, IEEE, Bursa, Turkey, November 2013. pp y [24] K. Rajagopal, V. T. Pham, F. R. Tahir, A. Akgul, H. R. Abdolmohammadi, and S. 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https://openalex.org/W2467995164	https://europepmc.org/articles/pmc4939040?pdf=render	English	null	Antioxidant activities and oxidative stress inhibitory effects of ethanol extracts from Cornus officinalis on raw 264.7 cells	BMC complementary and alternative medicine	2,016	cc-by	6,391	© 2016 The Author(s). 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. Hwang et al. BMC Complementary and Alternative Medicine (2016) 16:196 DOI 10.1186/s12906-016-1172-3 Hwang et al. BMC Complementary and Alternative Medicine (2016) 16:196 DOI 10.1186/s12906-016-1172-3 Abstract Background: Cornus officinalis, is a deciduous tree native to the eastern Asia, distributes mainly in (e.g. Korea, as well as China, and Japan). It is used as folk medicine to backache, polyuria, hypertension and nervous breakdown. Pharmacological studies have demonstrated that C. officinalis possess anti-oxidant, anti-hyperglycemic, and immune regulatory effects. However, reports on the antioxidant activity of C. officinalis have been limited to in vitro radical scavenging studies. Its mechanism of action within the cell at the genetic level especially has not yet been clearly defined. Therefore, we investigated the anti-antioxidant activities of C. officinalis in RAW 264.7 cells. Methods: The antioxidant activities and protective effects of C. officinalis ethanol extract on cell damage and the antioxidant enzyme system in lipopolysaccharide (LPS)-induced oxidative stress-damaged RAW 264.7 cells were assessed. To measure the effects of C. officinalis on antioxidant activities, we used the following methods: Total phenol and flavonoid contents, DPPH scavenging activity assay, ABTS scavenging activity assay, FRAP value measurement, xanthine oxidase activity assay, ROS generation measurement and real time PCR. Results: The total phenol and flavonoid contents of C. officinalis extracts were 27.04 mg GAE/g and 3.70 mg QE/g, respectively. The antioxidant activities of C. officinalis extracts increased in a dose-dependent manner: the IC50 values for DPPH and ABTS radical scavenging activities of C. officinalis extracts were 99.32 μg/mL and 138.51 μg/mL, respectively. C. officinalis extracts inhibited xanthine oxidase activity and reactive oxygen species generation. The expression of antioxidant enzymes, Cu/ZnSOD, MnSOD, catalase, and glutathione peroxidase increased upon treatment with C. officinalis extracts at 100 μg/mL, compared to that in the LPS-treated group. Conclusions: These results suggest the therapeutic potential of C. officinalis extract as an anti-oxidant agent. Conclusions: These results suggest the therapeutic potential of C. officinalis extract as an a Keywords: Cornus officinalis, Oxidative stress, Antioxidant activity Keywords: Cornus officinalis, Oxidative stress, Antioxidant activity Kyung-A Hwang, Yu-Jin Hwang and Jin Song Kyung-A Hwang, Yu-Jin Hwang and Jin Song Background The mechanism of inflammation injury partially involves the release of ROS from activated neutrophils and macro- phages. ROS propagate inflammation by stimulating the release of cytokines such as interleukin-1, tumor ne- crosis factor-α, and interferon-α, which stimulate re- cruitment of additional neutrophils and macrophages. Free radicals are important mediators that provoke or sustain inflammatory processes, and consequently, their neutralization by antioxidants and radical scav- engers can attenuate inflammation [6, 7]. Therefore, compounds that have scavenging activities toward these radicals could have therapeutic potential. Reactive oxygen species (ROS) are produced in oxidation processes essential to most living organisms and are es- sential to produce the energy required to fuel other bio- logical processes. However, excessive production of ROS is damaging to cells because ROS destroy molecules such as DNA and proteins. Thus, ROS play an important role in the pathogenesis of various serious diseases, such as neurodegenerative disorders, cancer, cardiovascular dis- eases, atherosclerosis, cataracts, and inflammation [1–5]. * Correspondence: kah366@korea.kr Department of Agrofood Resources, National Academy of Agricultural Science, Rural Development Administration, Wanju-gun, Jeollabuk-do 55365, Republic of Korea There are two methods of suppressing ROS. First, natural defense mechanisms present in the human/mammalian Hwang et al. BMC Complementary and Alternative Medicine (2016) 16:196 Page 2 of 9 Page 2 of 9 system counteract the potential deleterious effects of ROS. Normally, cells have antioxidant systems that protect against the harmful effects of ROS, including superoxide dismutase (SOD), which converts superoxide anions to hydrogen peroxide (H2O2) for rapid removal by detoxifying enzymes such as glutathione peroxidase (GPx). Similarly, glutathione (GSH) can reduce ROS for GPx-catalyzed H2O2 reductions [8, 9]. Second, functional components from the external environment such as flavonoids, L- ascorbic acid (Vit C), and α-tocopherol (Vit E), act as anti- oxidants [10, 11]. Therefore, a diet rich in antioxidants could help the body defend itself against the molecular effects of free radicals and ROS and hinder the develop- ment of many chronic diseases. Numerous studies have shown that natural dietary compounds can potently modu- late oxidative stress. Plant phenolics, flavonoids, tannins, and anthocyanins have useful properties such as antioxi- dant, immune, and anticancer activities. The presence of these antioxidants increases the efficacy of the protection system against ROS [12–14]. acid, acetic acid, lipopolysaccharide (LPS), 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as- corbic acid (Vit C), and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Total flavonoid content A sample solution was mixed with 100 % ethanol, 10 % aluminum chloride, 1 M potassium acetate, and distilled water. The reagents were thoroughly mixed and allowed to stand for 40 min at room temperature, and the ab- sorbance of the supernatant was measured at 415 nm [26]. Quercetin was used to plot a standard calibration curve, and the results are expressed as quercetin equiva- lents in milligram per gram (mg QE/g) of dried extract. Sample preparation and extraction p p p C. officianalis was purchased from Korea Medicine Herbal Association, which is under the jurisdiction of the Ministry of Agriculture, Food and Rural Affairs (Seoul, Korea). The plant was identified and authenticated by the Korea Medicine Herbal Association. Voucher specimens (NAAS-15-03) were deposited at the Department of Agro- food Resources Herbarium, National Academy of Agricul- tural Science, Korea. C. officianalis (20 g) was extracted twice with 70 % ethanol at 70 °C for 6 h. The 70 % ethanol extract was filtered using filter paper (Advantec, Tokyo, Japan). Subsequently, the filtrates were combined and evap- orated under vacuum (EYELA CCA-1110, Tokyo Rikakikai Co., Tokyo, Japan) and then lyophilized with a freeze dryer (Ilshine Lab, Suwon, Korea) at −70 °C under reduced pres- sure (<20 Pa). The dry residue was stored at −70 °C. For further analysis, the dry extract was reconstituted with di- methyl sulfoxide (DMSO). Cornus officinalis (Cornaceae) is a deciduous tree na- tive to eastern Asia (e.g., Korea, China, and Japan). The fruit of C. officinalis, known as “Sansuyu” in Korean, is mainly harvested in the central and southern regions of Korea [15]. C. officinalis is often included in traditional treatments for conditions such as backache, polyuria, hypertension, and nervous breakdown [16]. Pharmaco- logical studies have demonstrated that C. officinalis pos- sesses antioxidant [17], antihyperglycemic [18], immune regulatory [19] and anti-inflammatory effects [20]. Furthermore, many functional compounds such as ursolic acid, tartaric acid, malic acid, glucosides, and fatty acids are present in the fruit. Several studies have also reported that these compounds have antioxidant and anti-inflammatory effects [21–24]. Total phenolic content The total phenol content of C. officianalis extract was determined by the Folin-Ciocalteau method [25]. The extract was oxidized with Folin-Ciocalteau reagents, and then the reaction was neutralized with saturated sodium carbonate. After incubation at room temperature for 1 h, the absorbance of the reaction mixture was mea- sured at 725 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The total phenolic con- tent is expressed as gallic acid equivalents in milligrams per gram (mg GAE/g) of dry extract. Reports on the antioxidant activity of C. officinalis have been restricted to in vitro radical scavenging stud- ies. Its mechanism of action within the cell at the genetic level has not yet been clearly defined. Therefore, the aims of this study were to identify the effect of C. offici- nalis on antioxidant activity, inhibition of ROS produc- tion, and antioxidant-related gene expression in RAW 264.7 cells (murine macrophage cell line). This study suggests that the ethanol extract of C. officinalis could be used as a natural source of antioxidants in the food and pharmaceutical industries. Background Iron (II) sulfate hep- tahydrate (FeSO4) was purchased from Junsei (Tokyo, Japan). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin (P/S), and trypsin-EDTA were obtained from Gibco (Waltham, MA, USA). The xanthine oxidase (XO) assay kit was purchased from Abcam (Cambridge, MA, USA). The other reagents used were of analytical grade. ABTS radical cation-scavenging activity The ABTS assay was performed to evaluate the ability of the C. officianalis extract to scavenge the ABTS radical cation in comparison to that of a standard (Vit C) [28]. The radical cation was prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate (1:1 v/v) and leaving the mixture for 24 h until the reaction was completed and the absorbance was stable. The ABTS radical solu- tion was diluted with PBS to an absorbance of 0.7 (±0.02) at 732 nm. The assay was conducted with diluted ABTS radical solution mixed with samples, and the mea- surements were taken at 734 nm after 30 min. The anti- oxidative activity of the samples was calculated by determining the decrease in absorbance. The free radical-scavenging capacity was expressed by IC50. Real-time reverse transcription polymerase chain analysis To determine the expression levels of Cu/Zn SOD, Mn SOD, catalase, and GPx, real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using a real-time thermal cycler Qiagen Rotorgene Q (Valencia, CA, USA), in accordance with the manufac- turer’s instructions. The cells were treated with C. officia- nalis extracts and cultured for 24 h. Thereafter, cDNA was synthesized from the total RNA isolated from cells. The PCR reaction was performed using the 2X SYBR Green mix (Qiagen, Valencia, CA, USA). All results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The following primer sequences were used for real-time RT-PCR: GAPDH, 5′-GAG CCA AAA GGG TCA TCA TC-3′ (forward), 5′-TAA GCA GTT GGT GGT GCA GG-3′ (reverse); Cu/Zn SOD, 5′- CAG CAT GGG TTC CAC GTC CA-3′ (forward), 5′- CAC ATT GGC CAC ACC GTC CT-3′ (reverse); Mn SOD, 5′-GGG TTG GCT TGG TTT CAA TAA GGA A- 3′ (forward), 5′-AGG TAG TAA GCG TGC TCC CAC ACA T-3′ (reverse); catalase, 5′-AAG ACA ATG TCA CTC AGG TGC GGA-3′ (forward), 5′-GGC AAT GTT CTC ACA CAG GCG TTT-3′ (reverse); and GPx, 5′- CTC GGT TTC CCG TGC AAT CAG-3′ (forward), 5′- GTG CAG CCA GTA ATC ACC AAG-3′ (reverse). Ferric-reducing antioxidant power (FRAP) activity FRAP activity was determined using manual assay methods [29]. The working fluid was freshly prepared by mixing acetate buffer (300 mM, pH 3.6) with 10 mM TPTZ in HCl and 20 mM iron (III) chloride hexahy- drate. The sample solution or Vit C was added to work- ing fluid, and the mixture was left for 4 min at room temperature. Cell cytotoxicity assay RAW 264.7 cells were plated at 1 × 104 cells/well. The C. officianalis ethanol extract in DMSO was diluted in PBS to obtain final concentrations of 10, 50, and 100 μg/mL. Cells were treated with samples for 24 h and MTT solu- tion was added. After 4 h, the media were removed and DMSO was added to each well. The resulting absorb- ance was measured at 540 nm. DPPH radical-scavenging activity Briefly, RAW 264.7 cells were plated at 5 × 105 cells/well. After 4 h, the cells were treated with 10, 50, and 100 μg/ mL of C. officianalis for 24 h. The treated cell pellets were mixed with assay buffer and the supernatants were isolated. The working solutions were added to the super- natants and incubated at 37 °C. After 1 h, the absorb- ance was measured at 570 nm. The DPPH radical-scavenging activity was carried out according to the Blois method [27]. DPPH (0.3 mM) was added to each sample. After incubation for 30 min in the dark at room temperature, the absorbance was mea- sured at 518 nm using a microplate reader. Vit C was used as a positive control. The free radical-scavenging capacity was expressed by IC50. Intracellular reactive oxygen species scavenging activity RAW 264.7 cells were plated at 1 × 106 cells/well. After 4 h, the cells were treated with 10, 50, and 100 μg/mL C. officianalis and LPS for 24 h. After incubation, the cells were washed with PBS and harvested. The cells were then incubated with dichlorofluorescein diacetate (DCF- DA) (25 μM) for 30 min at 37 °C in the dark. After sev- eral washings with PBS, the fluorescence was captured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). DCF fluorescence was measured at an excitation wavelength of 488 nm and emission wave- length of 515–540 nm. Methods Reagents Folin-Denis reagent, sodium carbonate, aluminum chloride, potassium acetate, potassium persulfate, 1,1- diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinibis 3-ethyl benzothiazoline-6-sulfonic acid (ABTS), 2,4,6-tris(2-pyri- dyl)-s-triazine (TPTZ), iron(III) chloride hexahydrate, gallic Page 3 of 9 Hwang et al. BMC Complementary and Alternative Medicine (2016) 16:196 Page 3 of 9 Cells and culture RAW 264.7 cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). The cell lines were grown in DMEM with 10 % FBS and 1 % P/S, and incubated at 37 °C in 5 % CO2. ABTS radical cation-scavenging activity The absorbance was measured at 593 nm. The results are expressed as FeSO4 equivalents. Statistical analysis Statistical analysis was performed using SPSS (version 17.0; SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to calculate the mean and standard error of the mean (SEM). One-way analysis of variance was per- formed, and when significance (p < 0.05) was found, the Cell cytotoxicity assay h ff The cytotoxic effects of C. officianalis on RAW 264.7 cells were determined by exposing the cells to various concentrations of C. officianalis (10, 50, and 100 μg/mL) for 24 h. Our results showed that there was no cytotoxic effect on RAW 264.7 cells at the tested concentration (Fig. 2). Therefore, ethanol extracts of C. officianalis with concentrations from 10 to 100 μg/mL were selected for subsequent experiments. Xanthine oxidase inhibitory activity assay XO inhibitory activity was assayed using a commercial xanthine oxidase assay kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. Page 4 of 9 Page 4 of 9 Hwang et al. BMC Complementary and Alternative Medicine (2016) 16:196 differences of mean values were identified with Duncan’s multiple range tests. C. officianalis ethanol extract was shown to have scaven- ging activity in a dose-dependent manner; the scaven- ging activity was 40.7 % after exposure to 100 μg/mL of C. officianalis. Results and Discussion The FRAP assay measures total antioxidant activity via the reduction of the ferric tripyridyltriazine (Fe3+-TPTZ) complex to the ferrous form [30]. The ferric complex- reducing ability of C. officianalis was similar to the results obtained for the radical scavenging assay. C. offi- cianalis (100 μg/mL) demonstrated the highest FRAP value (241.5 mM), similar to that of Vit C (256.1 mM), the positive control. Total phenolic and flavonoid contents The total phenolic and flavonoid contents of the C. offi- cianalis extract were determined through a linear gallic acid and quercetin standard curve and are expressed as mg GAE/g and mg QE/g, respectively. As shown Table 1, the total phenolic and flavonoid contents of C. officiana- lis were 27.04 ± 0.61 mg GAE/g and 3.70 ± 0.08 mg QE/ g, respectively. According to Jeon et al. [30], the total phenolic and flavonoid contents of C. officianalis ethanol extract were 34.22 mg/g and 5.67 mg/g, respectively, similar to our findings. According to Hwang et al. [36], antioxidant activity, as observed in DPPH, ABTS, and FRAP assays, highly cor- relates with the total phenolic content. Therefore, in the present study, the excellent antioxidant activity of C. offi- cianalis indicates a high phenolic content. Phenolic compounds have been reported to be associ- ated with antioxidant activity, anticancer effects, and other biological functions, and may prevent the develop- ment of aging and disease [31–33]. Thus, our study re- sults suggest that C. officianalis extracts might have high antioxidant activities. Antioxidant capacities of C. officianalis ROS scavenging has been reported to be a very import- ant antioxidant mechanism in inhibiting lipid oxidation and aging in the human body [34]. The radical scavenging capacities, as determined by DPPH, ABTS and FRAP assays, are shown in Fig. 1. Upon interacting with DPPH, antioxidants transfer ei- ther an electron or a hydrogen atom to DPPH, thus neu- tralizing its free radical character. Therefore, absorption at 518 nm is proportional to the amount of residual DPPH [35]. The radical scavenging activity was found to be 28.4 %, 40.9 %, and 52.44 %, at 10, 50, and 100 μg/mL of C. officianalis ethanol extract (IC50 = 99.32 μg/mL), respectively. Our results (IC50 = 99.32 μg/mL) were higher than those obtained by Jeon et al. (IC50 = 154 μg/ mL) [31]. DPPH radical scavenging activity is hypothe- sized to depend on the growth and extraction conditions of C. officinalis. XO inhibitory activity XO XO, an enzyme present in significant concentrations in the gastrointestinal tract and liver, is responsible for the metabolism of hypoxanthine and xanthine to uric acid in the purine catabolic pathway, which produces superoxide radicals. XO is an important biological source of superox- ides and has been reported to be present in various patho- logical processes [37]. In recent years, a number of research groups have investigated potential XO inhibitors from a wide variety of traditional folk medicines. We confirmed the inhibitory effects of C. officianalis ethanol extract on XO activity. As shown in Fig. 3, XO activity decreased in a dose-dependent manner; the in- hibitory effects were 100.5 %, 85.0 %, and 57.0 % at 10, 50, and 100 μg/mL of C. officianalis ethanol extract, respectively. The ABTS assay measures the relative ability of an antioxidant to scavenge the ABTS generated in the aqueous phase by reacting a strong oxidizing agent (po- tassium persulfate) with ABTS salt. Reduction of the blue-green ABTS radical solution is measured [29]. The In a study by Kim et al. [38], plant polyphenols were found to inhibit enzyme activity by binding to the enzyme-substrate complex instead of directly binding to the active site of the enzyme. Therefore, the XO inhibi- tory activity observed by C. officianalis may be due to polyphenols in C. officianalis. Further studies based on the purification and identification of the polyphenol from C. officianalis extract is necessary to investigate the exact mechanism of XO inhibitory activity. Table 1 Total phenolic and flavonoids contents of Cornus officinalis ethanol extract Sample Total polyphenola (mg GAE/g) Total flavonoidb (mg QE/g) Cornus officinalis 27.04 ± 0.61 3.70 ± 0.08 aTotal phenolic content expressed in mg of gallic acid equivalent (GAE) per gram of dry weight of plants bTotal flavonoid content expressed in mg of quercetin equivalent (QE) per gram of dry weight of plants Table 1 Total phenolic and flavonoids contents of Cornus officinalis ethanol extract Hwang et al. BMC Complementary and Alternative Medicine (2016) 16:196 Page 5 of 9 Fig. 1 Antioxidant activities of various concentrations of Cornus officinalis. a DPPH scavenging activity. b ABTS radical scavenging activity. c FRAP value. Values are the mean ± SEM of experiments in triplicate (n = 3). Values expressed by different letters are significantly different at p < 0.05 Fig. 1 Antioxidant activities of various concentrations of Cornus officinalis. a DPPH scavenging activity. XO inhibitory activity XO b ABTS radical scavenging activity. c FRAP value. Values are the mean ± SEM of experiments in triplicate (n = 3) Values expressed by different letters are significantly different at p < 005 Fig. 1 Antioxidant activities of various concentrations of Cornus officinalis. a DPPH scavenging activity. b ABTS radical scaven Values are the mean ± SEM of experiments in triplicate (n = 3). Values expressed by different letters are significantly different a Intracellular reactive oxygen species scavenging activity To investigate the intracellular levels of ROS, the cell- permeable probe DCF-DA was utilized. Non-fluorescent DCF-DA, hydrolyzed to DCFH inside the cells, yields highly fluorescent DCF-DA in the presence of intracellu- lar hydrogen peroxide and related peroxides [39]. We Intracellular reactive oxygen species scavenging activity To investigate the intracellular levels of ROS, the cell- permeable probe DCF-DA was utilized. Non-fluorescent DCF-DA, hydrolyzed to DCFH inside the cells, yields highly fluorescent DCF-DA in the presence of intracellu- lar hydrogen peroxide and related peroxides [39]. We Intracellular reactive oxygen species scavenging activity To investigate the intracellular levels of ROS, the cell- permeable probe DCF-DA was utilized. Non-fluorescent DCF-DA, hydrolyzed to DCFH inside the cells, yields highly fluorescent DCF-DA in the presence of intracellu- lar hydrogen peroxide and related peroxides [39]. We examined whether C. officianalis extract inhibited LPS- induced ROS generation. As shown in Fig. 4, LPS treat- ment significantly increased ROS formation in RAW 264.7 cells, as determined by DCF fluorescence. How- ever, treatment with C. officianalis extract blocked LPS- induced ROS generation, similar to the results obtained Fig. 2 Cytotoxicity effect of Cornus officinalis on RAW 264.7 cells. Cells were treated with Cornus officinalis at different concentrations (10, 50, and 100 μg/mL). After treatment for 24 h, cell viability was measured with the MTT assay. Values are the mean ± SEM of experiments in triplicate (n = 3) Fig. 2 Cytotoxicity effect of Cornus officinalis on RAW 264.7 cells. Cells were treated with Cornus officinalis at different concentrations (10, 50, and 100 μg/mL). After treatment for 24 h, cell viability was measured with the MTT assay. Values are the mean ± SEM of experiments in triplicate (n = 3) Hwang et al. BMC Complementary and Alternative Medicine (2016) 16:196 Page 6 of 9 Fig. 3 Xanthine oxidase inhibitory effect of various concentrations of Cornus officinalis on RAW 264.7 cells. XO inhibitory activity XO Cells were treated with Cornus officinalis at different concentrations (10, 50, and 100 μg/mL). After treatment for 24 h, xanthine oxidase activity was measured with a commercial kit. Values are the mean ± SEM of experiments in triplicate. Values expressed by different letters are significantly different at p < 0.05 Fig. 3 Xanthine oxidase inhibitory effect of various concentrations of Cornus officinalis on RAW 264.7 cells. Cells were treated with Cornus officinalis at different concentrations (10, 50, and 100 μg/mL). After treatment for 24 h, xanthine oxidase activity was measured with a commercial kit. Values are the mean ± SEM of experiments in triplicate. Values expressed by different letters are significantly different at p < 0.05 from the antioxidant assays. This evidence suggests that C. officianalis extract may prevent the formation of ROS. (Additional file 1: Figure S1). In addition, we confirmed that C. officianalis ethanol extract was indeed respon- sible for the antioxidant activity in H2O2-induced RAW 264.7 cells by analyzing Cu/Zn-SOD, MnSOD, catalase, and GPx expression by immunoblotting. Similar to the gene expression results, the antioxidant enzyme levels increased in the C. officianalis-treated cells (Additional file 1: Figure S2). Genes and protein expression of antioxidant enzymes Genes and protein expression of antioxidant enzymes We assessed whether C. officianalis ethanol extract af- fected the expression of genes associated with the antioxidative system, such as Cu/Zn-SOD, Mn-SOD, catalase, and GPx genes, in RAW 264.7 cells. Our data show that C. officianalis ethanol extract significantly in- creased the expression of Cu/Zn-SOD, Mn-SOD, cata- lase, and GPx compared to that in the LPS-induced control group at 100 μg/mL (Fig. 5). To rule out general defects in oxidative stress in RAW 264.7 cells, the cells were treated with H2O2. The increased oxidative stress is related to an overproduction of free radicals or defi- ciency in the antioxidant defense system. The mRNA expression of antioxidant-related enzymes was reduced. However, the C. officianalis-treated group showed an in- crease in the antioxidant-related enzyme mRNA levels The SODs convert superoxide radicals into hydrogen peroxide and molecular oxygen (O2), while catalase and GPx convert hydrogen peroxide into water, and in the case of catalase, to oxygen and water. During homeosta- sis, SOD sufficiently inactivates superoxides. However, during pathological states such as oxidative stress and dis- eases, increased levels of superoxides are not inactivated by SOD in the cells and can result in ROS-induced damage. There are three SOD enzymes. MnSOD is localized in the mitochondria, Cu/ZnSOD is located in the cytoplasm and nucleus, and ECSOD is expressed Fig. 4 Effects of Cornus officinalis on LPS-induced intracellular ROS generation in RAW 264.7 cells. Cells were treated with Cornus officinalis extracts at different concentrations (10, 50, and 100 μg/mL). After treatment for 24 h, ROS generation was measured by DCFH-DA staining with flow cytometry analysis Fig. 4 Effects of Cornus officinalis on LPS-induced intracellular ROS generation in RAW 264.7 cells. Cells were treated with Cornus officinalis extracts at different concentrations (10, 50, and 100 μg/mL). After treatment for 24 h, ROS generation was measured by DCFH-DA staining with flow cytometry analysis Hwang et al. BMC Complementary and Alternative Medicine (2016) 16:196 Page 7 of 9 Fig. 5 Effect of Cornus officinalis extracts on the expression of Cu/Zn-SOD (a), Mn-SOD (b), catalase (c), and GPx (d) in RAW 264.7 cells. Cells were treated with Cornus officinalis extracts at different concentrations (10, 50, and 100 μg/mL). Values are the mean ± SEM of experiments in triplicate. p < 0.05 compared to the LPS-treated group. COE; Cornus officinalis ethanol extracts Fig. Genes and protein expression of antioxidant enzymes 5 Effect of Cornus officinalis extracts on the expression of Cu/Zn-SOD (a), Mn-SOD (b), catalase (c), and GPx (d) in RAW 264.7 cells. Cells were treated with Cornus officinalis extracts at different concentrations (10, 50, and 100 μg/mL). Values are the mean ± SEM of experiments in triplicate. p < 0.05 compared to the LPS-treated group. COE; Cornus officinalis ethanol extracts herbs and other dietary plants. Moreover, these results have also suggested that antioxidant enzyme activity is responsible for their antioxidant capacity [43–45]. In the present study, our data showed that C. officianalis extract has potent free radical-scavenging capacities for DPPH and ABTS radicals. Antioxidant capacities in C. officianalis were highly correlated with their antioxidant enzyme activities. High activity of SOD, catalase, and GPx increased the free radical-scavenging capacity of the C. officianalis-treated group. This study has shown that C. officianalis ethanol extract had significant antioxidant activity and increased antioxidant enzyme activity. Thus, we suggest that consuming C. officianalis fruit may be beneficial to human health. extracellularly in some tissues. Other antioxidant enzymes include catalase, which is found in peroxisomes and cytoplasm, and GPx, which can be found in many sub- cellular components including the mitochondria and nu- cleus [40–42]. In our results, the C. officianalis ethanol ex- tract suppressed oxidative stress by increasing antioxidant enzyme activities including those of SOD, catalase, and GPx. Thus, C. officianalis ethanol extract may have a strong antioxidant effect due to C. officianalis-induced ac- tivation of antioxidative enzymes at different locations within the cells. These antioxidant enzyme activities are positively correlated with antioxidant effects. The main function of antioxidant enzymes is to neutralize free radicals. Antioxidant enzymes may interrupt an oxidizing chain reaction to minimize the damage caused by free radi- cals. The risk of acquiring free-radical-related diseases could be reduced by decreasing exposure to free radi- cals and increasing the intake of antioxidants and anti- oxidant enzyme-rich foods. Many studies have shown a strong positive linear correlation between antioxi- dant capacity and antioxidant enzymes of medicinal Consent for publication p This information is not relevant. 24. Jang SE, Jeong JJ, Hyam SR, Han MJ, Kim DH. Ursolic acid isolated from the seed of Cornus officinalis ameliorates colitis in mice by inhibiting the binding of lipopolysaccharide to Toll-like receptor 4 on macrophages. J Agric Food Chem. 2014;62:9711–21. Conclusion The present study found that C. officianalis extract has strong antioxidant activity in cells via activation of the antioxidative enzyme system. Further studies of the mechanism of action of these compounds are under- way. Based on these results, C. officianalis appears to be a good natural antioxidant agent and could be of Page 8 of 9 Page 8 of 9 Hwang et al. BMC Complementary and Alternative Medicine (2016) 16:196 Page 8 of 9 significance in the food industry for treating various human and animal diseases. 5. Ghosh S, Bishayee K, Paul A, Mukherjee A, Sikdar S, Chakraborty D, Boujedaini N, Khuda-Bukhsh AR. Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation. J Integr Med. 2013;11:116–24. Abbreviations 11. Kim SM, Cho YS, Sung SL. The antioxidant ability and nitrite scavenging ability of plant extract. Korean Soc Food Sci Technol. 2001;33:626–32. ABTS, 2,2’-azinibis 3-ethyl benzothiazoline-6-sulfonic acid; DCFH-DA, dichlorofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DPPH, 1,1-diphenyl-1-picrylhydrazyl; FBS, fetal bovine serum; FeSO4, sulfate heptahydrate; GAE, gallic acid equivalent; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GPx, glutathione peroxidase; LPS, lipopolysaccharides; MTT, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; P/S, penicillin- streptomycin; QE, quercetin equivalent; ROS, reactive oxygen species; RT-PCR, real-time reverse transcription polymerase chain reaction; SEM, standard error of the mean; SOD, superoxide dismutase; TPTZ, 2,4,6-tris(2-pyridyl)-s-triazine; XO, xanthine oxidase 12. Khalafalla MM, Abdellatef E, Dafalla HM, Nassrallah AA, Aboul-Enein KM, Lightfoot DA. Active principle from Moringa oleifera Lam leaves effective against two leukemias and a hepatocarcinoma. Afr J Biotechnol. 2010;9: 8467–71. 13. Seog HM, Seo MS, Kim HM, Ahn MS, Lee YT. Anttioxidative activity of barley polyphenol extract (BPE) separated from pearling by-products. Korean Soc Food Sci Technol. 2002;34:889–92. 14. Heinonen M. Antioxidant activity of plant extracts containing phenolic compounds. J Agric Food Chem. 1999;47:3954–62. 15. Joo SJ, Park JH, Seo BI. Effects of Korean Corni fructus on treatment of osteoporosis in ovariectomized rats. The Korea J Herbology. 2007;22:83–95. Funding h g This project was financially supported by National Academy of Agricultural Science, Rural Development Administration, Korea. This project was financially supported by National Academy of Agricultural Science, Rural Development Administration, Korea. 18. Yamahara J, Mibu H, Sawada T, Fujimura J, Takino S. Antidiabetic principles of Corni fructus experimental diabetes induced by streptozotocin. Yakugaku Zasshi. 1981;101:86–90. Received: 7 January 2016 Accepted: 14 June 2016 Received: 7 January 2016 Accepted: 14 June 2016 25. Folin O, Denis W. On phosphotungastic-phosphomolybdic compounds as color reagents. J Biol Chem. 1912;12:239–49. 26. Moreno MIN, Isla MI, Sampietro AR, Vattuone MA. Comparison of the free radical scavenging activity of propolis from several region of Argentina. J Ethnopharmacol. 2000;71:109–14. Availability of data and materials The datasets supporting the conclusions of this article are included within the article and its additional files. 19. Dai Y, Hang B, Huang Z. Inhibition of Corni fructus on experimental inflammation. China J Chinese materia medica. 1992;17:307–9. 20. Akhavan N, Feresin R, Johnson S, Pourafshar S, Elam M, Hsieh YH, Arjmandi B. Cornus officinalis Modulates the Production of Pro- Inflammatory Molecules in Lipopolysaccharide-Activated RAW264.7 Macrophages. FASEB J. 2015;29:922–30. Acknowledgements This study was supported by the “Research Program for Agricultural Science & Technology Development (Project No. PJ010946),” National Academy of Agricultural Science, Rural Development Administration, Korea. 16. Seo KI, Lee SW, Yang KH. Antimicrobial and antioxidative activities of Corni fructus extracts. Korean J Food Preservation. 1999;6:99–103. 17. Lee NH, Seo CS, Lee HY, Jung DY, Lee JK, Lee JA, Song KY, Shin HK, Lee MY, Seo YB, Kim HK, Ha HK. Hepatoprotective and antioxidative activities of Cornus officinalis against acetaminophen-induced hepatotoxicity in Mice. Evid Based Complement Alternat Med. 2012;2012:804924. Additional file 6. Khansari N, Shakiba Y, Mahmoudi M. Chronic inflammation and oxidative stress as a major cause of age-related diseases and cancer. Recent Pat Inflamm Allergy Drug Discov. 2009;3:73–80. Additional file 1: Figure S1. Effect of Cornus officinalis extracts on the expression of Cu/Zn-SOD (a), Mn-SOD (b), Catalase (c), and GPx (d) genes in RAW 264.7 cells. Cells were treated with H2O2 (100 μM) and Cornus officinalis extracts at different concentrations (10, 50, and 100 μg/mL). Values are the mean ± SEM of experiments in triplicate. Values expressed by different letters are significantly different at p < 0.05. COE; Cornus officinalis ethanol extracts. Figure S2. Effect of Cornus officinalis extracts on the protein expression of antioxidant enzymes in RAW 264.7 cells. Cells were treated with Cornus officinalis extracts at different concentrations (10, 50, and 100 μg/mL). (PPTX 148 kb) 7. Delaporte RH, Sanchez GM, Cuellar AC. Anti-inflammatory activity and lipid peroxidation inhibition of iridoid lamiide isolated from Bouchea fluminensis (Vell.) Mold. (Verbenaceae). J Ethnopharmacol. 2002;82:127–30. 8. Feillet-Coudray C, Sutra T, Fouret G, Ramos J, Wrutniak-Cabello C, Cabello G, Cristol JP, Coudray C. 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W2017895326.txt	https://zenodo.org/records/1631399/files/article.pdf	de		XXIX. Über die otogene Pachymeningitis interna purulenta	European archives of oto-rhino-laryngology/European archives of oto-rhino-laryngology and head & neck	1,910	public-domain	14,424	IX. Mitteilung alas der Klinik ftir Ohren- und Halskrankheiten des Kommunehospitals in Kopenhagen. (Chef: Professor Dr. tI. ~Iygind). XXIX. Oberdie otogene Pachymeningitisinterna purulenta. Von N. Rh. Blegvad, Assistenzarzt der Klinik. (~it Tafel IX). Die otogene Paehymeningitis interna puru!enta wird im allgemeinen fiir eine seltene Krankheit angesehen, ihr wird daher yon den 0toehirurgen nur wenig Beachtung gesehenkt. Sie tritt indessen sieherlieh h~ufiger auf, als angenommen wird, und verclient aus diesem Grunde eine grSi~ere Beaehtung; sic bildet aller Wahrseheinlichkeit nach oft die Einleitung zu einer Leptomeningitis, die dureh reehtzeitiges Eingreifen wiirde ~erhtitet werden kiinnen, und viele F~ille yon sogenannter ,,zirkumskripter Meningitis" stellen sicher eine Pachymeningitis interna vor. K ii r n e r (20) unterscheidet die diffuse Pachymeningitis interna yon dem ,subduralen Abszei~ oder CortiealabszeS." Aus Griinden, die weiter unten angefiihrt werden, wird man diese Unterseheidung vermutlich ebenso gut unterlassen kiinnen, und daher werden hier alle Formen der otogenen Paehymeningitis interna purulenta zusammengefal~t behandelt werden, worunter ein Entziindungsprozei3 in der Dura mater zu verstehen ist, der eine Absonderung von Eiter auf die eerebrale Fl~ehe der Dura verursaeht. Das Leiden wird in der Regel mit einem ,intrameningealena und ,,subduralen" Abszel} synonym sein. Es werden nur die Formen yon Paehymeningitis in Betraeht gezogen, die direkt vom Ohrenleiden induziert werden, und nieht diejenige, die z. B. dadureh entsteht, da~ ein Gehirnabsze• in den Subduralraum hinausperforiert. Der Ausgangspunkt dieser Untersuehungen bildet die nachfolgende Gesehiehte eines Krankheitsfalles. 248 IX. N. RH. BLEGVAD, Ketty Kragh L., 10 Jahre alt, Tochter eines st~dtisehen Arbeiters, wurde am 13. Juni 1909 in die 0hren- und Halsklinik des Kommunalhospitals a u g genommen. A n a m n e s e : Mehrere Jahre hindurch periodischer Ausflul~ aus dem l i n k e n 0hre, oft. mit vorausgehenden 0hrensehmerzen. Im April d. J. t4 Tage lang Ausflu/~, sonst kein Ausflui} bis v e t vier Tagen, we sich wieder solcher aus dem linken 0hre einstellte, nachdem 24 Stunden lang Schmerzen im 0hre vorhanden gewesen waren. Drei Tage vorher war Patientin im Seebade gewesen. Am 9. Juni mugte sie, nachdem sie in der Nacht heftige Ohrenschmerzen gehabt hatte, wegen Kopfschmerzen, Erbreehen, und Schmerzen im linken Ohre die Schule vorzeitig verlassen, sie hatte subektive Febrilia, jedoch keinen Schiittelfrost. Patientin war seitdem bett~igerig, sie hatte Fieber, jedoch keinen Schiittelfrost, ab und zu Phantasieren, best~indig Erbrechen, Stirnkopfschmerzen, ab und zu Schmerzen im linken Ohre. Lag teilweise im ttalbschlummer. Am 11. Juni zeigto sich hinter dem linken Ohre eine Anschwellung, die langsam zunahm, die Schmel~en hSrten gleichzeitig auf; der Ausflul~ hat sich unver~ndert geh~lten. K e i n e Symptome der Respirationsorgane. Keine anderen cerebralen Symptome als die angefiihrten. Status praesens. Temp. 41,3, Puls 120, Resp. 16. Patientin ist stark kongestioniert im Gesicht mit recht starker Cyanose der Lippen; kein Herpes. Zunge belegt. Foetor ex ore. W~ihrend tier Untersuchung spricht sie wiederholt vollst~indig verwirrt, reagiert jedoch normal auf Aufforderungen, sich aufzurichten, die Zunge zu zeigen usw. Keine ,,slow cerebration". Keine Nackenstarre, kt'ine Km'nigs oder Babinskis Symptome. Kniereflexe zwar schwach, jedoch vorhanden. Keine Paresen. A u r. s i n.: Sehr reichlicber, br~iunlicher, stinkender Eiter im Geh6rgange. Nach der Reinigung war der Hintergrund fast ganz yon Granulationsmassen bedeekt, die sich von 0ben und yon vorn vorwSrtsschoben ; beim Driicken auf die Intumeszenz hinter dem 0hre entstr6m..t dem GeMirgange reichlicher Eiter. Regio m a s t . s i n . : Auf dem Ubergange zwischen der Auricula und dem Schiidel befindet sich aufwiirts eine leicht gespannte, fluktuierende Intumeszenz, die das Ohr ein wenig n.ach augen ddingt; die Haut ist etwas schmerzhaft, jedoch nicht brennend. Uber der Spitze des Prec. mast. etwas Infiltration, aber keine Druckempfindlichkeit. Auf beiden Seiten des Halses kleine, indolente Driiseu. -- Bei extremem BIick nach links (krankes 0hr) zeigen sich ein paar kleine, rotat.prisctie Nvstagmus-Bewegungen, worauf das A u g e zur Ruhe kommt; etwas Ahnliches, jedoch weniger ausgepr~gt, beim Blick nach rechts (gesundes 0tlr). Kalorische Probe: Beim Ausspiilen des linken 0hres mit 12 0 Wasser tritt rotatorischer Nystagmus sehr schnell ein F lii s t e r s t i m m e linkes Ohr 0, S p r e c h s t i m m e : 0,25 Meter, mit B a r a n y s Lfirmapp. ira rechten 0hre: 0,05 Meter. A u r. d x : Nach hinten groge, trockene, randst~indige Perf. P h a r y n x : Leichte akute RSte. S t e t . p u l m . : Etwas kurzer Ton in der linken Reg. axiUae. Auf beiden Riickfl~chen Rhonchi vorhanden, keine deutlichen Resp-Ver~nderungen. Kein Rassel- oder Reibungslaut. S t e t . c o r d i s n o r m a l . O p h t h a l m o s k o p i e (Herr Dr. med. C h r . F. B e n t z e n ) normal. U r i n normal. 0 p e r a t i o n I. Sofort nach der Einlieferung wird um 4 t/~ Uhr nachmittags unter C h 1 o r o f o r m n a r k o s e (mit Riicksicht auf eine mSgliche Pnemnonie) L u m p a I p u n k t u r mit Entleerung einer klarewFliissigkeit vorgenommen [die Mikroskopie des zentrifugierten Bodensatzes zeigte sp~tter nur ganz wenige cellultire Elemente, im wesentlichen mononukle/ire, kleino Lymphocyten. Keine Mikroben, keine GewKchse auf .~gar nach vier Tagen.] Darnach R e s e c t i o totalis a u r i s r e e d . s i n . Eswerdenca. 5Gramm dunklen, stinkenden Eiters aus einem subperiostalen Abszeg entleert. Corticalis nicht zerstSrt, aber unter dieser findet sich reichlicher stinkender Eiter, der yon einem perisinnSsen AbszeB pulsierend hervorstr6mt. [Im direkten Pr~iparat dieses Eiters sieht man zahlreiche Mikroben verschiedener Art Kokken und St~ibe), u.a. Streptekokken]. Das Mittelohr ist yon cholestea- Ueber die otogene Pachymeningitis interna purulenta. 249 tomatfsen Massen angefiillt, die bis ganz zur Dura hinaufreichen, die bloBgelegt und in grofier Ausdehnung auf der Aufienseitc nfit Granulationen besetzt ist. S i n u s t r a n s v , w i r d i n s e i n e m g a n z e n s e n k r e c h t e n Ver\| a u f e b 1 o B g e 1 e g t, belm obersten Knie wird Whiting-hleiers Tamponade angewendet. Sinus wird gespalten und seine laterale Wand exstirpiert. Ein recht frischer, flach gedrfickter Thrombus wird entfernt, der sich nach unten gegen den Bulbus fortsetzt, aafw~rts stiirzt beim Entfernen des Knietampons reichlich Blut hervor. Verlauf. 14. 6. Temp. 41,3--38,4--37,6--38,6. (Temp. 12 Uhr mittags: 41,3, 6 Uhr abends: 38,4, 12 Uhr Mitternacht: 37,6, am 14. 6. 6 Uhr frfih: 3S,(i.) Puls 106--96--144--129. Patientin befand sich ganz gut bis heute friih 5 Uhr, wo sich ein leichter Schiittelfrost einstellte, worauf die Temp. 39,4 war. Darauf war das Befinden der Patientin recht gut, sie war aber ersichtlich aufgeregt und zuweilen unklar, lm fibrigen keine Cerebralsymptome. U r i n normal. 15. 6. Temp. 38,7.-38--39,5--39,6. Puls 120--100--100--108. Lag den gr6Bten Tell des gestrigen Tags im Halbschlummer, war a b e t ganz vernfinftig, wenn sie wach war. Kein Schiittetfrost oder Cerebralsympt. bis heute 5 Uhr fl'iih, wo sie, nachdem sie die Nacht geschlafen hatte, sehr unruhig und unklar wurde und aufschrie. Darauf lag sm abwechselnd still im Halbschlummer oder mit den krmen um sich schlagend und Grimassen schneidend. Sie antwortete nicht auf Fragen, klagt, wenn man sie anrfhrt. Keine Nackensteifheit, beginnend K e r n i g (bei ca. 120 °) - ~ B a b i n s k i . O p p e n h e i m dorsal. Kein spontaner Nystagmus. Bei der L u m b a l punktion werden 10 ccm etwas unklare Flfissigkeit entzogen. [tin t~r~iparat des zentrifug. Bodensatzes sind reichliche Mengen Eiterk6rper zu sehen. Mononukle~re: polynukle~ire ungefi~hr ,= 1:5. Keine Mikroben. Auf A g a r kein Gew~ichse nach 5 Tagen]. Rp. Hexamethylentetramin ctgr 50 4 mal t~glich. O p e r a t i o n II. (2 Tage nach der ersten Operation.) In der _&thernarkose wird um 4~)2 Uhr nachm. C r a n i o t o m i a vorgenommen, indem man die bei der vorigen Operation gebildete Craniotomie6ffnung so erweitert, dab sie nngeffihr die Gr6fie einer Kinderhandfl~iche erh~lt. In die D u r a wird ein K r e u z s c h n i t t gelegt. Indem man die obersten Duralappen in die H6he hebt, str6mt eine reichliche Menge dfinnflieflenden, stinkenden Eiters heraus; und indem man einen Kupferstreifen zwischen Dura und Arachnoidea hinaufschiebt, dringt ununterbrochen Eiter hervor. [Bei der sp~iteren l ~ I i k r o s k o p i e dieses Eiters sind zahlreiche St~be und Kokken verschiedener Art zu sehen.] Man legt daher mit dem MeiBel ca. 2 ~/.~ cm h f h e r hinauf eine runde s e k u n d ~ r e Craniotomiefffnung, ungefiihr senkrecht fiber Proc. mast. Indem man nun mit einem Kupferstreifen unter die Dura geht, verfolgt man fiberail den Eiter, bis solcher schlieBlich nicht mehr hervorsickert, wenn man das Gehirn mit dem Streifen yon der Dura abdl~ickt. Es zeigt sich nun, daBt dieser gtofle, subdurale AbszeB sich erstreckt hat: nach vorn bis zum vordersten Rande der obersten Craniotomie5ffnung, nach oben bis zu 2 ~/.~ cm fiber deren oberen Rand, nach hinten 4 cm hinter deren hinteren Rand und nach unten bis ganz zum Tentorium. Indem man versucht, die Dura von der Arachnoidea zu 15sen, zeigt es sich, dab an vielen Stellen kleine bindegewebeartige Adh~renzen vorhanden sind. Die Innenfliiche der Dura ist an mehreren Stellen samtartiff und bier und da sind Andentungen yon fibrin6sen Membranen zu finden. Gyri sind leicht abgeflacht, und aie Leptomeningen sind an mehreren Stellen verdickt und weiBlich. Es werden nun zwischen Dura und Arachnoidea Jodoformgazestreifen in verschiedenen Richmngen sowohl in der oberen wie in der unteren Craniotomie6ffnung angebracht. 16.6. Temp. 37,8--38,7--39,6--39,7. Puls 112--112--104--108. Zustand nach der Operation ungefahr unver~indert. Keine erkennbare Nackensteifheit. K e r n i g deuflicher ausgepr'£gt. 17.6. Temp. 38,8--38~6 - 39,2 -39,3. Puls 120-- 112--] 20--100. Patientin 250 IX. N. RH. BLEGVAD. war gestern sehr unruhig und schlug mit Armen und Beinen um sich. Hat reichlichTgetrunken , die ganze :Nacht geschlafen. Heute ein kleiner Krampfanfall, ~ ~ihrend dessen Dauer sie am ganzen Kiirper steif war, mit den Ziihnen knirschte, schielte und den Kopf nach rechts drehte. Auf Anrede gab sie keine Antwort, aber tat, was man ihr hiel~, und liel~ Wasser auf Aufforderung. Bei der L u m b a l p u n k t i o n wurden 20 ccm FIiissigkeit gewonnen, die triiber ist als die vom 15. 6. [Sie enthi~lt eine reichliche Menge polynuklei~rer, jedoch nur ganz wenige mononukle~ire Leukocyten. Mikroben sind nicht zu sehen, huf A g a r Gew~ichse nach zwei Tagen.] 18. 6. Temp. 38,7--41,3--40,2--37,5. Puls 96--136--120--112. Den grSBten Tell des gestl4gen Tages mit Halbschlummer und Schlaf verbracht. Nur hin und wieder etwas unruhig. Hat reichlich Milch genossen, und der Zustand war w~hrend der letzten 24 Stunden im ganzen ersichtlich besser. Kein Schiittelfrost. Keine anderen Cerebralsympt., als Mattigkeit. 5~euer V e r b a n d a n g e l e g t . Hierbei erwacht sie und ja~amert, sagt aber nichts. Der Verband riecht weniger als bisher, und die Wunden sehen reiner aus. Es ist nur geringer Prolaps und nur in der unteren CraniotomieSffnung. Jodoformgaze. Rp. Phenosalylumschlag, der zweimal t~iglich erneuert wird. O p h t h a l m a s k o p i e normal. 19. 6. Temp. 37,2--37,4--38,5--38,1. Puls 104-102--120--116. Lag gestern im IIalbschlummer. Gestern abend 7 ~!~ Uhr recht heftiger Anfall yon Schiittelfrost yon einer Viertelstunde mit Cyanose. Temp. darauf 40,3. W~hrend des Schilttelfrostes u n d bis eine halbe Stunde darnach Grimmassierung beider Gesichtsseiten. Dicses hat sich letzte Nacht fiinfmal wiederholt. Sie schrie auch auf. In der A t h e r n a r k o s e wird L u m b a l punktion ausgefiihrt, Entleerung yon 20 ccm nur wenig unklarer, schwach gelblicher Fliissigkeit die bedeutend weniger unklar ist als bei der letzten Punktur. [Der centrifug. Bodensatz enth~lt nur ganz wenige degenerierte Zellen, deren Kerne sich nicht distinkt fiirben lassen (24 Stunden Dauer.) Die mononukle~ren Leukocyten scheinen jedoch verh~ltnism~l~ig reichlich vertreten zu sein. Mi.kroben sind nicht zu sehen.] D a r a u f w i r d d e r V e r b a n d g e w e c h s e l t . UberaU stinkende Suppuration. Gehirnprolaps etwas grSfler mit Nekrose auf der Oberfl~iche. Tampons im subduralen Raum ohne Geruch, und hier scheint kein Eiter zu stagnieren. D i e s e T a m pons werden erneuert. Phenosalylumschlag. ICp. Chlorkalkwa.~serumschlag. 20. 6. Temp. 37,3--40,5--38,8--39,8. Puls 100--128--120--160. Liegt abwechselnd d~immernd und unruhig. Gestern abend gegen 5 Uhr heftiger Schiittelfrost mit starkem horizontalem Nystagmus zum rechten (gesunden) Ohre; gleichzeitig chronische Zuckungen der Muskeln der rechten Gesichtsh~lfte, besonders Orbicularis oris. Am Morgen abermals zwei SchiittelfrostanfMle mit Nystagmus und Gesichtszuckungen. Keine Zuckungen in den Armen und Beinen. K e r n i g sehr stark, bei ca. 100 °. Es zeigt sich unzweifelhaft weniger starke lnnervation des rechten Facialis, besonders des unteren Zweiges. Kaum Extremiti~tenparese. 21. 6. Temp. 37,6--38,3--40--38,5. Puls 112--I04--120--I~6. Zustand unver~indert. Nachts 12 Uhr Schiittelfrost. Liegt mit stark zuriickgebogenem Kopfe; ausgesprochene 57ackensteifheit. Starker K e r n i g. Deutlicher B a b i n s k i. V e r b a n d e r n e u e r t. Die ganze Wundenkavit~t yon nekrotischen Granulationen bedeckt. Prolaps bedeutend grSfler mit zunehmender Nekrose. Sekret stark stinkend. 22. 6. Temp. 41,1~-40--37,7--3~. Puls 150--160--104--90. Gestern zweimal, heute morgen einmal Schiittelfrost. Zustand unver~ndert; nicht eigentlich unklar, jedoch d~immernd. Prolaps jetzt so grofi wie eine &pfelsine und vSllig schwarz. 23. 6. Temp. 39,7--38,9--40,1--40. Puls 128--112--132--128. Zustand fast etwas besser. Antwortet durch IWicken. Patientin hat in den letzten 24 Stunden die rechten Ober- und UnterextremitRten nicht bewegt. Zwei Schiittelfrostanfiille. Ueber die otogene Pachymeningitis interna purutenta. 251 24. 6. Temo. 38,6--39,2--40--39,5. Puls 120--112--120--116. Stumpfsinniger als friiher. Einmai Schiittelfrost. Unzweifelhaft ]?arese des rechten Armes, anscheinend auch des rechten Beines. Recht starke Gangraen des Frolaps. 25.6. Temp.40,2--38,3-39,6--40,5. Puls 136--72--80--120. Zunehmende Somnolenz. Zweimal Scbiittelfrost. 26. 6. Temp. 40,2--39,5--40,6--31. Puls 136--136--124--136. Einmal Sehiittelfrost. Liegt vSllig teilnahmslos darnieder. Genierit nichts. Der gangriinSse Prolaps halb abflieriend, an GrSrie zugenommen. 27. 6. Temp. 41--41,1. Fuls 136--100. Kollabierte und starb gestera Abend 113/4 Uhr. Sektion des Seh~dels. In der liuken Temporalgegend ist im SchSdel eine ()ffnung yon der GrSrie einer Kinderhandfl~che zu sehen, die teilweise dem Proc. mast. entspricht. 21/2cm welter hinauf ist eine TrepanationsSff.nung yon der Gr0rie eines Zweimarkstllckes vorhanden, und aus.. beiden 0ffnungen quillt das gangr~in~ise Gehirngewebe heraus. Bei der Offnung des Sch~dels zeigt die Dura auf der linken Seite in hohem Marie schlechte Farbe und zwischen Dura und Cerebrum befindet sich ein grorier Abszeri mit um'einem, sehr stinkendem Eiter; entsprechend diesem Abszeri sieht man aufdem Cerebrum eine bedeutende Depression des Oceipitullappens. Die Oberfl~iehe des Gehims ist hier mit einer schleehffarbigen Eiterschicht bedeckt, u n d e s ist eine purulente Infiltration der Pin vorhanden. Aurier auf dem Occipitaltappen liegt auch auf dem linken Frontallappen Eiter, wo auch eine geringere Depression des Gehirns Vorhanden ist, sowie auch auf der Oberseite der linken Seite des Tentoriums (s. Fig. 1), Auf der Medialseite der linken Hemisph~ire befindet sich nach hinten eine purulente Decke. - - Auf der rechten Hemisphere ist die Pin makroskopisch natfirlich, jedoch ist im Subduralraum etwas diinne purulente Fliissigkeit, und auf der Inneitseite der Dura sind zum Teii diinne, purulente Membranen vorhanden. Mikroskopische Untersuchung (Herr D r . . m e d . . E l l e r m a n m . D u r a s i n. (you der Occipitalgegend) (s. Fig. 2) ist stark verdickt bei bedeutender Leukocyteninflltration des Gewebes. In den Venen sind Leukoten oder Thromben im Lumen. Auf der Innenseite eine dicke Schicht brin mit mehr oder weniger hinfiflligen Leukocyten. P i a s i n. (vonder Frontalgegend). Eine starke Anschwellung ist vorhanden, die ihre Ursache hat in der Aufh~iufung van Fibrin und polynukle~lren Leukocyten zwischen den Bindegewebsbiindeln. In den feinen Gef~Ben fast nur Leukocyten. Auf der Aurienseite eine m~ichtige Schicbt yon Eiterzellen und Fibrin. Die Eiterzellen sind oft degeneriert. Das Himgewebe ist natiirlich ohne Leukocyteninfiltration. D u r a d ext. (you der Temporalgegend) (s. Fig. 3). Auf der Innenseite eine Schicht yon EiterzeUen, die yon Fibrinmassen zusammengehalten werden. Die innerste Schicht der Dura fast iiberall mit Eiterzellen infiltriert, und diese Infiltration dringt an manchen Stellen reeht tief in das Gewebe ein. Aurierdem sind an vielen Stellen eiuzel liegende polynukle~re Leukocyten zu sehen. Die Arterien haben natiirliches Aussehen. In den Venen sind verh~iltnism~iriigviele Leukocyten. P i a d e x t. (yon der Temporalgegend) ist nicht angeschwollen. Kein Exsudat um die Gef~ii~e. Das Gewebe ist vielleieht etwas mehr zelienreieh als normal, doch ist keine Infiltration mit Leukocyten zu bemerken. Im bakteriengef~rbten Schnitte sind iiberalI, wo Exsndat vorhanden ist, tells St~be, tells Kokken und zwar sowohl Diplokokken, als haufenweise geordnete Kokken, zu sehen. ~Tbrige Sektion zeigt nichts Abnormes. ~ii 252 IX. N. RH. BLEGVAD. Epikrise. Hier handelt es sich um ein zehnjahriges M~dehen, bet dem sieh wiihrend des Verlaufes ether chronischen Mittelohreiterung Benommenheit, Unklarheit, Erbrechen~ Fieber, Kopfsehmerzen und Ohrensehmerzen sowie ein subperiostaler AbszeI~ hinter dem Ohre z e i g e n . Bet der Operation zeigt sich, daft Cholesteatom and Thrombose des Sinus transversus sowie Caries des Tegmen antri vorhanden ist, so dal~ die Dura blo~gelegt und mit Granulationen bedeckt ist. Man hiilt bet der Dura inne r weil keine fokalen tiirnsymptome da sind~ und man erwartet, dal3 die diffusen ttirnsymptome sieh verlieren werden, naehdem der prim~ir infizierte Focus entfernt ist. Indessen stellen sich naeh der Operation SchiittelfrSste ein~ und zwei Tage sprite r ist das Lumbalpunktat~ das friiher klar war~ unklar und enth~lt polynukle~ire Leukocyten und es zeigt sich beginnender K e r n i g. Daher wird sofort Craniotomie vorgenommen, wobei sich herausstellte, dab sich eine sehr groBe E i t e r a n s a m m l u n g u n t e r d e r D u r a befindet, die verschiedene St~ibe und Kokken~ ebenso wie der Eiter vom Proe. mast. enthiilt. Es wird sine grol~e und eine kleine Craniotomie5ffnung angelegt; der Eiter wird entfernt~ und es werden Jodoformgazem6chen hineingelegt. In der darauffolgenden Zeit verschlimmert sieh der Zustand, es treten andauernd SehiittelfrSste auf~ hin und wieder auch Konvulsionen, Benommenheit~ sensorische Aphasie, zunehmender K e r n i g . Das Lumbalpunktat ist zwei Tage nach der letzten Operation noch unklarer und mit zahlreichen polynuklefiren Leukocyten, jedoch keinen Bakterien 7 angefiillt; vier Tage nach der Operation ist es indessen nut ganz wen~g unklar, und es enthiilt nut ganz wenige zelluliire Elemente7 im wesentlichen mononukleiire Leukocyten. Trotzdem ist keine Besserung im Zustande der Patientin festzustellen. Eine Retention seheint im Subduralraume nicht vorhanden zu sein, es hat sich aber ein grol~er ttirnprolaps gebildet, der in den folgenden Tagen gangr~Lniert. Die cerebralen Symptome nehmen jetzt zu: es treten auf lqaekenstarre 7 B a b i n s k i ~ Parese der kontralateralen Extremit~iten und Gesichtsh~lfte~ andauernde SehiittelfrSste and hohe Temperatur Ibis 41,1). Man nimmt an, dab eine progredierende Encephalitis vorhanden set, und sieht daher yon weiteren Eingriffen ab. Patientin stirbt 14 Tage naeh der Mittelohrresekfion7 t2 Tage nach der Craniotomie. Wie sieh aus der Sekfion ergab, war auf beiden Seiten eine diffuse Paehymeningitis interna purulenta vorhanden, die auf der Ueber die otogene Pachymeningitis interna purulenta. 253 linkea Seite zur Bildung von groflen subduralen Eiteransammlungen gefiihrt una bier zugleich eine purulente Leptomeningifis mit sich gefiihrt hatte. Die Verh~iltnisse auf der reehten Seite zeigen augenscheinlich einen Prozel~, analog demjenigen auf der linken Seite, jedoeh an einem friiheren Zeitpunkt tier Entwicklung. Die Entziindung ist hier auf die Dura lokalisiert, ohne dal~ - - weder makro- noch mikroskopiseh -- irgendwelehe Entziindungsphiinomene in der Arachnoidea oder in der Pia naehgewiesen w erden kSnnen. In der Literatur habe ieh 43 F~lle yon Paehymeningitis interna purulenta otitischen Ursprungs gefunden, yon denen jedoch nur 27 zur Beleuehtung des Krankheitsbildes Verwendung finden kSnnen. Die iibrigen waren mir tells nur als kurzgefafSte Referate zugiinglich, tells war deren Besehreibung zu kurz gefagt oder mangelhaft, und tells waren die F~ille der Art~ dab die Diagnose nieht mit Sicherheit gestellt werden konnte. Dieses gilt z. B. yon G. Kiiirs (18) Falle; er ist Abseessus lobi frontalis genannt, aber aus der Besehreibung" geht nur das eine mit Sieherheit hervor, dab Eiter unter der Dura war, u n d e r gleicht vSllig anderen Fiillen yon Pachymeningitis interna. Einige franz5sisehe Verfasser, z. B. C h a v a s s e (6), bezeichnen mit ,,Absc~s sous-dure-m6rien" augenscheinlich oft epidurale Abszesse, und in einzelnen Fi~llen, z. B. L a n n o i s & C o r n e l o u p (23), ist es nach den Krankheitsgesehichten unmSglich zu entscheiden, ob der AbszeI~ unter oder auf der Dura lag. B e e o s (4) Fall 2 ist sichertich ein Abszel~ im Gehirn. Auf der Grundlage der 27 in der Literatur mitgeteilten F~ille in Verbindung mit dem in der Ohrenklinik des Kopenhagener Kommunehospitals beobaehteten soll hier versueht werden, das Krankheitsbild aufzurollen. Atiologie. Von den Erkrankten waren 15 Mii,nner, 5 Frauen und 6 Kinder zwischen 1'/2 und 15 Jahren. Ein Patient war 42 Jahre, ein anderer 48 Jahre alt, fiir die iibrigen stellte sieh das Alter zwischen 17 und 39 Jahren. Der Ausgangspunkt war fast immer eine e h r o n i s c h e S u p p u r a t i o n , oft mit Cholesteatom verbunden (6 Fiille). Dieses Verh~ltnis hiingt wahrscheinlich damit zusammen, da[~ das Leiden in der Regel nur entsteht, wenn eine ausgedehnte Destruktion Archly f, Ohrenheilkunde. Bd, 83, t7 254 IX. N. RH. BLEGVAD. des Knoehens vorhanden ist. ~ur in 2 Fallen, U f f e n o r d e s (36) Fall 1 und M a c e w e n s (28) Fall 13, war das Ohrenleiden akut bzw. subakut. Nur 6 F~ille sind bakteriologisch untersucht. Bei zweien wurden versehiedene Kokken und Sffibchen (F~ulnisbakterien) gefunden, in zwei anderen land man Staphylokokken, in einem Streptokokken, in einem Streptococcus mucosus. Pathogenese. Die Dura wird fast immer per continuitatem angegriffen. Durch eine info]ge eines destruktiven Prozesses hervorgerufene ()ffnung oder eine Dehiszenz in dem Tegmen antri oder tympani oder an einer anderen Stelle des Os temporis pflanzt sich die Entziindung zur Dura welter, die dabei in der Regel zuerst auf der Au[~enseite angegriffen wird, so dal~ sie hier yon fibriniisen Ablagerungen (Pachymeningitis externa) besetzt wird, oder es entsteht ein epiduraler Abszel~ (ll F~ille), zuweilen gleichzeitig eine Sinusthrombose. Von der Aul~enseite der Dura schreitet die Entziindung danach welter zur Innenseite, und in verschiedenen Fiillen zeigte sich yon der Dura infolge des Prozesses ein grSLferer oder geringerer Tell destruiert, so dab eine grSfiere Offnung oder Fistel zur Eiteransammlung im Subduralraum fiihrend gefunden wurde. Jedoch sind F~tlle verSffentiicht: D e l s t a n c h e (8)~ C h e v a l (7) und A l e x a n d e r (2), bei denenes scheint, als ob die Entziindung auf der Innenseite der Dura entstanden w~ire, ohne dal~ die Au~enseite zuerst angegriffen war, da die Dura an der Stelle, die bei der Operation blol~gelegt wurd% ein natiirliches Aussehen zeigte. Dal~ sich in zwei von diesen F~illen, D e l s t a n c h e (8), C h e v a l (7), eine kleine Fistel in der Dura fand, spricht nieht absolut geg'en diese Pathogenese~ da man sehr wohl annehmen kann, dai~ eine solche Fistel sekundiir yon der subduralen Pusansammlung" entstehen kann. In keinem dieser beiden Fi~lle liegt Sektion vor, so dab es nicht ausgeschlossen ist7 dal~ die Entztindung sich doch an irgend einer Stelle der Dura, die bei der Operation nicht exponiert wurde, sich per continuitatem verbreitet hat. Und diese Pathogenese ist jedenfalls die allerh~tufigste. A l e x a n d e r (2) hSlt fiir die wahrscheinlichste Pathogenese seines Fal\|es~ da[~ sich die Entziinclung' l~ngs der kleinen Venen dutch die Dura ausbreitete, will aber auch nicht eine Infektion auf metastatischem Wege yon einem vorhandenero Lungenabszel~ als vSllig ausgeschlossen betrachten. Ueber die otogene Pachymeningitis interna purulenta. 255 Pathologische Anatomic. In der Literatur gibt es nur einen Fall, derjenige yon Uff e n o r d e (36), der mikroskopisch untersucht ist; andererseits gibt abet der oben referierte Fall ganz besonders gute Beitr~ge zur Beleuchtung der pathologisehen Anatomie der Krankheit. Bei der beginnenden Paehymeningitis interna purulenta wird die Dura auf ihrer cerebralen Fl~ehe yon einer Schicht Eiterzellen bedeckt~ die yon Fibrin zusammengekittet sind, und die innerste Schicht der Dura wird mit Leukocyten infiltriert. In gleichem Mai3e wie der Proze$ vorwfirtssehrcitet, dringen nach und nach Bakterien in das Gewebe ein, das mit Eiterk~Jrperchen gefiitlt wird; diese greifen die Venen an, die thrombosieren. Sehliei~lich ist die ganze Dura mit Leukocyten infiltriert, stark angeschwolten, mit dicken~ purulenten Membranen auf der Innenseite, die aus Fibrin und hinf~lligen Eiterkiirperchen bestehen, bedeckt. Sehr oft bilden sich Adh~renzen zwischen der Dura und der Arachnoidea, und diese kiinnen sieh so ausbreiten und so stark werden, daI~ der Eiter im Subduralraum eingekapselt und der 1)rozel3 dadurch begrenzt wird, jedenfalls fiir einige Zeit; aber dieser Umstand bedingt in patbologisch-anatomischer Beziehung keinen entscheidenden Unterschied, so dal~ kaum ein Grund besteht dafiir, die Trennung zwischen einer diffusen und einer zirkumskripten Pachymeningitis in zwei versehiedene Krankheiten aufrecht zu erhalten; die zirkumskripte Pachymeningitis kann jeden Augenb!ick diffus werden. Bilden sich zeitig starke Adhiirenzen, so wird die Eiteransammlung im Subduralraum ganz gering; zuweiten aber werden m~ichtige Eitermengen gefunden, i/3 Liter, (Guder 10), so dab das darunterliegende Hirngewebe stark komprimiert ist. Vom Subduralraum aus findet der Eiter oft Abflul~ auf die Oberfl£che des Schi~dels entweder durch das Ohr oder an einer Stelle, die welt vom Ohre entfernt ist. Wie bereits erw~hnt, wird die Innenseite der Dura fast immer yon dem primiiren Focus aus per continuitatem angegriffen, und kS kann daher nicht Wunder nehmen~ daft der Eiter aus dem Subduralraum einen Weg zum Mittelohr linden kann. Eigentiimlieher ist es, dal3 der Eiter sich an einer Stelle fern yore Ohre einen Weg aus dem Subduralraum dutch die Dura in den Epiduralraum brieht, um danach den Knochen anzugreifen, ( L e h r 24~ Fall 16), ja sogar dutch den Knochen hindurchbrieht und kuf der Oberfl~tehe des Sch~dels einen groBen AbszeB bildet, (Ceci 5), 17" 256 IX. N. RH. BLEGVAD. Die Verh/iltnisse der Pia und Arachnoidea. Bei der obengenannt'en Patientin zeigte der Sektionsbefund auf der rechten Seite die interessante Tatsache, dab die Arachnoidea jedenfalls einige Zeit fang yon Eiter, der reich an Mikroben ist, umspiilt werden kann, ohne davon angegriffen zu warden; selbst nicht durch Mikroskopie konnten Entzfindungsph~nomene in den Leptomeningen naehgewiesen werden, l~berliigt man den Prozeg sieh selbst, so wird die Entziindung natiirlieh schlielSlich die Leptomeningen angreifen, und es zeigt sich dann hier ein Prozeg ganz analog demjenigeu in der Dura: Infiltration des Gewebes und der Gef~ige mit Leukocyten, Ablagerung yon Fibrin auf der Auf~enseite der Arachnoidea. Die Eitermembranen auf der Oberfl/iche der Araehnoidea kiinnen aui~erordenflieh dick werden, und die Arachnoidea kann doeh aueh ganz normal aussehen, wenn man sie abstreicht, so dag man nur durch Mikroskopie die Entzfindung in den Leptomeningen konstafieren kann, ( U f f e n o r d e 36). Hat der Prozeg l~ngere Zeit bestanden, so kann sich auf der AulSenseite der Arachnoidea eine dicke Schicht Granulationen bilden (Lch r 24). Wenn die sekundi£r entstandene Leptomeningitis sich fiber griii~ere Teile der Itirnoberfl/~che erstreckt, so wird der Tod des Patienten eine weitere Ausbreitung des Prozesses bald hemmen; ist aber die Entzfindung in der Dura oder den Leptomeningen yon Adhfirenzen begrenzt, so wird sie yon der Pia ins Gehirn dringen, so dab eine Encephalitis mit oberfl/~chlicher Nekrose des Hirngewebes entsteht: es bildet sich ein ,Corticalabszeg" (,,RindenabszeB", ,,ulceration of the brain"); abet dieses ist nut eine weitere Entwickelung des Prozesses, und es besteht vermeintlich kein Grund~ diesen Zustand mit M a c e w e n (25) und K S r n e r (')0) als eine besondere Form der Pachymeningitis interna zu unterscheiden. Wenn K S r n e r (20) meint, dal.i sieh im Subduralraum eine diffuse Suppuration befindet, die die Leptomeningen n i c h t angreift, da diese yon einer Sehicht Granulationen beschfitzt werden, so ist hiergegen einzuwenden, daft bisher kein Fall verSffentlicht worden ist, der diese Behauptung unterstiitzt. L e h r s (24) und H S l s c h e r s ( 1 6 ) Fall kann hierfiir nicht angefiihrt werden~ da bei ibm keine Mikroskopie vorgenommen wurde. U f f e n o r d e s (36) und mein Fall zeigen dagegen deutlieh~ dab sich Leptomeningen, die mit Granulationen oder Pseudomembranen bedeckt waren, bei der mikroskopisehen Untersuehung als entzfindet erwiesen. Nach allem Vorliegenden mug man sagen, dag die Pachymeningitis interna ohne Mitergriffensein der Leptome- Ueber die otogene Pachymeningitis interna purulenta. 257 ningen nur ein Ubergangsstadium ist entweder zur Heilung oder zur Leptomening'itis, eventuetl Encephalitis. Man hat aueh Beispieie dafiir, dab yon der Pachymeningitis ein Hirnabszel~ induziert werden kann, der vom Ohre weir entfernt gelegen sein kann, ( G u d e r 19, M a e e w e n 28). Ist ein Abflu[5 zum Mittelohr oder zur Oberfl/iehe des Seh/tdels hergestellt~ kann der Prozel~ sieh lunge halten~ ohne sieh auszudehnen. Symptome. In der meisten Fiillen war die Pachymeningitis mit verschiedenen anderen Leiden (Osteitis proc. mast., Sinusthrombose, epiduralem Abszefi, Itirnabszel~ usw.) kompliziert, so daft das Krankheitsbild yon Symptomen iiberdeckt und verwischt wurde, die auf diese Leiden zuriiekzuffihren sind. Eine bestimmte Symptomatologie aufzustellen ist daher unm5glich. In den meisten F/illen traten nur Symptome auf, die im allgemeinen das Vorhandensein einer diffusen intrakraniellen Komplikation angaben: Unklarhei~, Delirien, Benommenheit, SehlKfrigkeit, Schwindel, Erbreehen, Neuritis optica, N~ekensteifheit, K e r n i g s Ph~nomen, Konvulsionen, und man hat dabei sehr oft die Diagnose Leptomeningitis gestellt. Es ist natiirlich anzunehmen, daft diese Symptome wirklich yon einer - - serSsen oder purulenten, toxischen oder bakteriellen)--Leptomeningitis her{iihren, die yon der Pachymeningitis induziert ist. Von f o k a l e n S y m p t o m e n wurdeu beobachtet Hemianopsie, Doppelsehen, Pupillendifferenz, Ptosis, J a c k s o n s Epilepsie, sowie verh/~ltnism/ifiig hiiufig amnesfisehe Aphasie (sieben F/ille) und Pa,'esen der kontralateralen Extremit/iten und Gesiehtsh/ilfte; in einem Falle (Mil b u r g 31) Parese der kontralateralen Extremit~ten und der homelateralen Gesichtsh/ilfte. Diese fokaleu Symptome riihren wahrscheinlich yon der Pachymeningitis her, indem man annehmen kann, dug die Eiterausammlung das Hirn komprimiert hat, oder es ist anzunehmen, d a { ~ bei einer kleinen subduralen Eiteransammlung - - ein ()dem der Hirnsubstanz entsteht ( [ I e i n e l 4 ) . Selbstverst~indlich sind die genaunten Symptome keineswegs fiir die Paehymeningitis pathognomonisch, da sie ebenso gut z. B. yon einem HirnabszeB herriihreu kSnnen. Sehr hiiufig war F i e b e r vorhandeu, in einigeu F/illen jedoch ( B e e o 4 Fall 2, L e h r 2 4 ) war die Temperatur normal. Der P u l s ging in der Regel sehnell, nur in einzelnen F/illen ( L u c a e 26, ~ a c e w e n 28) wurde ausgepr~gter Hirndruekspuls festgestellt. 258 Ix. N. RH. BLEGVAD. Verlauf u n d P r o g n o s e . Von spontan geheilter Pachymeningitis liegt keine Mitteilung vor. Dagegen wurden v e r s e h i e d e n e - t 4 durch Operation geheilt, w~hrend neun kiirzere oder l~ngere Zeit naeh der Operation an Leptomeningitis starben, drei iiberstanden die Operation gut, starben aber danach an einem anderen Leiden, und einer starb, ehe ein operativer Eingriff vorgenommen werden konnte. Die Prognose ist daher bei reehtzeitigem operativem Eingriff recht gut, zumal in Betraeht zu ziehen ist, wie ernst eine Eiteransammlung gerade auf der Leptomening 5 anzusehen ist. Es scheint ein schlechtes prognostisches Zeichen zu sein, wenn Naekenstarre d. b. beginnende Leptomeningitis auftritt, doch gibt es Fi~lle, wo der Patient trotz einer anwesenden Leptomeningitis (unklare Cerebrospinalflfissigkeit, Oh e v a l 7) gerettet wurde, und einen Fall ( G u d e r l 0 ) , wo die Leptomeningitis spontan vor tier Operation zurfickging (die Cerebrospinalflfissigkeit~ die zuerst Leukozyten enthatten hatte, wurde wieder frei yon zellul~ren Elementen). Diagnose. Infolge der obigen Ausfiihrungen fiber die Symptome ist die Diagnose der Pachymeningitis interna purulenta sehr schwierig. Die vorhandenen Symptome kSnnen in den meisten Fi~llen ebenso gut yon einer Leptomeningitis oder einem Hirnabszel~ wie yon einer Paehymeningitis interna stammen. Zu dem Zeitpunkt des Verlaufes, wo sich Symptome einer diffusen intrakraniellen Komplikatlon zeigen, ist es oft die die Pachymeningitis begleitende diffuse Leptomeningitis, die die Symptome hervorruft. Indessen wiirde die Diagnose der Pachymeningitis zu einem friiheren Zeitpunkt sehr zu wiinschen sein. Die Krankheit ist unzweifelhaft h/iufiger, als angenommen wird; in den F~llen, wo eine chronische Suppuration sich vom Mittelohr nach innen zur Dura verpflanzt hat, findet sich sicherlich nicht selten auch eine Pachymeningitis interna, (die wahrscheinlieh oft spontan heilen kann, wenn der prim~re Focus entfernt wird), und in den F~llen, wo eine Leptomeningitis yon einer Ohreneiterung per continuitatem entsteht, ist tier U~bergang unzweifelhaft eine Paehymeningitis und interna externa; kSnnte diese zu einem friiheren Zeitpunkt entdeckt und angegriffen werden, so wiirde viel gewonnen sein. In den Fi£11en~ wo sich fokale Hirnsymptome finden~ ist eine Trennung zwischen Hirnabsze[~ und Pachymeningitis interna un- Ueber die otogene Pachymeningitis interna purutenta. 259 mSglich. Finder man in solchen F/illen bei der Operation in der D u r a eine Fistel, yon welcher Eiter aus dem Snbduralranm fiiel~t, and sieht man nach der Inzision der Dura die normale Kontur der Hirnwindungen auf dam Grunde, oder ist die Dura auf der Innenseite mit einer Eitermembran bedeckt, so ist die Diagnose Pachymeningitis leicht. Aber ist die Dura auf der Oberfl~che natiiriich, so haben wir bis jetzt kein anderes Mittel zur Diagnose als Punktur oder Inzision der Dura. Die Verh~ltnisse der Cerebrospinalfliissigkeit. Leider enthalten nur ganz wenige der ver~iffentlichten F/~lle ein Resultat der Lumbalpunktion. Es gibt aber drei besonders instruktive F~lte ( H e i n e 14, Fall 2, G u d e r 10~ C h a v a s s e and M ah u 6), bei denen trotz grol~er subduraler Eiteransammlungen (der eine derselben ist yon einer Sinuitis frontalis induziert (C h a v a s s e u. M ah u 6)) eine klare Cerebrospinalfltissigkeit ohne zellul/ire Elemente gefunden wird; auch in dem oben mitgeteilten Falle ist die Cerebrospinalfliissigkeit vor der ersten Operation klar, obgleiehdie vondickenMembranen umgebenegrol~esubduraleEiteransammlung, die zwei Tage sp~ter gefunden wird, sicherlich schon an diesem Zeitpunkt vorhanden gewesen ist. In den Fiillen~ wo die Cerebrospinatfliissigkeit unklar gewesen ist, waren gleichzeitig auch andereZeichenvondiffuserLeptomeningitis 1) vorhanden. Mt~nkann vermutlich daher den Schlu~ ziehen, daft P a c h y m e n i n g i t i s interna purulenta ohne gleichzeitig vorhandene diffuse Leptomeningitis k e i n e z e l l u l / i r e n E l e m e n t e in der Cerebrospinalfltissigkeit aufweist. Tlierapie. Um die Pachymeningitis interna purulenta heilen zu k5nnen, ist die erste Bedingung, dal~ man dem Eiter Ablauf sehafft. Und es ist bewiesen 7 daft man, um ein giinstiges l~esultat zu erzielen, reiehliehe Drainage sehaffe/~, oder a u s g e d e h rite K r a n i o t o m i e 5 f f n u n g e n anlegen mu[~. W i t z e l (37) legte in einem Falle eine 1) HS]schers Fall (16)scheint hierffegen zu sprechen, da eine Woche vor Mors unklare Cerebrospinalfliissigkeit ohne meningitische Symptome gefunden wird. Bei dcr Sektion ist ,Pia ganz frei", also keine Leptomeningitis; aber es wird gleichzeitig mitgeteilt, dab auf der Hirnoberfl~tche dicke purulente BelEge gefunden wurden; Mikroskopie wurde nicht vorgenommen, undes rout] gerade aus diesem Grunde -- in Analogie mit Uffenordes und meinem Fall -- angenommen werden, dal~Leptomeningitis vorhanden war. 260 IX. N. RH. BLEG-VAD. Trepanationsiiffnung an, die grSl~er war als eine Handfliiche. Danach mul~ man eine grol~e Inzision in die Dura machen, entweder Kreuzsehnitt oder - - wie F e d o r K r a u s e (19) empfiehlt-- Lappenschnitt. Die Dura zu exstirpieren, wie A l e x a n d e r ( 2 ) empfiehlt, ist kaum notwendig und kann vielleicht sogar schKdlich sein. Fiir die Drainage empfiehlt U f f e n o r d e (36) weite GlasrShren, durctl die ein Jodoformgazestreifen gefiihrt ist. W i t z e l (37) drainiert mit gro[Sen Gazem~chen, die mit physiologischer KoehsalzauflSsung getr~nkt sind, und schreckt nicht davor zuriick, da5 dadureh eine Kontusion des Hirngewebes hervorgerufen werden konnte. Wie bei aller tIirnchirurgie ist auch bier Puncture saliens, dais man friih z e i t i g o p e r i e r t . Wenn es gelingt eine beginnende Pachymeningitis interna zu drainieren, w i r d u n z w e i f e l h a f t oft d i e deleti~re L e p t o m e n i n g i t i s v e r m i e d e n w e r d e n k S n n e n , und selbst in Fiitlen~ wo die Pachymeningitis bereits eine Leptomeningitis induziert hat, gliickt es zuweilen, den Patienten zu retten 1). Da aber die Paehymeningitis keine priizisen Symptome gibt, ist man oft genStigt, sehon allein auf den blot3e a V e r d a e h t hin zu i n z i d i e r e n . Einen so grol~en Eingriff nimmt man natiirlieh nur dann vor, wenn man vor oder wiihrend tier Operation einen s t a r k e n V e r d a c h t erhalten hat, dal~ ein subdurales Leiden vorhanden ist. Wie ausgefiihrt, gleieht die Pachymeningitis in ihrem Auftreten bald einem Gehirnabszel~, bald einer Leptomeningitis diffusa, mit welehen beiden Leiden sie aueh oft kompliziert ist. Zeigen sich stark auftretende fokale Symptome (die also entweder yon einem Gehirnabszei3 oder eincr Pachymeningitis herrtihren kSnnen), so ist die Art des Vorgehens bei der Operation klar: Entleerung des prim~tren Focus im Sehliifenbein, Kraniotomi 5 Spattung der Dura usw. Zeigen sieh aber nur Zeichen einer diffusen intrakraniellen Komplikation (Leptomeningitis, Pachymeningitis int.), so ist die Lage sehwieriger. Wenn es sich - nachdem man die Aufmeil~elung vorgenommen und nachdem maa die Dura dutch Kraniotomie blol~gelegt hat - - zeigt, dal~ die Dura gangr~inSs event, fistulSs durchbrochen ist, so ist die Dura zu inzidieren, der Subduralraum zu untersuchen und, falls sieh hier Eiter vorfindet, dureh eine groBe Kraniotomieiiffnung zu drainieren. Ist die Dura dagegen nicht gangr~tnSs oder fistulSs durchbrochen, so h/ingt das Vorgehen davon ab~ ob das Lure1) Siehe z. B. meinen F~ll 2 INancy C). Ueber die otogene Pachymeningitis interna purulenta. 261 b a l p u n k t a t k l a r i s t o d e r nicht. Ist die Cerebrospinalfliissigkeit ktar, so kann man einige Tage lang abwarten; dauern dann aber die Hirnsymptome an, s o ist die Dura zu inzidieren; denn dann handelt es sieh entweder um eine progredierende Leptomeningitis, auf deren Verlauf eine BIoNegung des Cerebrum eine seh~dlicbe Wirkung nicht haben wiirde, oder um das Vorhandensein einer subduralen oder cerebralen Eiteransammlung. Ist die Cerebrospinalflfissigkeit dagegen unklar, so ist die Frage, wie man operieren soll, schwieriger zu entseheiden, wie yon M y g i n d (32) hervorgehoben wird. Man l~iuft n~mlich Gefahr, sowohl wenn man die Dura 5ffnet, als aueh, wenn man das unterl~l~t. Offnet man n~mlieh die Dura nicht, so riskiert man, dat/ im Subduralraum oder im Cerebrum ein Eiterherd zuriiekgeIassen wird, der die Ursache zur Lept0meningitis gewesen ist, die sieh dann im Laufe yon 1--2 Tagen naeh der Operation welter ausbreiten und fiber das Stadium kommen kann, wo sie wieder zuriickgehen kann. Und inzidiert man die Dura, ohne dal~ sieh solcher Eiterherd vorfindet, riskiert man, dal~ das Gehirn infiziert wird, oder dal~ sieh sekundiir ein Hirnprolaps bildet, der, wie die Erfahrung in der Klinik wiederhott bewiesen hat, zu verschiedenen Komplikationen ffihren kann, zum Tell vpn lebensgef~hrlieher Natur. Da in kurzem vom Chef der Klinik ein Berieht erscheinen wird fiber die Erfahrungen, die mit Bezug auf die operative Behandlung der otogenen Leptomeningitis in dem erw~thnten Punkte gewonnen wurden~ will ich mich mit dem Hinweis darauf beschr~nken. Der oben referierte Fall fordert auf jeden Fall gewissermafien zu einem radikaleren Vorgehen auf. Bei der ersten Operation wurden nut diffuse, nicht besonders starke Hirnsympton~e gefunden, und die Cerebrospinalfliissigkeit war ktar, und doch war - - w i e oben erw~ihnt - - unzweifelhaft schon an diesem Zeitpunkt eine subdurale Eiteransammlung vorhanden. Das Resultat w~re mSglicherweise gfinstiger gewesen, wenn man sehon zu diesem Zeitpunkt radikal vorgegangen w~tre. Die Inzision der Dura ist der Punktur vorzuziehen, tells desbalb, welt man Beispiele daftir besitzt, wo durch die Punktur das Vorhandensein einer subduralen Eiteransammlung nicht konstatiert wurde, namentlich aber deshalb, well die Gefahr einer Infektion der Leptomeninges bei der Inzision geringer ist als bei der Punktur. Dem Chef der Klinik~ Herrn Prof. Dr. med. M y g i n d , spreche 262 IX. N. RIt. BLEGVAD. ieh h i e r m i t m e i n e n b e s t e n D a n k a u s fiir seine A n r e g u n g zu dieser Arbeit, f e r n e r fiir das Interesse, das er m e i n e r Arbeit e n t g e g e n . brachte, u n d fiir die E r l a u b n i s , d a s J o u r n a l m a t e r i a l der K l i n i k veriiffentlichen zu diirfen. N a e h d e m die o b i g e n A u s f i i h r u n g e n u i e d e r g e s e h r i e b e n w a r e n , h a t t e n w i r i n der K l i n i k Gelegenheit, n o e h e i n e n i n t e r e s s a n t e n u n d i n s t r u k t i v e n F a l l y o n o t o g e n e r P a e h y m e n i n g i t i s i n t e r n a zu verfolgen. D e r P a t i e n t w u r d e d u t c h die O p e r a t i o n geheilt u n d in der S i t z u n g der D i i n i s c h e n o t o l a r y n g o l o g i s c h e n Gesellsehaft v o m N o v e m b e r 1909 d e m o n s t r i e r t . ~ N a n c y C., 11 Jahre alt, unverheiratete Aufw~_rterin. Wurde in die O h r e n - u n d HMsklinik des Kommunehospitals am 26. Oktober 1909 eingeliefert. 3. n a m n e s e. Vorher inlmer gesund gewesen. In den letzten beiden Jahren AusfluB aus beiden Ohren; aus jedem Ohre sollen viermal Polypen herausgezogen worden sein. In den letzten sechs Tagen starke Sehmerzen im linken Ohre und iiber dem 0hre sowie auf der linken Kopfseite; zu~leich Fieber. An den letzten drei Tagen best~indiges Erbrechen, kein Sehiitteifrost, Schwindel oder Kdimpfe. Wurde behande]t mit Bettliegen und warmen Umschl~igen. Die Mutter teilte mit, dab Pat. immer etwas stumpfsinnig gewesen sei, die Stumpfl~eit habe aber in den letzten beideu Monaten stark zugenommen, gleichzeitig habe sic sich bestiindig m~ide und schl~ifrig gefiihlt. Vor sechs Tagen habe die Mutter die Pat. besucht und dabei den Eindruck gewonnen, dai] sic teilweise unklar sei. Sic sei aul~er Bette gewesen und babe ihre Arbeit verriehtet, habe aber vie] fiber Ohrenschmerzen, Kopfschmerzen, Ubelkeit und Erbrechen geklagt. Status pr~isens. Temp. 38,3. Puls li2. Bei der Einlieferung sah Pat. etwas angegriffen aus; sic gibt etwas langsam Antworten auf Fra~en, was zum Teil auf lhre Schwerhongke~t zurfickzufiihr~n 1st, doch hat man den bestimmten Eindruck~ da~ zugleich ,.slow ccrebration" vorhanden ist. Keine Nackenstarre, keine K e r n i g oder B a b i n s k i, kein Sehielen, keine Facialis- oder andere Paresen. Schneller, feiner, iiberwiegend rotatorischer Nystagmus bei extremen Btickrichtungen, besunders beim Blick naeh rechts gesundes 0hr) ; d a s Auge kommt jedoch nach cinigcr Zeit Fixation zur Ruhe. A u r i s s i n.: Sehr stark stinkender Eiter. Die Hintergrund ist yon chotesteatomat5sen Masseu ang'effillt. R e g i o m a st o i d e a s i n. Um den ganzeu Processus mastoideus bedeutende 5demat5se Schwellung. "Dber dem 0hre erstreckt sich bis ganz vorn zur Orbita und hinauf zur Line~ temp. sup. eine fluktuierende Intumeszenz yon der GrSBo einer Kinderhandfl~iche, bedeekt mit sehr empfindlicheL nicht brennender Haut yon natiirlicher Farbe. Einzelne geschwollene Driisen auf der Seite des Halses. A u r i s d e x t. Reichlicher Eiter ohne iiblen Geruch. In der Tiefe des GehSrgauges cholesteatomatSse Massen. l:tegio m a s t o i d e a dext. Niehts Besonderes. Cav. n a s i } Leichte akute RSte. Pharynx 0,50 . 0.25 Sprechstimme O,5-0" FliistersUmme ()\10 mit B ~ r ~ n y s L~irmapparat im ad aurem anderen 0hre ad aurem" Ueber die otogene Pachymeningitis interna purulenta. 263 K a l o r i s c h e r N y s t a g m u s Ibei Ausspiilung mit 120 Wasser) tritt sehr sp~it ein, aber unzweifelhaft auf beiden Seiten. S t e t h o s k o p i e normal. O p e r a t i o n I. Bei Athernarkose erfolgt um 4 Uhr nachmittags L u m b a l p u n k t i o n mit nieht vSllig kristallklarer Fliissigkeit, worin zahh'eiche kleine weiBe Schuppen [ M i k r o s k o p i e (die Untersuchung erst 18 Stunden nach der Entleerung vorgenommen). Im Prfiparat des zentrifugierten Bodensatzes ist eine recht reichliche Menge, ziemlich hinf~illiger weif~er BlutkSrperchen zu sehen, deren Kern sictl nur schlecht f~rbt Verll~ltnis zwischen den mononukle~ren und den polynukIe'Xren Leukocyten ungefiihr 1 : 4. Keine roten BlutkSrperchen. Keine Mikl:oben. Keine Gew~ichse auf A g a r nach 24 Stunden]. Danach wird R e s e c t i o t o t a l i s a u r i s m e d i a e s i n i s t r a e vorgenommen. Nachdem man durch die stark geschwollenen Weichteile und das stark verdickte Periost gedrungen ist, kommt man zu einer grol~en, mit ekelhaft s~inkendem Eiter gefiillten Abszeffh6hle, die namentlich fiber dem obersten Teile des Proc. mast. und in der Regio temp. liegt. Corticalis iiberall elfenbeinweiB, und erst ca. I cm tief werden im gr6gten Teil des Proc. mast., jedoch nicht in seiner Spitze, zahlreiche Kavit~iten gefunden, die mit Eiter, Granulationeu und stinkenden ctmlesteatomatSsen Massen geftillt sind. Vom Antrum dringt eine Menge stinkender Eiter hervor, der yon einem grol~en epiduralen Abszel~ kommt, der mit dem Antrum durch einen ca. erbsengro~en Defekt in der Decke in Verbindung steht. [Bei der Mikroskopie des di~ekten Pr~iparats yore Eiter sind zahlreiche Kokken und St~ibchen verschiedener AVe zu sehen ] Tegmen antri wird entfernt, und die Dura ist hier nicht vorgewSlbt, schwach pulsierend, verdickt, auf der Aul~enseite mit samtartigen Membranen bekleidet, die hier und da von schlechter Farbe sind. Da man annimmt, dai~ die vorhandenen Cerebralsymptome yon dem groBen epiduralen Abszefi herriihren, 5ffnet man nietLt den Subduralraum. Sinus transv, wird bloBgelegt; sein Aussehen ist natiirlich. Die TrommelhShle ist mit Cholesteatommassen gefiillt, in denen kleine Uberreste yon Malleus und Incus sind. Hinterste, membran6se GehSrgangswand wird reseziert. Man legt Jodoformgaze zur Drainage des Epiduratraums und des Mittelohres ein. 27. 10 38,1--38,7. Puls 104--96. War seit der Operation sehr unruhig, hat aber nach Guttae roseae ziemlieh gut geschlafen. Hat best~ndig geschwatzt, indem sie ununterbroehen deuselben Satz wiederholte. Antwortet auf Fragen verwirrt; deutlich sensorische Aphasie. da sie einen Nagel ,,Finger" nennt, fiir Portemonaie keine Bezeietmung linden kann u. a. Ophthalmoskopie (durch Herrn Dr. reed. B e n t z e n ) normal. Kein Schreien, Z~ihneknirschen oder Schielen. Keine Naekenstarre oder K e r n i g . Auf fast der ganzen Oberfliicbe des Sch~idels ist eine sehr bedeutend 6dematSse Schwellung, am st~rksten auftretend auf dem rechten Tuber parietale. O p e r a t i o n IL In der Athernarkose wird um 5 Uhr nachmittags Lumbalpunktion (trocken) vorgenommen, danach eine E r w e i t e r u n g der KraniotomieSffnung. Das Periost ist in grol~er Ausdehnung um die Operationskavit~t stark verdickt, 6dematSs, leicbt 15sbar yore Knochen: auf der dem Sch~ideI zugewendeten Fl~iche ist es yon schlechter F~rbung, mit stinkendem Eiter bedeekt. Die Oberfl~che des Knochens ist an mehreren Stellen, namentlich zur Sutura oecipitomast, bin, grfiulich, an einigen Stellen schw~irzlich. Die Dura wird in einer Ausdehnung yon 5 c m v o n der einen Seite zur anderen und yon 51/~ c m v o n oben nach unten blol~gelegt, lhre Oberfl~che ist auf einer Pattie, die nach hinten und nach oben von gesundem Duragewebe durch eine scharf gebogene Linie begrenzt wird, verdickt, graugriin und uneben. Naeh vorn vel~olgt man die ver[~nderte Dura nicbt ganz, da man befiirehtet, bei der Kraniotomie die Art. meningea reed. zu li~dieren. Auf diese Weise bekommt man nicht zu sehen, wie welt sich die Pachymeningitis ext. nach vorn erstreckt. Man macht darauf aus tier Dura einen 264 IX. N. RH. BLEGVAD. Lappen mit (ler Basis nach unten, indem man die Grenze des Lappens ca. 1 cm yon der Peripherie der Kraniotomieiiffnung tegt. Der Lappen wird heruntergekiappt, wobei sich die lnnenseite der Dura mit einer dicken, ~Stiichen Membran bekleidet zeigt. Die Dura ist fast tiberall an die Arachnoidea mit ziemlich starken Adhiirenzen festgeheftet. Leptomeningen selbst sind verdickt and milehig. Es werden Inzisionen im Gehirn naeh drei verschiedenen Richtungen mit negativem R e s u l t a t vorg'enommen. Jodoformgazetamponade. Phenosalytumschlag. Zur M i k r o s k o p i e wird ein Stiick Dura yon der einen Ecke des Lappens genau am L~bergange zum normal anssehenden Duragewebe entfernt. Die mikroskopisehe Untersuchung zeigt: Dura durch und durch infiltriert mit polynukle~ren Leukocyten. Anf der a u s w e n d i g e n Fl~che tier Dura finder sind eine ziemlich dicke Schicht Fibrin mit reichlicher Leukecyteneinlagerung. An einzelnen Stellen ist oben auf der Fibrinschicht eine dichte, zusammenh~ingende Schicht rater BlutkSrper (artifizieLle Blutung?) zu sehen. Auf der i n w e n d i g e n Fl~iche der Dura ist eine sehr dicke 5cbicht Fibrin, worin zahlreiche Leukocyten sowie eine Anzahl roter BlntkSrperchen lagern, die an einigen Stellen, namentlich zwischen der Fibrinschicht und der D u r a , m grol~en Haufen liegen. Auch yon den L e p t o m e n i n g e n wird ein Stiick exzidiert, das bei der Mikroskopie zeigt, dal~ die Leptomeningen verdickt und stark mit Rundzellen infiltriert sind. Einige Stellen im Pr~iparat sind sic mit einer SchichtFibrin bedeckt, in der sich eine Anzahl Leukoeyten und zahlreiche rote BlutkSrperchen befinden. Im []irngewcbe keine Zelienvermehrung. 28. 10. 12 Uhr mittags. 27. lq,. Temp. 38,0. Puls 108. 8 Uhr abends. 27. 10. Temp. 38,4. Puls 96. t2 UhrNacbts. 27. i0. Temp. 38~8. Puts 88. 6 Uhr morgens. 28. 10. Temp. 38,6. Pals 76. Gestern Abend his I2 Uhr sehr unruhig; bekam dann Guttae roseae, worauf Schlaf. Am Morg'cn sehr anklar, wiederholt stets densclben Satz, singt. Operationswunde leicht riechend, dnnketgefiirbt mit etwas Prolaps vom ltirngewebe. ()dem der Integumentcn auf dem Kopfe fast geschwunden. 29. 10. Temp. 39--39,5--39,5--39,7. Pals 96--100-86~-98. Lag den grSfiten Tell des gestrigen Tages im Ha]bschlummer, genofi n u t wenig. Bestiindig amnestiscbe Aphasie Liegt heute morgen teilnahmslos, scheint aber die Umgebung zu kennen und nennt bestSndig den Arzt ,Fr~hflein." Reichliche stinkende Eitersekretion aus der Wunde. Der Prolaps ist so grol~ wie eine Wainul], ganz schwarz auf der OberflSche. in den Sabduralraum werden Jodoformgazetampons gelegt (man kann jedoch nicht welt hincinkommen). Rp. Chlorkalkwasserumschlag. 30. 10. Temp. 3%9--39,9--39~4--38,5. Puls 110--100--94--80. Zustand unvedindert. O p e r a t i o n !iI. In der Athernarkose wird um 10~/.., Uhr vormittags L u m b a 1p u n k t i o n (trocken) vorgenommen. Darauf wird die K r a n i o tomieSffnung nach oben erweitert, bis man das 0s parietale erreicht, und ca. 3 cm nach vorn, Ms man die Dura soweit bloligelegt hat, daft iiberall ca. I cm normal aussehende gl~nzende Dura innerhalb der Peripherie der KraniotomicSffnung zu sehen ist. Danaeh exzidiert man die pathologisch veriinderte Dura nach oben und nach vorn. Hierauf werden drei Jodoformgazem~chen zwischen Dura und Arachnoiden nach oben, nach yore und nach hinten eingefiihrt. Rp. Chtorkalkwasserumschlag. [Die M i k r o s k o p i e des exzidierten Durastiickes zeigt, da~ die Dura sehr stark yon polynukleSren Leukocyten durchsetzt ist, die auch die Gef~l]w~inde infiltrieren. Auf der a u s w e n d i g e n Fl~che der Dura ist eine rccht dicke Schicht leukocyteninfiltriertes Fibrin, das stark im Begriffe ist, sich zu organisieren, da sich iiberall zwisehen der Dura und der Fibrinschicht eine Schicht Granulationsgewebe befindet. Auf der i n w e n d i g e n Fl~ichc der Ueber die otogene Pachymeningitis interna purulenta. 265 Dura ist eine miichtige Schicht Fibrin (bedeutend dmker als diejenige welche auf dem am 27. I0. entfernte~ Durastficke gefunden wurde) ; sic enthfilt namentich in den peripherischen Teilen groBe Mengen Leukoeyten.] 31. 10. Temp. 38--38,7--37,7--37,8. Puls 80--96--90--90. Zustand ungeffihr unvedindert. DreimaI dfinner Stubl. Brieht am Morgen alles wieder aus, was sic ~eniel~t. Seit der 0peragon ist in sehr reichlichen Massen Cerebrospinalflfissigkeit ausgeflossen, so dal~ das Kopfkissen verschiedene Male gewechselt werden muBte, Keine ~-aekenstarre. Der Umschlag wird geweehselt, jedoch bleiben die Jodoformgazetampons unberiihrt. 1. tl. Temp. 37,3--38,1--38,1--3~,1. Puls 88--84--90--90. Sehr h~ufiges Erbreehen, im fibrigen war der Zustand eher etwas besser zu nennen. Sie ist ab und zu klar. K e r n i g ? B a b i n s k i ? Keine Nackenstarre. Geringere Aktion des r e c h t e n Nerv. facialis. 2. 11. Temp, 3S,6--39,4--38,5--38,3. Puls 100--10S--S6--S0. 123/, Uhr nachm. (1. 11.) bekam Pat. einen Kdimpfeanfall, der 11/4 Stunden dauerte. Sie wurde yon der Krankenpflefferin beobachtet, die angibt, dab die Zuckungen auf der l i n k e n (kranken) Seite des Mundes begannem danaeh gingen die klonisehen Kr~mpfe in beide Arme und sehliel]lich in die Beine. besonders links. Sic war stark cyanotisch und schwitzend Naeh dem Anfall sehlief sie und war stark kongestioniert im Gesicht. Darauf trat ein reiehliches Aussickern von Cerebrospinatfl[issigkeit ein. Um 5 Uhr naehmittags wh, d der Verband erneuert. Die Tampons werden herausgenommen. Der Prolaps hat so zugenommen, d ~ er jetzt apfelgrol3 ist; er ist schwarz auf der Oberft~ehe. Es gelingt nur nach oben und nach ifinten Tampons in den Subduralraum hineinzufiihren. Pat. hat in der Nacht gut geschlafen und war am Morgen klar; als sie h6rt, da$ hente der 2. ~ovember ist, weil~ sic, dat3 ihr Geburtstag ist. Andauernd etwas sensorische Aphasic. 3. I1. Temp. 3%7--38,2--37~7--37,2. Puls 100--88- 88-80. W a r h e u t e ziemlich erregt, jedoch ab und zu klar Keine Erbrechen oder andere meningitisehe Symptome. 4. 1I. Temp. 37fi--37,8--37,8--3%8. Puls ~6--80--80--78. Zustand unver~indert. 5.11. Temp. 37,2:-38,6--37,8--37,8. Pals 80--94--80--S0. Heute klarer. K e r n i g nnzweifelhaft. Keine Nackenstarre Prolaps unver~ndert Man kann den Subduralraum noch naeh oben drainieren. 6. 11. Temp. 3%6-37,4, Puls 78--74. In den letzten 24 Stunden fast vollst~indig klar. Hat etwas iiber Kopfschmerzen geklagt. Aphasie geringer. Ist etwas erregt, aber sonst klar. 7. 11. Temp. 37,9--37,3. Puls 88--84. 8. 11. 37,6--37,4. Puls 96--80, Befindet sich w o h l Wundensekret ohne iiblen Gerueh. Prolaps unvedindert. 9. 11. 37,8--37,5. Puls 88--76. Letzte Naeht reeht reichliches Aussickern yon CerebrospinalflSssigkeit. 10. II. Temp. 37,9--37,7. Puts 88--84. Sehr reiehliehes Aussickern yon Cerebrospinalflfissigkeit. Ist jetzt v~illig klar, redet nur ein wenig dummes Zeug (kongen~tale Imbezitlitiit). Keine Aphasie mehr. Es zeig't sich. da$ die Cerebrospinalflfissigkeit im wesenfliehen aus einer klemen (}ffhung an der Spitze des Prolaps kommt, aus der sie in elnem Str,~hte schiel~en kann, wenn Patientin aufschreit oder wenn man auf den Prolaps dri~ckt. 15. I L Temp. 37,7--37,5. Puls 88--80. Wohlbefinden. Noch immer ab und zu etwas erregt. Bestfindig Aussickern yon Cerebrospinalfliissiglzcit. Prolaps hat nicht zngenommen und ist fast iiberall mit guten Granulationen bedeckt. Der Subduralraum kann nieht mehr drainiert werden. ~- K e r n i ~. In der folgenden Zeit befindet sich Pat. v~)llig wohl, die Temperatur ist subfebril. 37,7--38,6 des Abends, 3%4--37,5 des Morgens, Puls 100 bis 266 IX. N. RH. BLEGVAD. 80. Der Prolaps nimmt etwas an GrSl~e zu; hin und wieder ist Pat. eigen und miirrisch, Stimmung empfindlich, sie ist abet vollst~ndig klar; kaum eine Spur yon Aphasie. Man versucht, den Prolaps mit dem Verbande zu komprimieren, aber er scheint dadurch sogar grSl~er zu werden. 7. 12. Temp. 37,5--37. Puls 84--80. 8. 12. Temp. 37,5--36,$. Puls 84--76. In den letzten drei Wochen kein Anssickern yon Cerebrospinalfliissigkeit mehr. 18. t. 1910. Andauen~d Wohlbefinden. Der Prolaps hat in der letzten Zeit gut abgenommen; er hat jetzt nur noch knapp einen Umkreis yon der GrSBe einer Kinderhand und prominiert nur ganz wenig fiber das Niveau des Sch~dels; Epidermis f~ngt an, sich an den R~ndern vorzuschieben. 15. 3. Der Prolaps ist ganz geschwunden, die CraniotomieSffnung ist yon normal aussehender Epidermis bedeckt; die gesamte Partie pulsiert reeht stark und schmerzt nichL wenn man darauf drfickt. Das Mittelohr ist fast vollst~ndig epidermisiert. Epikrise. Auch in diesem Falle handelt es sich um ein junges M~idchen mit einer chronischer Mittelohrsuppuration. Es treten Schmerzen auf im Ohre und im Kopfe, Fieber, Erbrechen, Benommenheit, ,,slow cerebration" und ~ystagmus, sowie ein grol~er subperiostaler Abszel~ fiber Regio temp. und mastoidea. Bei der Operation zeig't sieh Cholesteatom im Mittelohre, Defekt in der Decke des Antrnm, ein groi~er epiduraler Abszet~; Dura fiber Antrum mit einer dicken, fibriniisen Membran bekleidet. D a der gefundene epidutale Abszcl~ sehr wohl die cercbralen Symptome crkl~iren kann, liil~t man die Dura unberiihrt; aber da sich nach der Operation sensorische Aphasie, hohe Temperatur mit verh~ltnism~ii~ig langsamem Puls (38,7--96) zeigen, wird 24 Stunden nach der ersten Operation Kraniotomie mit Bloi~legung der Dura in einer Ausdehnung yon ca. 30 qcm, entsprechend der Fossa cranii media~ vorgenommen. In ungef~hr dieser ganzen Ausdehnung wird eine bedeutende Pachymeningitis externa gefunden. Man macht einen Lappenschnitt in die Dura, die auch auf ihrer Innenseite mit einer fibrinSser Membran bekleidet ist und bei der Mikroskopie sich in ihrer ganzen Dicke entziindet zeigt. Auch in der Leptomeningen ist - - sowoht makro- wie mikroskopisch - - Entziindnng' vorhanden. Die Dura ist adherent an die Arachnoidea, jedoch wird, soweit man sehen kann, freier Eiter im Subduralraum nicht gefunden. Drei Inzisionen des Gehirns haben ein negatives Resultat. In den folgenden Tagen steigt die Temperatur, wiihrend der Puls andauernd relativ langsam ist; Pat. ist best~indig unklar, schl~ifrig, aphatisch. Ca. 60 Stunden nach der zweiten Operation wird die KraniotomieSffnung erweitert, und man exzidiert soviel yon der Ueber die otogene Pachymeningitis interna purulenta. 267 Dura naeh vorn, dab sieh um den ganzen inneren Rand der Kraniotomie~ffnung ein Streifen Dura befindet, die auf der AuBenseite yon normatem Aussehen ist. Dann werden drei Jodoformgazem@hen in den Subduralraum gefiihrt, worauf ein Chlorkalkwasserumsehlag angelegt wird. In der folgenden Zeit wird der Subduralraum bes~ndig mit Gazemeehen drainiert, mit Intermissionen fliefft andauernd in sehr reiehliehen Massen Cerebrospinalfliissigkeit aus; es ensteht ein Hirnprolaps, der naeh und naeh so grol~ wie eine Mandarine wird und sieh mit Granulationen bedeekt. Am Anfang ist best~ndig hohes Fieber und Hirnpuls vorhanden, die Patientin ist in der Regel unklar, apbatisch, hin und wieder Konvulsion, oft Erbrechen; aber vier Tage naeh der Operation besteht nur sehr wenig Fieber, und in der darauffolg'enden Zeit bessert sieh der Zustand bestiindig. Aphasie und Unklarheit schwinden, die Temperatur ist nur ganz wenig erhSht und einen Monat naeh der letzten Operation normal; Pat. befinder sich wohl, ist ganz normal mit einem leiehten imbezillen Anstrieh (so wie sie nach der Aussage der Mutter vor der Operation war). Pat. kann vermutlieh als geheilt betraehtet werden, die Kraniot0mieSffnung ist ganz mit Epidermis iiberzogen und der Hirnprolaps ist geschwunden. Die Cerebrospinalfltissigkeit war bei der Einlieferung ungefiihr klar, zeigte aber bei der Mikroskopie, daft sie polynukleiire Leukocyten enthielt, abet keine Bakterien; trotz wiederholter spiiterer Versuehe gliiekte es leider nieht, dureh die Lumbalpunkfion Cerebrospinalfliissigkeit zu entlehren, da die Punktion stets ,,troeken" war. Der eytologische Fund bei der ersten Untersuehung ist vermutlich auf die vorhandene Leptomeningitis zuriickzuftihren, die bei der Operation am Tage danach festgestellt wurde, da, wie oben angefiihrt, angenommen werden muff, daii die unkomplizierte Paehymeningitis eine klare Cerebrospinalfltissigkeit ohne polynukleiire Leukoeyten liefert. 268 IX. N. RH. BLEGVAD. Tabellm'ische ~bersicht iiber die in der Literatur verSffentdes i Namen Verfassers V.erha t-[ tische Ver- t . Externe Alter msse der [httttnisse der Sltz usw. der Fliiehe der Jahr der Ohren- [Paehymenin. / Paehymemngltis Dura Publikation ~ Jahre eiterung gifts I ChroMetastase Auf der Innenseite der Tin'ombose 1 A l e x a n d e r weibl. 42 nische yon derLungc Dura 6 ovale, l ' / 2 x 2 ' / 2 des Anus 1902 aus ~?) o d e r cm Flecken, gelblich-transv., sons~ Supp. holcste ~- Infektion yon weig, yon fibrinOsen, nichts Abciner Sinus- purulenten Koagulis benormes tom thrombose deckt mit chagrinierter ausgehend, Oberfliiche. den Gef~lten folgend Nr. _~ ~ I 19 Chroniseh¢ Supp. Tegmen Eiter im Subduralraum. aditus ad Die 0berfl~iche des Ceantr. cafiSs rebellums gelblichgrau. Beco 1902 m~inn]. 7'/~ Chronisehe SuPl). Cheval 1904 mSnnl. 32 Chronische Supp. Tegmcn antri Groger AbszeB untcr Nit Granudestruiert der Dura, sich in die lationen beOberfliiche des Ccrc sctzt. Kleiner: cpiduralcr bcllums erstreckend. Al)szel~, Der Finger kmm t0 cm tief in die AbszeBhShle spiiter Fistel I hineinget'iihrt werden. in der Dura Tegmenantri' ')00 Gramm f6tider Sieht gesund destruiert E~ter unter d e r Dura. aus (es kommt abet ein Tropfen Eiter hervor auf der Stelle, wo d. Tegmcn destruicrt ist) 39 Chronische Supl). 2 Beco 1902 3 4 /m~mnl. EpiduralabszeB [ 5 Ceci m~innl. und 0 n e t t i 1886 ] 60 Gramm fStider !Fistel durch Eiter unter der Dura. ]die Dm'a und [ Der Abszel~ li~,gt auf I den ~chfidel dem Tentoriuin. ! Ueber die otogene Pachymeningitis interna purulenta. 269 lichten F~tlle yon Pachymeningitis interna purulenta. S3-mptome Tem- "~ ~"~ ~ terien pera- tur Normal (makroskopisch) dereAn" Diffuse SymHirnptome sympt, i Fokale Hirnsympt. Operation 38.6 /14010hren- Kopf- 1 Schiit-ttel_i Isehmerzensehmerzen fr6ste i n n s - Radikal?om- operation ose unen- szel ,,scheint intakt zu sein" (makroskopisch) A. yon nekrotischen ViUosit:£ten bedeckt 39.0 110 Sehiit telfrSstc Be- I nommenheit ddu der tbzel3 37.9 Somno- ddu tier kbzeg lenz ft Sta104Fazja-~tauungs ?aresede Triibe phylo- 39"6i ] . / papille contra- /Cerebrokok- I P i i a- ErhShte lateralen I spinalke~e:n / Sehnen-Extremi- [ fiiissigk. reflexe tgten m. polyCere- I nuel.Leu bospikocyten nal-I und Sta fliissig[ phylokeit kokken. 12 Tage I naeh de: I Operation klarq i Flfissigkeit DerBoden )4 Schwin-I Paras- / des Abdel, Kopf- I thesien u./ szesses schmerzen Schmerwird yon zenin den den eiteri Unter" 1 bedeckten ~extremi- I Ix t~iten 1 Hirnwindungen Pupillen- ] gebildet differenz Cerebellare / Ataxie L (An Lungenabszefl.) Die Pachym wird erst bei der Sektion gefunden 5" PlStzlich vor der Operation Zwei Op. Heilung. Der sub- Eingrol~er ~luraleAb. Prolaps szel~ wire bleibtbeerst bei stehen der 2. 0p. I gefunden / In einer Heilung Sitzung Resektio~ les Proe mast. um Inzision der Dur~ AufHeilung meil~eIung des Proc. mast. 1 Jahr nachher Trepanation und Inzision der Dura I Archly ftir 0hrenheilkaade. 83. Baad. Aus- gang t8 270 IX. N. RH. BLEGVAD. Namen des Verfassers Nr. Jahr der Pubtikation Verhalt- t tische Vet- 1 Alter ~msse der lh~iltnisse derl i Ohren-IP ehymen n-I Jahre ~elterung I 6 D e l s t a n c h e m~tnnl. jilt]. 1898 gifts Chronische Supp. Flstel der Wand des Antrums Sitz u,w der Pach menin " t i s Y Externe Fl~ehe der I)il a I Gro~er subduraler Ab- Fistel dureh sze~, 1/4 Liter iibel- die Dura, dis riechenden Eiters ent- sonst norma haltend. aussieht f I 7 Donath 1903 weibl. 26 Chronische Supp. LaminavitreaI Auf der Unterseite des Dura sieht des Aditus 1Cerebrums ein hasel- normal a~s, verdfinnt, die] nul~groger, flaeher, in- ist weicher Diplo~zellen Jltramenin~ealer Abszel~. als normal entzfindet t Gegentiber dem Porus anzut'tihlen aeust, int. auf derInnenseite der Dura eine gelbe, diehte, fibrinopurulente Sehieht yon i--2 mm Dicke, wm~n Acusticus und Facialis. Guder 1903 m~innl. 22 Chronisehe Supp. Dura liegt entblSl]t Heine 1903 'mnnlI 26 Chronische Supp. 1/a Liter Eiter in einer Dilra ohne enormen H6hle anf der Pulsation Unterseite des Gehirns. Bei der Sektion ein groBer ,intracerebraler' Abszel~, der den ganzen zentralen Tell des Gehirns destruiert hat. Vermutlich nach der Operation entstanden. Bei der Operation wird Kleine Fistel ein Abszel~ in der Ober- in der Dura auf der fl~iche des Cerebellums gefunden (@r6t]e wie Riickseite 1/2 Kirsche): :Bei der der Pars peSektion anf der Innen- trosa. Die seite der Dura fiber dem Dura fiber vorderen Tell des Ge- dem Cere hirns auf beiden Seiten bellum mil~farbig Igelbliche Ablagerungen /mit roten Punkten und 1Streifen. Ueber die otogene Pachymeningitis interna purulenta. 271 g-~=~.=................................................................................................................................ L I Symptome d'~ BakTern-] ~ An. [terlen / pera-I_~ 2 ere l>"~< t ~~ / tur il ptome Diffuse Hirnsympt, Strep- 39.6 tokok-I ken Sympt. leiner Leptomeningifts ~~ ~-4,-~ Fokale Hirnsymt. .~~ ~ ~ g ~ .. vperanon ~~ Ausgang Heilung / ti Pia in den In der/Fieber Abszeg Cere- I einge- brospi-t lagert, nalfl. ] mm dick, Strep- I mit einer tokok-I dicken, ken u. I byogenen Pro- ] Membran teus t bedeckt vutg. In der Dura Sta/phyl 2 t Fazia- Kopf- und lis- ] Nackenpara-]schmerzen lyse Erh6hte Kniereflexe Besserung nach der Op. Dot]ion- stration in einer reed. Gesei]schaft. An demselben Tage Cerebralia Nach 7 Tagen Partus und Mors i coeeu~ citr. e albus P. undA. wieeine Sehleimhaut anzufiihlen 39.6 A. bei der Sektion SdematSs. Gehirn normal 38.3 -- 392 I i I Fazia- Kernig lisKopf- i parese sehmel~eniI-arwe uer Kt311~ nllr~: ISchwindel] kontra- vielen, 'Erbrechen lateralen polyKonvul- Extrenukl. sionen mifiiten Leukocy~en. Sp~iter (vor ~er Op.) klare Fliissigkeit ohne Leukocyten Ohren-/ Kopfschm:r Is;hbm:~'~e¢~ Ns'tCke: Bemmmenhot bei 1. 0p. gefunden (Punktion). 3 Monate nachher eine subdurale Eiteransammlung + RadikalOp. Der Sp~ter an AbszeI~ Lungenwird erst tuberbei einer kutose 0peratiol 8 Tage ,p~ter ge funden 18" 272 IX. N. RH. BLEGVAD I PathogeneVerh~ilt- I ~ . . . . ~/el'- t r~isene ~.. . Alter msse • (~er dei• . . . . ~l~Z usw [hattmsse derl ........ 0hrenvacnymenlngms ., [ Pachymenin-I Jahre el~erung I gitis__ _ I . . . . . . . . . . . . . . . . . . . . . . . i Namen des VerfasSers ~ Nr. _l Jahr der Publikation r~xterne ,, .. ~lache der Dura 10 Heine 1905 Im~innl. 35 Chronische Supp. 11 Heine m~innl. 35 19o5 i Chro- Kleine Dehis- Bei der Sektion werden Perforation nische zenz (nicht r 2 grofie, subdurale der Dura, det~ Supp. Caries} des I Abszesse (mehrere El~- Dehiszenz int CholeTegmens 16ffel Eiter) gefunden Tegmen ent~ steatom Membranen auf der sprechend Dura. 3 tiefe Impressionen auf der 0berfl~che des Gehirns. Auf diesen Stellen dicke Membranen. 19 Chronische Supp. 12 Hinsberg t901 m~nnl. Bohnengrol]er, subdu- Dura infilraler AbszefL 1/2 Jahr U'iert, gelb spiiter wird ein zweiter grol~er Abszel] welter nach hinten gefunden. Cochlea se- 1I Bei der Sektion wird questriert. ein alter, abgekapselter, Dadurch eine I meningitischer Focus Fistel an der r in der Gegend des Riickseite der; Porus acustic, int. gePars petrosa, i funden. [ 13 HSlscher1905 m ~ n l . i 21 14 HSIscher 1905 48 weibl, ]Fistel in der[i Reichlicher Eiter auf~erunterenWand halb der medialen Wand des Sinus d e s Sinus transv. Bei transv, der Sektion 5 Tage spiiter kein Eiter im Subduratraume. Wieder- Tegmen antri I Bei der Sektion findet Die Auitenholte An- fehlt m emem} man auf mehreren seitederDur: fiille yon Umfange v o n Stellen der Hemimit kleinen Otitis 1 qcm 3h~irendicken, griinen, Granulanicht fliissigen Eiter tionen bezwischen der Arach- setzt, 14Tag noidea und der Dura sp~iter miB. farbig gelagert. Ueber die otogene Pachymeningitis interna purulenta. 273 J Symptome AnBak- Tem- ® ' dere terien pera-tur Symptom( 40:0[ SchiittelfrSste Diffuse Hirnsympt. Fokate Hirnsympt. Kopfseh~lerz., Amne- stische Aphasie nofflmen- heit / Nackenstarre, Be. P. und A. ~hit dicken Membranen bedeckt, ~ach deren Enffernung P. normal aussieht GehirnSdematSs l]onlnl PB- i -~- Fiebet" Leptomeningitis diffusa heit Operation He]lung (die amnestische Aphasie h~ilt sich [ lange) DieCere- szel~ Radikal- / d" Op. Der 6 Tage brospi- [ mbdurale [ nach der nal- [ Abszel~ ]Operation fliissigwird erst keit ]st bei der ] klar J Sektion gefunden Grol~er [ epidu- Stauungspapille, N aekenstarre, Sehwindel + Leptomeningitis, i Keine Adh~irenzen zwischen ~ler Dura und der Arach noidea Purulente Strep- norAufla,~er. tokok- mal ~uf d. Ge- ken in Ihirnober- der ~l~iche. Da- CereIrunter ]st brospiPia ganz nap fliisfrei sigkeil E gangAUS" 5 Tage nach der Operation tl'ansv. Nystag- I Senso- Die Cerenus, Kopf- rische / brospiund I Aphasie nal Nacken- ] I fliissigschmerz., [ keit Schwind. i leicht sp~iter ~ I getriibt Konvulmit sionen. [ Streptokokken [ Aufmeil3eiung 2 real Die mit einem meningit. Zwischen- Sympt. raum v 8Mortschwinden 14Tg. sp~ttor D~llv~t-,n Kraniotom., ~l~..~v~. Punktion : 6 'lage triibe, serSse nachher Fliissigk. die I~,~,~r,~l_ Dura wird / ~ " . . . . . . ges[,alten; i s l o n e n ~ es entleert t Aphasie. I sich wenig . . . . getriibte I l~raI~.101~" i Fliissigk. [ e t c , ~ ± v l o r s 274 Namen des Verfassers Jahre der Publikation 15 Jansen 1895 iX. N. RH. BLEGVAD. r I Path°gene" [ Veh~lt- ti h V r ! ] Alter l nisse d~r I s e e e Sitz usw der [ Externe $ ~~ ~ i hiiitnisse der " . . Ohren• Paehymemngms / Fl~ehe der 1 . !I aehymeninDura _ J a h r e ! elte!m!g I ......g ' i t i s ........± . . . . . . . . . . . . . . . . . . . . . . . . . .Z. . . . . . . . . . . . . ~m~nnl. i 31 Chronisehe Supp. [ J Fistel im Bei der Punktion der [ Dura grauTegmen antri Dura kommt ein I gelb, Tropfen Eiter hervor, t gangr~in6s Dura gelblicl~griin, mit Pin und Araehnoidea verklebt. I i I 16 Kiimmel 1904 weibl. 11 Chronisehe Supp. Cholesteatom L7 Lehr 1899 weibl. 36 ~8 Lueae 1899 Dura von Chro- Ausgedehnte I Im Subduralraume Granulanisehe Ostitis, Sinus- zwischen den 2 unteren tionen gethrombose. Sehl~tfenwindungen Supp. deckt, teil1/2 TeelSffei Eiter. Chole- Nekrose der weise nekromedialen steatom tisch Sinuswand und groBcr nekrotiseher Defekt dcr Dura. Dm'a grauLinsengroBe Dutch eine ganChrogriin, Fistel im gr~nierte Pattie der nisehe Supp. Tegmen antri Dura dringt eine Sonde gangr:~inSs etwa 1 cm hinein; Eiter CholeflieBt hervor; bei lnsteatom zision der Data kommt abet nichts. 14 i _)urn grfin'~ Bei der I. Operation lich, eine wird niehtsim~ubduralraume gefunden. Einen ! 'istel ist nichV [Monat spEter wird die] zu sehen Patientin kranker, die Dura tiber dem Antrum i ist hervorgewSlbt; sie wird iuzidiert, wodurch 2 EtllSffel ,,milehige Cerebrospinalflfissigkeit" entleert werden. K. meint, dab dieser Abszef; "9"011einer Infektion dureh die erste Inzision herrfihrt, Ueber die otogene Pachymeningitis interna purulenta. v ~ ~ ~= ~ ' ~ ~ymptome Bak- Tern-I ~ 1 An- 1 Diffuse ~ terien pera. i~i de,tel Hirn~.,~ .~ . fur i Iptome/ symp~, Leptome-[ nlnges I gelb!jchgrun 38.2 92 I I ~ g ~ t ~ ~ t~ Hirn- N~ N~ symp~. q Aus- iUpera~°n gang I EpiduDie / Heilung raier Pachy- t Abszel~ meningit. mit wird bei ] l ~istel Jder 1. 0p. in das lentdeckt. Cere-I bruin i On. FaziaI lisIparese Die Corticalsubstanz ist in 1.5 cm Tiefe zerstSrt 380 --40° P. u. A. yon Granulationen iiberzogen 36.8 76 0hreu- Neuritis ischmer optica, zen Nystagnus, Be- dt, len nomlxlen belt, ~chwind., Taumelnder Gang. 38.5 99 Fokale: SeaSchmerzenbeiBe-] sorische wegungen I Aphasie d. Kopfes. I Delirien I Appetitlosigkeit, Obsti- I i : 275 Kopfscbmerz., Nackenstarre Laby- Resektion Heilung rinth- desMittel~uppu-iohres. Inration zisiou der brutus mitl negat. Resultat, l 1. Monat spiiter In- / zision u. Drainage I des sub- f duralen I Abszesses i GroBer Der Ab- Heilung epidu- szeI~ wird raler bei 1.0p. k Ab- gefundenr I szet$. [ Throm bose / des i f Sinus t trausv. I 1 Der Ab-I Heilung sze8 wird/ bei i. 0p. gefunden. In der n~ichsten I Zeit neh- I men die I Him- J ~ympt. zu ;L de schwin / den erst, als veto Subdural- t raum j reichlicher Sekretion I ausgeht. 276 F :Namen des ~Nr, Veffassers Jahr der Publikation 19 M a c e w e n (Fall X) 1893 2O M a e e w e n (Fall XI) 1893 21 Maeewen 22 ~Iacewen (Fall XI[I) 1893 23 Milburg 1899 IX. N. RH. BLEGVAD. Verh~tltExterne Alter nisse der tisehe Ver- I Sitz usw. der Fl~iche der Ohren- h~ltnisse der Pachymeningitis Dura eiterung Paehymenin! Jahre gifts DasTe.g..men 10 g Eiter im SubduralDura n~innl, 19 Chro- I canos, raume mit Suppuration ulzeriert, ernisehe I der Hirnobeffl~iche weicht, Sups; I oberhalb des Antnlms, (1/~ ,Janr) 1/4 Zoll in Circumferenz. weibl. 91/2 D as Tegm en Subduraler AbszeI~ m i t Dura mit Granulageringer Eitermenge, desWdiert. yon zusammengelSteten lationen beMeningen begrenzt; der deckt; in AbszeB steht mit dem deren Mitte Cavum tympani dureh eine Fistel. eine Fistel der Dura und des Tegmens in Verbindung. 22/12 Chronisehe Supp. Ausgedehnte Destruktion der Pars petrosa, epiduraler AbszelL Kleine Eitermenge im Subduralraum. 2 Dauer der Eiterung 4 Monate do do weibl, 33 Chronische Supp. Caries mit Grol~er subduraler AbSequestrie- szei$, dem sequestrier- t rung eines ten Teile des Mittel- / Teiles des ohres entsprechend; Mittelohres. die Cavitiit geht 11 cm naeh unten-vorn-innen. m~nnl. 17 Chronische Supp. Der Prozeg Bei der Sektion werden Dura in der Mitte des auf der entgegendureh epiduralen Tegmen antri gesetzten (gesunden) verpflanzt. Seite Abszesse auf der Abzesses destruiert. Innenseite der Dura gefunden. Sie enthalten ein plastisches z~hes Exsudat und sind fiber die ganze Basis antri zerstreut. (Fall XII) lb93 ~Dura er- weicht aufgefasert erodiert und perforiert. do Ueber die otogene Paehymeningitis interna purulenta. Symptome Bak- Tern- ~ An- Diffusc d ere terien peruSym- Hirntur ptome Sympt. Suppuration der Hirnoberfl~che. 36.6 Suppura-! tiou der Hirnober- i Niche. Fie-] ber .$ Operation AUS* gang Heilung. sehnlerz., BewuBtlosigkeit, Konvulsionen. Symptome "~ an ttlberkulSs. IEnteritis ] 47 Tage naeh der Operation. einer tuberku- [6sen Mei ningitis. } Suppuration der l IIirnober- t fliiehe. i Fokale Hirnsympt. Neuritis optiea Kopf- 62 277 -~ Fieber Ohren-[ sehmer L Heilung. zen~ 1 ; para- 1 flY se. ] do Pia rot, getriibt, prolabierend. @ Fieber @ Fieber, Schfit-! tel-l frSste H eilung. paralyse. Neuritis Parese deri optica,Be- homolatenommen- ralen Ge- L I heit, Er- sichts[ brechen, hNfte und der kon- / / tralatera" i len Extre-[ mit~iten, / amnestische Aphasie. Ostei- Der Ab- Heilung, jedoeh tis b l e i bi e n d e d e r acuta szegbeiwird ersten leiehte proeesstlS Operation Aphasie. gefunden. t mast. SomnoPtosis, ]lenz, Ver- Paralyse wirrung, des Sehwind., Armes. Nackenstarre. Gro6. Bei 1 )p. Die Symepidu- wird e r ptome raler epidm .tei nehmen Ab- Absz ~ zu und sze6 gefun~ m, Patient erst b( d. stirbt Sekti a einen Tag werd. ie nach der sub& ~f. Operation. Absz. I 278 IX. N. RH. BLEGVAD. PathogeneVerhiiltAlter nisse der tiscbe Verh~iltnisse der Ohren - PaehymeninJahre eiterung gitis Akute Fistel ira 25 U f f e n o r d e ]m~innl.I 30 Supp. ~egmen antfi. 1908 Nr. Namen des Verfassers Jahre der Publi!~ation 26 27 U f f e n o r d e in~innl. 1908 Witzel 1901 25 Chronisehe Supp. Sitz etc. der Pachymeningitis Externe Fl~iche der Dura Ein wenig Eiter im Dura gelb, Subduralramne, dem weniger reepiduralen Abszesse sistent; Epientsprechend. Bei der duralabzeB Sektion wird all der yon GranuBasis des Occipital- lationen um\|appens ein grofier geben. Keine subduraler AbszeB Fistel in der Dura. gefunden, mit erstgenanntem AbszeB durch eine Rinne verbunden. Fistel hinter Dura auf der Innen- Dura gelb, der Radix are. seite dem epiduralen epiduraler zygomatic. AbszeB entsprechend hbszeB yon bach der verdickt. Ganz vorne einem GranuFossa cranii eine Fistel in der Dura lationswalle media und dieser entsprechend umgebem fiihrend. ein groBer AbszeB im Keine Fistel zu sehen. Schf~ifenlappen. Triibe, serSse Flfissigkeit aus dem Subduralraum fiber der moterisehen Region. Ueber die otogene Paehymeningitis interna purulenta. "279 --;=---~ ......... -°--= ............:::: :==::-== . . . . . . . . . . . . . =............. - - - = : ~ = : - - L = : = : ; ..... a ~ ~ Svmptome i t I "= ~ "~'= An .. ~ ~~,okale o ] ~ , ~~ ' Aus~ ~ Bak - TernDiffuse "~ . ~: ~ ~ ~ terien pera- ~ ! dere HirnHim"~ i ~ ~ Operatmn gang ~_~l ........... tu~ Auf A. ]Strepeine dickel tot~lbnn - I coccus schicht; [ pyo~ der Sub-i genes duralraum / erweitert, I mit Eiter I u. Rand-I zellen ge- / gefiillt. --] Fibrinab-I lagerun- I gen in Pial u. im Ge-i him, den] GefiiBen ] entlang, i Makro-i skopisch i nichtsAb-I normes in] Pig od. iml Gehira. i A. scheintl Ver:~9 intakt sehiezu sein. dene St~bchen u. ! Kokk. (F~tul-I nisbak i Iterien) . . . . I . !ptonle . sympt. ~" [ -- Kopfschmerzen, Erbrechen, Respirations[~hmung~ Coma. IEpidu- Totalauf -t- Kurz raler meiBelun nach der Ab- I1. subdm Operation szel~ Abszell an Leptowird meninw~illrend gifts d. Operau bei Inzision der t Dura ge-I funden. ] 2. Abszefl wird erst bei der Sektion gefunden KopfLeichte sehmer- iAbduzens- Grog. Totalauf Heilung epidu- meitlelung raler i Inzision Absz. der Din-a, Absz. I Drainage im (mit GlastSchlii- Idrain) des i , J sympt, zon~ 1)arese, Mattigkeit. leichte. amnemsche lAphasie. ]Naeh derl Operation Paragraphie t il at¢tipen abHs~r~ses r Oin'e'i i eat" / i fernt, VOln I subduralen ] r Absz./ indu- 1 ziert A.in einem 1 Mark grollen Abszel~ verwandelt, wovon Aus- l~iufer yon Eiter in den Su]ci Die Sympt I entstand, l apoplektiform / nach der I 0peration i des Pro-! cessus mast ' Hand- -~- an Meningitis tellergrotle 12 Taffe Kranioto- nach der mie, Jodo- Operation formgazet~mponade 280 IX. N. RH. BLEGVAD. ]irkllirung der Abbildungen auf Tafel IX. .Fig'. I. Man sieht die grol~e Depression des Frontal- und Occipitallappens. F i g . 2. D u r a s i n i s t r a . Man sieht auf der Oberfl~che (Innenseite) der Dnra die dicke Schicht von Fibrin mit Leukocyten, die teilweise degeneriert sind; darunter die stark leukozyteninfiltrierte Dura. Nur ein Bruchstiick der Dura ist abgebildet, etwa ~/~ der ganzen Dicke, Fig'3. Dura dextra. Auf der 0beffFziche (Innenseite) der Darn eine recht dicke Schicht yon Fibrin mit Rundzellen iniiltriert, darunter Dura mit Leukocyteninfiltration. Literaturverzeichnis. 1. A l e x a n d e r : Zur Pathologic und patbologischen Anatomic der Pachymeningitis interna in Fiillen otogencr Py~imie. Monatsschr. f. Ohrenh. 1905. S. 105. 2. A l e x a n d e r : Ein Fall von otogener Py~imie mit frischer Pachymeningitis interna circumscripta. Verh. d. 5ste~T. oto]. Ges. 24. Nov. 02. Ref. Centralbl. f. Ohrenh. i903 S. 316. 3 B a l l a n c e , C h a r l e s A.: Some Points in the Surgery of the Brai~ and its Membranes. London 1.qi~L S. 50. 4. B e c o , L.: Trois cas de suppuration intra-durale d'origine otique. La presse oto-laryngologique Beige. 1902. S. 131. 5. C e c i u. O n e t t i : Ascesso intercranico, Craniotomia esplorativa della clinica chirurg, della Universit~ di Geneva 18S6. Ref. v. B e r g m a n n . Die chirurg. Beh. v. ttirnkrankh. [L Aufl 1889. S. 80. 6. C h a v a s s e u. Mahu~ De la ponction lombaire duns les complications endocraniennes des otites. - - Soc. fram;, d'otolgie etc. Okt. 1903. S. 1. 7. Ch e v a l : lntraduralabszel~. - - Otolaryngolog. Klinik. Er~iffnungvorlesung. H. 1. 1904. Ref. Centralblatt f. 0hrenh. 1904. S, 511. 8. D e l s t a n c h e jan.: Bulletin de la Soc. Belge d'otologie, de lalTngologie etderhinologie. 1898. H. 1. S. :~8. R e f K 6 r n e r , 9. D o n a t h : Beitr~ge zur eitrigen Meningitis cerebrospinalis, Wiener klin. Wochenschr. 1903. S. 759. 10. G u d e r, Un cas de Pachymeningitis interna (A bsc~s intradvral) d'origine otique. Ann. des real. de l'oreille etc. 1903. S. 64. 11. G r o l ~ m a n n , Kasuistisches zur Lumbalpunktion und zirkumskripten Meningitis. Archly f. Ohrenh. Bd, 64. S. 24. 12. H e i n e , B.: Zirkmnskripte Gangr~n der Dura and subdm'aler Abszel~ in der hinteren Schlidelgrube infolge chronischer Mittelohreiterung. Deutsche med. Wochenschrift 1903. S. 119. 13. H e i n e , B.: Ein Fall yon zirkumskripter Gangrfin der Kleinhirndura. Berliner otol. Ges. 13. I. 03. Ref. Monatsschr. f. Ohrenh. 1903. S. 311. 14. H e i n e , B.: Zur Kenntnis der subduralen Eiterungen. Festschrift fiir Lucae. 1905. S. 34l. 15. H i n s b e r g , Zur Therapie and Diagnose der otogenen Meningitis. Zeitschr. f. 0hrenh. 1901. Bd. ~8. S. 126. Fall I. Ueber die otogene Pachymeningitls interna purulenta. 281 16. H S l s c h e r : Die otogenen Erkrankungen der Hirnh~iute. II. Die Erkrankungen im Subdurah'aum und die eiterige Entziindung der weiclmn Hirnh~iute. Halle. Marhold. 1905. 17. J a n s e n: 0ptische Aphasie bei einer otitischen, eitrigen Entziindung der ttirnh~ute am Iinken Sehl~fenlappen mit Ausgang in Heilung. Berl. kiln. Woehenschr. 1895. H. 35. S. 763. 18. KiAr, G o t t l i e b : Abszess. epidural, reg. tempor, et Abszess. lobi frontal, cerebri. Operation. Helbredelse. - - Ugeskrift f. LSger 1905. H. 45. 19. K r a u s e , F e d o r : Chirurgie des Geliirns und Riickennlarks. 1908. 20. K S r n e r , O t t o : Die otitischen Erkrankungen des Itirnes, tier Hirnhiiute und der Btutteiter. Dritte Auft. Wiesbaden t902. 21. K S r n e r , O t t o : Nachtr:,ige zur dritten Auft. der otitischen Erkrankungen des Hirns etc. Wiesbaden 1908. 22. K i i m m e l , W.: Uber infektiSse Labyrintherkrankungen. Zeitschr. f. klin. Med. Bd. 55. 1904. S. 373. Fall 5, 23. L a n n o i s et C o r n e t o u p : Absc~s sous-dure-merien aigu ouvert spontan~ment au niveau de l'occipitale. Soc. franc, d'oto-rhino-laryngolog. 19--21. Okt. 1903. S. 124 24. L e h r , G.: Beitr~ge zur Kenntnis der otitischen Erkranknngen des Hirns, der Hirnh~iute und der Blutleiter. Zeitschr. f. 0hrenh. Bd. 35. 1899. S. 12. Fall 16. 25. L e i d z y n s k i : Ref. Neurologisches Centralblatt 1899 und K S r n e r . 26. L u c a e , A.: Operative Heilung eines ])alles yon otitiseher Meuingitis purulenta. Berl. klin. Wochenschr. 1899. Nr. 23. 27. Mac C o y , J o h n : Fall yon Mastoiditis mit Interduralabszei] und Durchbrucb dutch die Sutura parieto-occ, mit enormem Hautabszel~. Otolog. sect. N. Y. Acad. of Med. i6. 0kt. 1907. Ref. Centralblatt f. Ohrenh. i908. Seite 342 28. M a c e w e n : Pyogenic infective diseases of the Brain and Spinal Cord. 1893. S. 75. 29. M a n a s s e : Uber die operative Behandlung der otitisehen Meningitis. Zeitsehr. f klin. Me(]. 55. Bd. 1904. S. 315. 39. M e i e r , E d g a r : Uber einen geheilten Fall von otitischer Py~mie, Meningitis und zwei Hirnabszessen. Miinch. med. Wochensch 1900. ~, 813. 31. M i l b u r g , S.: HirnabszeI~ infolge chronischen Ohreneiternng, Neuritis optica, ()ffnung yon Warze und ScbSdel, Iteilung. Journal of Eye, Ear, Throat-Diseases. tIL 1. Ref. Centralblatt f. Ohrenh. Bd. 46. I899. S. 70. 32. M y g i n d , H o l g e r : Den otogene Meningitis reed shrligt Hensyn til dens operative Belaandling. Ugeskrift f. L~iger. 1909. S. 269. 32. P o l i t z e r , A d a m : Lehrbuch der 0hrenheilkunde. Fiinfte A u f l Stuttgart 1908. 34. S u c k s t o r f f u. H e n r i c i : Beitr~ge zur Kenntnis der otitischeu Erkranknngen des Hirns, der Hirnh~ute uud der Blutleiter. Zeitschr. f. 0hrenh Bd. 44. 1903. S. 161, Fall 57. 35. S z e n e s , S i g m u n d : Empyema .a.ntri mast. und Subduralabszel~ mit meningitischen Symptomen. Budapester Arzteverein. 21. M~rz 1903. Ref. Centralbtatt f. Ohrenh. 1904. S. 230. 36. U f f e n o r d e : Zwei FSolle yon subduralem Abszel~ mit Demonstration. 17. Vers. d. deutschen otol. Ges. in Heidelberg. 1908. S. 232. 37. W i t z e l , O s k a r : Die operative Behandlung der phlegmon6sen Meningitis Mitteilungea aus den Grenzgebieten der Mediz n u. Chirurgie. Bd. 8. 1901. S. 385. Archly ftir O h r e n h e i l k u n d e . 83. Bd. Tafel IX. Fig. 1. Fig. 2. Fig. 8. Dura sinfstra. Man sieht auf der Oberfl~iche die dieke Sehicht yon Fibrin mit - - teilweise degenerierten - Leukozyten Darunter die stark rundzelleninfiltrierte Dura, yon dieser ist nur 1/~ der ganzen Dicke gezeiehnet. Blegvad. Dura dextra. Auf der Oberfi~iche elne recht dicke Schicht rundzelleninfiltrier tes Fibrin. Darnnterdie Dura mit recbt starker Infiltration yon Leukozyten. Verlag yon F. C. W. Vogel in Leipzig. ~,o,,rd,.h, (,, Otto),L.rp,~g.
https://openalex.org/W4362535095	https://figshare.com/articles/journal_contribution/Supplementary_Figure_S3_from_Simvastatin-Induced_Apoptosis_in_Osteosarcoma_Cells_A_Key_Role_of_RhoA-AMPK_p38_MAPK_Signaling_in_Antitumor_Activity/22508217/1/files/39970035.pdf	Latin	null	Supplementary Figure S1 from Simvastatin-Induced Apoptosis in Osteosarcoma Cells: A Key Role of RhoA-AMPK/p38 MAPK Signaling in Antitumor Activity	null	2,023	cc-by	304	amel et al. Supplementary Figure S3 C A TOFA (µg/ml) 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1.0 1.2 Relative cell viability Day 1 Day 2 Control 0 0.5 1.5 Tumor weight (g) (N=14) 1.0 NS Fat-free diet (N=13) B 1.0 1.2 Control 0 0.2 0.4 0.6 0.8 Tumor weight (g) Simvastatin (N=10) (N=10) NS Control 0 0.5 1.5 2.0 Tumor weight (g) (N=16) 1.0 NS simv 80 (N=16) D E amel et al. Supplementary Figure S3 C A TOFA (µg/ml) 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1.0 1.2 Relative cell viability Day 1 Day 2 Control 0 0.5 1.5 Tumor weight (g) (N=14) 1.0 NS Fat-free diet (N=13) B 1.0 1.2 Control 0 0.2 0.4 0.6 0.8 Tumor weight (g) Simvastatin (N=10) (N=10) NS Control 0 0.5 1.5 2.0 Tumor weight (g) (N=16) 1.0 NS simv 80 (N=16) D E amel et al. Supplementary Figure S3 C A TOFA (µg/ml) 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1.0 1.2 Relative cell viability Day 1 Day 2 Control 0 0.5 1.5 Tumor weight (g) (N=14) 1.0 NS Fat-free diet (N=13) B 1.0 1.2 Control 0 0.2 0.4 0.6 0.8 Tumor weight (g) Simvastatin (N=10) (N=10) NS Control 0 0.5 1.5 2.0 Tumor weight (g) (N=16) 1.0 NS simv 80 (N=16) D Kamel et al. Supplementary Figure S3 Kamel et al. Supplementary Figure S3 B Control 0 0.5 1.5 2.0 Tumor weight (g) (N=16) 1.0 NS simv 80 (N=16) D B A 1.0 1.2 Control 0 0.2 0.4 0.6 0.8 Tumor weight (g) Simvastatin (N=10) (N=10) NS C TOFA (µg/ml) 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1.0 1.2 Relative cell viability Day 1 Day 2 (N=10) (N=10) D C D C (N 14) (N 13) E E
https://openalex.org/W4387058478	https://www.frontiersin.org/articles/10.3389/fsoc.2023.1267706/pdf?isPublishedV2=False	English	null	Neither entrepreneurship nor intrapreneurship: a review of how to become an innovative split-off start-up	Frontiers in sociology	2,023	cc-by	13,499	TYPE Review PUBLISHED 25 September 2023 DOI 10.3389/fsoc.2023.1267706 TYPE Review PUBLISHED 25 September 2023 DOI 10.3389/fsoc.2023.1267706 COPYRIGHT © 2023 Knossalla and Carbon. 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. entrepreneurship, corporate start-up, intrapreneurship, split-offs, spin-offs, innovation, leadership OPEN ACCESS OPEN ACCESS EDITED BY Antonio La Sala, Sapienza University of Rome, Italy REVIEWED BY Ziska Fields, University of Johannesburg, South Africa Piero Formica, Maynooth University, Ireland CORRESPONDENCE Claus-Christian Carbon ccc@uni-bamberg.de RECEIVED 31 July 2023 ACCEPTED 05 September 2023 PUBLISHED 25 September 2023 CITATION Knossalla CE and Carbon C-C (2023) Neither entrepreneurship nor intrapreneurship: a review of how to become an innovative split-off start-up. Front. Sociol. 8:1267706. doi: 10.3389/fsoc.2023.1267706 Christina Elisabeth Knossalla 1 and Claus-Christian Carbon 1,2 1 Department of General Psychology and Methodology, University of Bamberg, Bamberg, Bavaria, Germany, 2 Research Group EPÆG (Ergonomics, Psychological Æsthetics, Gestalt), Bamberg, Bavaria, Germany Splitting off departments from corporations in order to establish corporate start- ups has become of strategic importance for the performance and innovation of corporations. While the settlement process is widely practiced, there is a lack of knowledge of how entrepreneurship may exist in such split-offs. The main aim of this study was to explore how entrepreneurship in corporate start-ups can exist in order to contribute to corporate performance. Based on a systematic literature review from 2021 to 2023, which resulted in a total of 1,516 scientific, English- language articles in economic journals, a total of 150 articles were analyzed in-depth. Our research shows that it is of crucial importance that corporations position leaders with an appropriate mindset and behavior at all levels as early as starting the split-off process, which is, however, neither entrepreneurship nor intrapreneurship. The niche corporative start-up area shows that entrepreneurship is a continuum and requires a new definition of corporate start- up entrepreneurship (CSE). For corporate start-ups to be successful, we revealed that there needs to be (1) the appropriate legal form, which ensures ownership but also the risk of the leaders, (2) an explorative business rather than exploitation, (3) variable compensation rather than fixed and (4) corporate entrepreneurs rather than employees and managers. Implications of the findings for entrepreneurial leadership theory development and future research are discussed. Front. Sociol. 8:1267706. doi: 10.3389/fsoc.2023.1267706 Conceptual model To close the research gap regarding what is CSE and which antecedents must be given in advance we adapted the conceptual model for corporate entrepreneurship from Urbano et al. (2022) (see Figure 1). The rationale behind this is, as far as we know, it is one of the latest SLR results focusing on all three levels of antecedents, individual, organizational and environmental, with a ground basis of literature. With our understanding of entrepreneurship aligning with the findings from Schumpeter, the adaption of the model for our investigation is necessary, as we understand entrepreneurship is solely possible with new business venturing plus, in this research, we focus on the niche of corporate start-ups. For the sake of completeness, Urbano et al. also mention the dimensions of strategic intrapreneurship (no new business addition but rather restructuring and organizational rejuvenation) and intrapreneurship, in addition to the dimension of new business venturing. Both dimensions are out of focus for our work and, therefore, removed from the original model, as shown below. Lastly, the consequences of new business venturing shall be innovation and not, as Urbano et al. put it, strategic and financial. Nevertheless, the adapted model allows to provide a systematic content analysis and organizing framework for the SLR we undertook. The importance of entrepreneurship in new venture creation (e.g., start-ups) and the effect on innovation and economic success is confirmed widely (Baron, 2007; Sambasivan et al., 2009). However, many scientists focus on intrapreneurship, which cannot lead to innovation (see Schumpeter). What is missing scientifically and in practice is which antecedents for corporate start-up entrepreneurship (CSE) must be given so that it can prevail in this niche organizational framework and then, in turn, pay off in terms of innovation. Accordingly, the main objective of this article is to enhance the understanding of how entrepreneurship may exist in a corporate start-up context. Therefore, our first requirement is to define a uniform framework for what can be understood by the term corporate start-up. To date, there has been no uniform definition of this. The second requirement is to derive corporate start-up entrepreneurship (CSE), which is needed for innovation in split-offs. We did so through a comparison between entrepreneurship and intrapreneurship. A descriptive research approach was chosen for this purpose. Our third requirement is to assess the antecedents for CSE based on the conceptual model for corporate entrepreneurship. Introduction Fastly growing start-ups that get to the heart of customer requirements pose a formidable challenge for existing corporations. On the one hand, there are start-ups, which, due to their small independent organizational form, can quickly adapt to the ever-changing market and customer requirements, bring innovations to the market quickly, thus being fast in execution and using new technologies, and ultimately take a large market share from established corporations (Mishra, 2020). On the other hand, large corporations struggle due to their entrenched structures, often struggling with change, rigid organizational structures with numerous, settled employees, being slower in execution than their start-up competitors, proceeding business models that are largely related to exploiting working business models and products rather than exploring new, innovative business areas. Start-ups are the biggest Frontiers in Sociology 01 frontiersin.org 10.3389/fsoc.2023.1267706 Knossalla and Carbon start-up context and which antecedents have to be established when splitting off. competitors for corporations that try to be innovative (Pfennig, 2021). As a result, organizations split up individual divisions of the corporation to create the fictitious space of a start-up under the umbrella of the parent corporation (= corporate start-up; Dalton and Dalton, 2006). What they neglect through pure organizational reorganization of split-offs is Schumpeter’s findings from 1912: Entrepreneurial innovation is central to economic development and the driving force for competitiveness. However, innovation does not come from growth in the context of core business. This is because concentrating on the core business brings optimization up to a certain point but excludes the possibility of releasing resources to devote to research and development and, thus, to innovation. Furthermore, in the innovation process, the entrepreneur is the central innovator because they are the only ones carrying out innovation intelligently, risk-taking, breaking up old and creating new traditions by shareholding their ideas (Schumpeter, 1912). In the split-off process, corporations fill their leadership positions with managers already hired in the corporation rather than with hiring entrepreneurs. These ambitions to introduce entrepreneurship into corporations lead, at most, to intrapreneurship but not entrepreneurship. This leads to the fact that introducing innovation fails, and all efforts are short-lived with relatively little seriousness (Mishra, 2020). The research for reliable figures regarding the success of corporate start-ups shows that articles provide widely diverging success values or that no figures are provided at all. Introduction What is uniform, however, is the statement that business start-ups must be carried out with better performance (Jung et al., 2015; Hundt, 2018). Concerning start-up success in general, the 3–30 rule is often applied: within 3 years, 30% of all newly founded businesses terminate their business activities (Held, 2019). f To answer the research question of which organizational antecedents have to be established to create CSE in corporate start-ups, this study is organized in five main sections. The first section focuses on the conceptual model (1) which was used to structure the systematic literature review (SLR). We then described the method (2) and steps used in our SLR. In our results (3) section we delineate the framework and the specifics of split-offs from corporate groups, which shall be referred to as corporate start-ups and external start-ups, to derive the particular context that plays an essential role concerning corporate entrepreneurship. Similarities and differences between corporate and external start-ups are highlighted in the result section to clarify what to look out for when entrepreneurship is to be achieved in corporate start-ups. Moreover, the results of the SLR show a comparison of entrepreneurship and intrapreneurship in order to derive corporate start-up entrepreneurship thereupon. Finally, the limitations and implications of the conclusive results are discussed (4) and concluded (5). Conceptual model We developed our line of argument on a model that has been derived by Urbano et al. (2022), giving an overview of individual, organizational and environmental antecedents for CE but with a broader perspective of new ventures as well as no new business additions (Urbano et al., 2022). All three requirements contribute to a more transparent view of how corporations can achieve innovation with corporate start-ups as new ventures. We elaborate on the current research status of the fields: start-ups, corporate start-ups, entrepreneurship, and intrapreneurship. With this, we contribute to enhancing the understanding of how entrepreneurship may exist in a corporate Frontiers in Sociology frontiersin.org Data collection Through a systematic literature review (SLR), including the steps of Tranfield et al. (2003), this study aimed to gain deeper insights into the impact of original entrepreneurship as a leadership style in corporate start-ups. The literature search was performed through two main procedures displayed in Figure 2: preliminary data analysis through forward database screening and secondary screening through a snowball systematic. We started with identifying the most relevant keywords of the research area, proceeded with the selection of studies, assessment of the quality of the papers, data extraction and concluded with the data synthesis as a result. Frontiers in Sociology Definition of new business venturing In the present paper, we understand new business venturing as adding new (innovative) business to the firm by growing organically (Harzing, 2002). Organic growth is defined as prosperity resulting from within and with internal resources (e.g., increasing sales or service volume or broadening the customer range; Dalton and Dalton, 2006). Organic growth can be divided into three areas: invest, meaning to reallocate resources to a focus area; perform, which means to excel at commercial functions; and create, which is understood as creating new products or services. As we focus on split-offs, we address the lever of create. This lever is understood as achieving innovation when a certain level of growth and stability prevails, making the most significant difference in outperforming the competition (Ahuja et al., 2019). In this paper, focusing on split-offs, 02 frontiersin.org Knossalla and Carbon 10.3389/fsoc.2023.1267706 FIGURE 1 Conceptual model for corporate entrepreneurship in new business venturing (own representation based on Urbano et al., 2022). FIGURE 2 Data collection and article selection process (own representation). FIGURE 1 Conceptual model for corporate entrepreneurship in new business venturing (own representation based on Urbano et al., 2022). FIGURE 2 Data collection and article selection process (own representation). FIGURE 2 Data collection and article selection process (own representation). Methods we do not consider inorganic growth, which is defined as external business growth (e.g., acquisitions, takeovers, or mergers; Dalton and Dalton, 2006) or any hybrid growth strategies blending the previously mentioned two and meaning that in parts resources are borrowed or purchased from the market and in portions growth is achieved from the corporation’s own resources (Agnihotri, 2014). frontiersin.org Definition of innovation We understand innovation along the classic definition of Schumpeter as “doing of new things or the doing of things that are already done in a new way” (Schumpeter, 1947, p. 14), which includes the creation of a new product, a new production method, the development of a new market, a new source of raw materials or a new organization. As a prerequisite, he states that the innovators are entrepreneurs who must design and implement the new thing themselves. Thus, they are a shareholder of the innovation and willing to take risks since they are entering the new and uncertain (Schumpeter, 1912). The first analysis (forward screening) is based on EBSCO database and took place between 2021 and 2023. EBSCO was chosen due to its coverage of articles that focus on management and new venture topics. Supplementary Tables S1a, S1b, show which keywords were used to start the elaboration. We decided on the following keyword 03 frontiersin.org 10.3389/fsoc.2023.1267706 Knossalla and Carbon FIGURE 3 Number of publications with reference to our corporate start-up entrepreneurship (CSE) keywords per year. FIGURE 3 Number of publications with reference to our corporate start-up entrepreneurship (CSE) keywords per year. FIGURE 3 Number of publications with reference to our corporate start-up entrepreneurship (CSE) keywords per year. The second analysis, a snowball search systematic, was applied to identify the most recent and most cited literature based on the previously systematically reviewed literature. The most recent and directly linked literature was identified through a related search for relevant most cited authors. For this purpose, the literature indexes from the 83 articles of our first step of SLR were screened, and the frequency of author mentions was analyzed. Thus, another 87 articles were added for a full paper check analysis. Supplementary Table S2 identifies the authors who were previously cited more than once among the 83 articles. The second part shows which authors and papers have emerged from step two of the analysis. The systematic literature review was therefore extended by further monographs in order to display a solid, qualitative, and comprehensive picture of the definitions and the most current range of publications. combinations as there are the most commonly used terms in the literature to describe entrepreneurship in corporate start-ups: “corporate start-up,” “corporate start-ups and entrepreneurial leadership,” “corporate start-ups and intrapreneurship,” “corporate split-off” and “corporate split-off leadership.” For each keyword combination, it can be deduced how many articles were searched. Results The trustworthiness of the research findings was ensured by focusing on scientific, peer-reviewed work only. Therefore, only a filter was applied to all search engines to display peer-reviewed publications. A further filter was set on the language setting, English and German only, due to the limited authors’ linguistic expertise. The search for English-language articles appearing in the EBSCO database published up to January 2023 in scientific journals returned 83 peer-reviewed articles which were all included in a full paper analysis to assess the relevance. The combinations yielded 1,516 articles via the EBSCO platform. All of these articles were further analyzed for titles and their corresponding keywords. If the title and keywords matched the topic of the study, the abstract was analyzed in a further step. Articles that did not focus on entrepreneurship/intrapreneurship in corporations were excluded. These were articles focusing on family firms, gender differences, venture capital analyses only, scholar and learning and cultural differences. One hundred thirty articles were further analyzed concerning the relevance of their abstract. Relevant, matching research papers were then stored in our reference managing software for further and detailed review. The sum of the saved and pre-screened papers corresponds to a total amount of 83 papers. Frontiers in Sociology Definition of innovation A combination of keywords had to be chosen, as the individual terms would have yielded too many results in terms of quantity and too many deviating results in terms of content. We used Boolean logic (using “AND” or “OR”) to connect the keywords. The combinations consisted of one term from the organizational setting and one term from the area of entrepreneurship in order to link the two research areas. We searched for these words in the title, abstract, keywords, and in the body text and did not limit our search to any specific period of time. Based on this extensive systematic literature research, the terms corporate start-up and corporate start-up entrepreneurship are defined. The definitions rely on established definitions and new findings on entrepreneurship and intrapreneurship specified by the organizational framework of the corporate setting. *Leadership: initially, entrepreneurial leadership was included in the search. However, in the course of the research, it became apparent that the term leadership was too generalist, and too many results were obtained. Accordingly, the focus was subsequently placed only on entrepreneurship. frontiersin.org Descriptive analysis To objectivize the descriptive analysis, we conducted this analysis utilizing a standardized spreadsheet, which was developed a priori. We gathered information regarding the author(s), title, year of publication, journal, findings, critique, and respective keyword combinations. The oldest paper dates back to 1987, and the most recent was published in 2023 (the search ended in January 2023), as shown in Figure 3. Over the years, the number of articles increased 04 frontiersin.org 10.3389/fsoc.2023.1267706 Knossalla and Carbon respect. Journal Number of articles Small Business Economics 6 Entrepreneurship Theory & Practice 3 Frontiers in Psychology 3 International Entrepreneurship & Management Journal 3 Journal of Entrepreneurship in Emerging Economies 3 Strategic Management Journal 3 Foundations & Trends in Entrepreneurship 2 IEEE Potentials 2 International Journal of Innovation Management 2 International Small Business Journal 2 Journal of Management 2 Journal of Small Business & Enterprise Development 2 Management Decision 2 Psychology Research & Behavior Management 2 Review of Managerial Science 2 Sustainability 2 Number of other journals that published an article in the direction of CSE within our SLR only once. 42 respect. Journal Number of articles Small Business Economics 6 Entrepreneurship Theory & Practice 3 Frontiers in Psychology 3 International Entrepreneurship & Management Journal 3 Journal of Entrepreneurship in Emerging Economies 3 Strategic Management Journal 3 Foundations & Trends in Entrepreneurship 2 IEEE Potentials 2 International Journal of Innovation Management 2 International Small Business Journal 2 Journal of Management 2 Journal of Small Business & Enterprise Development 2 Management Decision 2 Psychology Research & Behavior Management 2 Review of Managerial Science 2 Sustainability 2 Number of other journals that published an article in the direction of CSE within our SLR only once. 42 Number of other journals that published an article in the direction of CSE within our SLR only once. ensure a common understanding, the following parameters are derived from our SLR and used for a definition and delimitation in this paper: The word start-up is derived from the English term “to start up something.” It is thus the symbol for the foundation of a new corporation. The Gabler Economic Encyclopedia describes start-up as a newly emerging, not yet established corporation that wants to realize an innovative business idea (Achleitner, 2018). Frontiers in Sociology Descriptive analysis The German Start-up Monitor shows similar attributes and defines a start-up based on the following three characteristics: younger than 10 years, highly innovative with significant employee and/or revenue growth, and the founding team mostly knows each other through university or friendships (Kollmann et al., 2021; Sternberg et al., 2021a). Furthermore, global definitions also refer to start-ups as new businesses by entrepreneurs (Low and Mac Millan, 1988). Also, while some researchers already define start-ups as the business area that describes entrepreneurial independence, others define more narrowing factors by saying that start-ups are those businesses in which new forms of business are founded, and the founders are understood as those who have significantly shaped, but also carried out, the start-up life cycle process (Held, 2019), which is associated with different characteristics, opportunities, and responsibilities. The life cycle with its six phases shows that start-ups are young corporations with innovative, scalable business ideas. They start with finding new ideas and making them feasible in the pre-seed phase, create business plans and market analyses as well as decide on the legal form in the seed phase, and preparing their product or service for the market launch in the start-up phase. In the development phase, the first sales are generated, and the business focuses on operational business capability, processes, and structures are professionalized. If the start-up continues to grow, this phase will bring a change in the team and not all founders will remain loyal to the start-up. In the final phase, start-ups orient themselves to future markets, acquisitions, and mergers (Freiling and Harima, 2019). significantly, from mostly 1 to 2 articles per year in the beginning to a much larger number only recently. The articles and authors were predominantly from the fields of economics and human sciences.hf The selected articles were published in 58 different journals. Sixteen of these journals published more than one article in this field (see Table 1). Small Business Economics was by far the journal that represented the most relevant articles (6 publications of the 83 total papers). Based on this extensive systematic literature research, the term corporate start-up entrepreneurship is defined. It is based on the definitions and findings on entrepreneurship and intrapreneurship specified by the organizational framework of the corporate setting “start-up niche in an enterprise,” which explains the importance of the prefix “corporate” and the distinction from intrapreneurship. Qualitative analysis Our qualitative analysis is split into the sections of (1) corporate start-ups and (2) corporate start-up entrepreneurship. Corporate start-ups A comparison of the term start-up, especially with the goal of innovation, with the term split-off has shown that we need to adapt and recommend certain framework conditions so that entrepreneurship, especially ownership and risk-taking, as well as entrepreneurial motivation, can prevail. Accordingly, as a result of the comparison in column three, Table 2, we have defined corporate start-ups more explicitly. Start-ups There is no uniform, common definition of start-ups available, especially in the scientific field. Accordingly, for this paper and to 05 frontiersin.org 10.3389/fsoc.2023.1267706 Knossalla and Carbon TABLE 2 Comparison of start-up and corporate start-up framework, based on literature review. Start-up Split-off Corporate start-up Scope of action Entrepreneur sets off own ideas; mainly exploration (Schumpeter, 1912; Low and Mac Millan, 1988; Szyperski and Nathusius, 1999; Held, 2019) Mostly, business ideas are followed (Kollmann, 2007; Li et al., 2020), sometimes also strategic entrepreneurship or restructuring, mostly exploitation (Szyperski and Nathusius, 1999; Jung et al., 2015) Entrepreneur rises own idea or creates a sense of ownership for business idea; has to be exploration not solely exploitation Risks and ownership Risk and ownership as key attributes for true entrepreneurs (Schumpeter, 1912; Pinchot, 1987) Management of split-off is usually still employed by the parent corporation, which limits risks and ownership (Pinchot, 1987; Li et al., 2020) Risk and ownership lie within leader(s) of the corporate start-up Motive Entrepreneurial motivation (Naffziger et al., 1994; Shane et al., 2003); Desire to realize one’s own concepts and creativity, striving for greater decision-making freedom, independence, demand for higher income, existing frustration in the current workplace, lack of career opportunities or current unemployment, maintaining a family tradition, etc. (Held, 2019; Sternberg et al., 2021a,b) Management or research and development divisions see innovative business ideas and splits- off a respective unit with their respective personnel. Personnel is not assessed or hired concerning entrepreneurial motivation; best case, employees are given a certain amount of time and budget to devote to new innovations (Pryor and Shays, 1993; Li et al., 2020) Entrepreneurial motivation (need for achievement, locus of control, vision, desire for independence, passion, drive, goal setting, self-efficacy) and seeing business opportunities beyond the core business Start-up life cycle Go through the complete start-up life cycle process independently (Held, 2019) Dependence on the parent corporation given at least as long as the split-off performs in terms of revenue and growth (Li et al., 2020) Pre-seed and seed phases can be completed within parent corporation as long as the entrepreneur owns shares and takes risks Legal form All legal forms possible (Hornsby et al., 1993; Bundesministerium für Wirtschaft und Klimaschutz, 2021) Differs by country: in the U.S., corporations (Co/ Inc) are mostly represented under split-offs; in Europe, limited liability corporations (LLC) are more common (Statistisches Bundesamt, 2022; U.S. Frontiers in Sociology Split-off With our SLR, we gathered what is generally understood and practiced by split-offs. When a corporate group decides to split-off an entire division of a corporation or a department, some researchers and practitioners speak of corporate start-ups (Klepper and Sleeper, 2005; Jung et al., 2015). We found out that some researchers refer to either split-offs or spin-offs, differentiating whether the divestiture is in the interest of the parent corporation or not. For us, a distinction regarding the starting point as to what degree the divestiture is wanted is irrelevant. Accordingly, we refer to split-offs in the following, regardless of whether they are intentional or unintentional divestments. We found out that a majority of the ideas that lead to divestments are generated in the dedicated research and development department of a group. Corporations decide to find start-up corporations if the new business field is not part of the core field and core products but still holds promising business prospects (Kollmann, 2007; Klepper, 2009; Li et al., 2020). New ideas may not grow in the established processes of a hierarchical group and may perish, primarily if the idea is not related to the core business. Also, divestments rather than keeping the innovative business as part of the original organizational structure take place because the existing customers and the core business must continue to be served and must continue to exist, but this usually leaves little budget and personnel to deal with ideas and technologies that do not yet generate revenue at this point, but do expend resources. The desired entrepreneurship is difficult to release in established, hierarchical organizations but possible in newly created corporations (Fröndhoff, 2007). The leading team is characterized to be intrapreneurial (Stevenson and Jarillo, 1990; Li et al., 2020). In general, corporate start-ups take over employees from the parent corporation, leading to employees being only hired from the external market in exceptional cases. In our SLR, we found out that some authors say the higher the level of resources taken over and ongoing co-determination from the parent corporation, the lower the degree of freedom and scope for shaping the corporate start-up (Kollmann, 2007). Also in split-offs, personnel resources are seldom selected because the split-off employees excel in entrepreneurship. In some countries, the works council, if present in the corporation, helps shape such transformation processes and influences personnel transfers. Split-off Employees do not always have the choice of remaining in the parent corporation or joining the corporate start-up. It is instead a decision whether to be joining the split-off or terminate (Fröndhoff, 2007; Klepper, 2009; McKendrick et al., 2009; Li et al., 2020). The degree of autonomy of the split-offs differs significantly in practice. While some split-offs are entirely autonomous from the parent corporation, others generally only have legal autonomy. The economic dependency may vary and be subject to restrictions (Zahra, 1996; Kollmann, 2007; Li et al., 2020). Furthermore, the dependency of the split-off also relates to the provision of resources. In some cases, the parent corporation at least provides consultancy input; in other cases also substantially supports and co-determination, as well as provision of resources, is practiced by the parent corporation, thereby limiting Start-ups Small Business Administration, 2023) A legal form which provides ownership of the entrepreneur, e.g., a corporation/ stock corporation Finance/resources Employee growth comes only with rising revenue growth or funding (Chrisman et al., 1998; Song et al., 2008) Personnel and financial resources are fully provided upfront; sometimes competition for resources among other innovative business areas (Li et al., 2020) Personnel and financial resources might be provided to start and boost the business idea. Employee growth should come through own performance Management and leadership Entrepreneur (Chrisman et al., 1998; Chorev and Anderson, 2006; Kühnapfel, 2015; Malik, 2016) Management or intrapreneurs (Li et al., 2020) Corporate Start-up Entrepreneur Processes Legal and economic independence depending on legal form and ownership (Pinchot, 1987; Hornsby et al., 1993) Full range from little co-determination to overall co-determination and monitoring (Zahra, 1996; Li et al., 2020) Legal and economic independence Products and services New venture (innovation) (Schumpeter, 1912; Kollmann et al., 2021) New venture or strategic entrepreneurship (Zahra, 1996) New venture (innovation) Competition/market access Innovative business field; market is new to entrepreneur (Sandberg and Hofer, 1987; Malik, 2016) The not core business field of the corporation; sometimes proximity to core business and customers (Li et al., 2020) Not the core business field of the corporation Team The founding team knows each other through friendships or from university (Kollmann et al., 2021) Organizational affiliations affect team composition. Sometimes whole divisions are split- off in terms of restructuring rather than entrepreneurial motivation (McKendrick et al., 2009; Li et al., 2020) Best case, staff should be chosen in means of entrepreneurial motivation regarding the new venture business idea. Being an entrepreneur is imperative for the objective of innovation Seniority Younger than 10 years (Kollmann et al., 2021) No numbers available Younger than 10 years Also, there is research rising on the topic of entrepreneurial motivation as a key success factor for start-ups. Entrepreneurial motivation is understood as those drivers of an entrepreneur that influence the direction and intensity of the entrepreneur’s activities and move him/her to a higher level of performance to achieve the goals (Naffziger et al., 1994; Shane et al., 2003). Looking at the legal form and ownership, mostly independent forms are chosen (Hornsby et al., 1993; Bundesministerium für Wirtschaft und Klimaschutz, 2021). Start-ups The industry sector accounts for the largest share of the split-off volume, followed by the consumer and tech sectors (Emrick et al., 2017). The legal forms mostly represented are, e.g., corporations (Co/Inc) in the United States or limited liability corporations (LLC), e.g., in Europe (Statistisches Bundesamt, 2022; U.S. Small Business Administration, 2023). all new ventures aim to be innovative (Kollmann et al., 2021; Metzger, 2021). We found that the characteristic common to all definitions is the characterization of start-ups as emerging, innovative corporations that primarily pursue innovative growth objectives independently going through the start-up life cycle in whole or part. Frontiers in Sociology Start-ups The life cycle and definitions show how start-ups can be distinguished from new ventures; for instance, not 06 frontiersin.org 10.3389/fsoc.2023.1267706 10.3389/fsoc.2023.1267706 Knossalla and Carbon the business risk of the split-off entity (Li et al., 2020). The continuing interdependence aims to profit in the different phases of the start-up life cycle from both sides. The new unit essentially benefits from the parent corporation at the pre-seed and seed phase due to reduced risk and resource provision (budget and personnel). In the course of time, the relationship tilts in the event of successful management so that technology and knowledge transfer take place bilaterally, and the parent corporation ultimately also participates in expertise, market share, and earnings/revenue. Furthermore, in some cases, the option of selling or reintegrating the split-off is also relevant for the parent corporation (Kollmann, 2007). Regarding the number of split-offs, we found out that according to a 2017 study, between 2001 and 2005, less than 10% of the corporations proposed divestitures or split-offs, while the percentage between 2011 and 2015 was already at 25% (Emrick et al., 2017). The industry sector accounts for the largest share of the split-off volume, followed by the consumer and tech sectors (Emrick et al., 2017). The legal forms mostly represented are, e.g., corporations (Co/Inc) in the United States or limited liability corporations (LLC), e.g., in Europe (Statistisches Bundesamt, 2022; U.S. Small Business Administration, 2023). the business risk of the split-off entity (Li et al., 2020). The continuing interdependence aims to profit in the different phases of the start-up life cycle from both sides. The new unit essentially benefits from the parent corporation at the pre-seed and seed phase due to reduced risk and resource provision (budget and personnel). In the course of time, the relationship tilts in the event of successful management so that technology and knowledge transfer take place bilaterally, and the parent corporation ultimately also participates in expertise, market share, and earnings/revenue. Furthermore, in some cases, the option of selling or reintegrating the split-off is also relevant for the parent corporation (Kollmann, 2007). Regarding the number of split-offs, we found out that according to a 2017 study, between 2001 and 2005, less than 10% of the corporations proposed divestitures or split-offs, while the percentage between 2011 and 2015 was already at 25% (Emrick et al., 2017). Synthesis This makes them innovators and catalysts for change in the business world (Kuratko and Hornsby, 2021). in the success of the firm (Schumpeter, 1934; Goldstein and Mayer, 1962; Sandberg and Hofer, 1987; Brüderl and Schüssler, 1990; Chrisman et al., 1998; Gartner et al., 1999; Chorev and Anderson, 2006; Gilbert et al., 2006; Meinhart, 2007; Storey, 2011). Some studies even identify the role of the entrepreneur as the most important driver in terms of start-up success (Goldstein and Mayer, 1962; Gartner et al., 1999; Chorev and Anderson, 2006) and innovation (Fueglistaller et al., 2012; Kuratko and Hornsby, 2021). An overview of the analyzed definitions, which shows the multilevel nature, can be found in Supplementary Tables S3a, S3b. We anticipate that we found that no definition was used more than once in the respective articles of our first SLR loop. One of the first definitions of entrepreneurship can be traced back to Schumpeter, who said that entrepreneurship concerns the whole economy and focuses on innovation. In his view, it is about combining resources in a new combination to disrupt the current state of the market. Furthermore, he was one of the first to say that entrepreneurs do not have to be self- employed, but that there are also entrepreneurs in an organization as long as they have a certain majority of shares to exercise innovation and disruption (Schumpeter, 1934). We point out that Stevenson and Jarillo (1990) essentially define entrepreneurship by saying that entrepreneurs are the people who seize opportunities and find ways to make them happen—regardless of the resources and processes for which they are responsible. With this, they confirm that entrepreneurs may exist in start-ups and corporations (Stevenson and Jarillo, 1990). Further, Czarniawska-Joerges and Wolff (1991) refer to entrepreneurship as leadership in contexts that are novel and where leaders cannot react with routine. According to them, entrepreneurship can be defined as leadership in challenging situations (Czarniawska-Joerges and Wolff, 1991). This is why we also looked at the definitions of entrepreneurial leadership and their respective characteristics. Kuratko and Hornsby (2021) elaborated on entrepreneurial literature by defining entrepreneurs as those people who master the task of identifying and developing ideas, testing them for practicability, marketability, benefits and competition, and deploying them in accordance with the resources required and adapted to the scope. This makes them innovators and catalysts for change in the business world (Kuratko and Hornsby, 2021). Synthesis the desire for higher income, existing frustration at the current job, lack of career opportunities or current unemployment (Held, 2019) and maintaining a family tradition (Kollmann et al., 2021), to name a few. Many of these motives are not even possible in a split-off, e.g., independence from existing structures or escape from frustration in current employment. So we see that scope in the sense of ownership and risk, as well as the motive for founding, have an indispensable influence. In addition, start-ups exist in any legal form. They start with an average of 2.5 people (full-time equivalent) and the founding team knows each other through friendships or from university (Kollmann et al., 2021). Unlike start-ups, split-offs are usually founded as limited liability corporations (Europe) or corporations (U.S.). The legal form already shows that the managing directors of the split-off are restricted in their scope of action. From Schumpeter we learned that ownership, responsibility, and risk are prerequisites for entrepreneurship which is why we state that corporate start-ups need an entrepreneur who has the entrepreneurial motivation to pursue the business idea as well as ownership and risks, performing in a legal form which supports this (e.g., corporation). From a financial perspective, in most cases, there is financial dependency on split-offs. But start-up founders especially want financial independence (Kollmann et al., 2021). The financial dependencies influence the decision-making ability of the corporation owner and, thus, the processes and the extent to which the management of a start-up or a split-off determines the strategy. In start-ups, the freedom to shape the strategy is generally much greater than in split- offs. In addition, in the case of split-offs, entire divisions are also spun off. These are rarely small and consist of several employees. In contrast, the teams in successful start-ups start small and hire as they grow, which regularly brings influence and knowledge from outside into the corporation. Split-offs rarely start small and rapidly grow, thus having little claim to recruit personnel from outside. Besides, in corporate groups there is more of a staff reduction than a staff increase. This is often also transferred to the split-off. As a result, the growth in knowledge and the external perspective are sometimes entirely denied. Frontiers in Sociology Corporate start-up entrepreneurship To elaborate on corporate start-up entrepreneurship, we compared the definitions of entrepreneurship (in start-ups) and intrapreneurship (in corporations). Antecedents Supplementary Table S4 shows an overview of the antecedents mentioned by the authors in our SLR. They are clustered according to individual, organizational, and environmental antecedents and show the corresponding author behind it as well as the number of how many articles addressed the antecedent (also mentioned in the brackets below). Synthesis This is why we derive that corporate start-ups should have entrepreneurs at least on leadership level and should firstly, not automatically take over entire divisions since they are part of the organization; secondly, pay attention to entrepreneurial motivation when forming the team; thirdly, make sure that hiring and external influence, as well as exchange, are possible. To sum it up, in all definitions, there is consensus that there must be an opportunity as well as a certain degree of creation, innovation, and riskiness from the entrepreneur. Most definitions also have in common that entrepreneurs have the will to go their own way and take advantage of opportunities (Stevenson and Jarillo, 1990). Regarding the factor of resources, whether owning them or obtaining them from elsewhere, as well as organizational form, whether entrepreneurs are self-employed vs. can also be employed, there is the highest disagreement in science (Fueglistaller et al., 2012). An essential part of the team and a significant influencing factor on new venture performance is the corporation founder/owner. Suppose it is clear that the founder, his attitude, his experience and his leadership have an influence on success. In that case, it is also decisive whether the founder is an entrepreneur (start-up) or a manager/ leader/intrapreneur who may have been active in the corporation before and was not selected based on entrepreneurial skills/ motivation. That is why we contribute a separate section on corporate start-up entrepreneurship. Synthesis The findings of our SLR indicate that a comparison of both terminologies, “start-up” and “split-off,” is essential and leads us to merge both and introduce the term corporate start-up. First of all, the tabular comparison is aligned with the start-up ecosystem model by Kollmann et al. (2021) who characterize start-ups along management and team, competition/market access, finance, products and services, and processes. We have found that corporate start-ups cannot be categorized entirely as either start-ups or split-offs, so a separate definition must be established. Typical differences can be found in terms of action, risk and ownership, motives, start-up life cycle, legal form, resources, management and leadership, processes, products and services, market, team and potentially seniority.hi Thus, we define corporate start-ups as follows: A corporate start-up is defined as a decentralized part of a corporate group with the aim of innovation and growth in a new market. The characteristics of corporate start-ups are shown in Table 2. We can already see differences if we look at the emergence of start-ups vs. that of split-offs. While a start-up is an original foundation that grows from its own resources, independent growth is not a given because it is usually a derivative foundation, which means that the split-off grows from existing structures and resources. The type of start-up has a decisive influence on the development of the new corporation, its goal and vision, and thus on its entrepreneurship vs. intrapreneurship and innovation (Szyperski and Nathusius, 1999). If a start-up can grow from its own resources and is less dependent because it can create more itself but also has more risk because it has full responsibility, the innovation process is less affected. Start-ups are usually founded with innovation as the main goal, while split-offs can also be created through restructuring and keeping the option open to sell the split-off if it is not successful. Innovation is not always the main goal of split-offs (Jung et al., 2015). The personal motives for founding a start-up differ greatly from those of a corporate start-up. Synthesis For start-up founders, the list includes the desire to realize one’s own concepts and creativity, the pursuit of more decision-making freedom, independence, 07 frontiersin.org 10.3389/fsoc.2023.1267706 Knossalla and Carbon in the success of the firm (Schumpeter, 1934; Goldstein and Mayer, 1962; Sandberg and Hofer, 1987; Brüderl and Schüssler, 1990; Chrisman et al., 1998; Gartner et al., 1999; Chorev and Anderson, 2006; Gilbert et al., 2006; Meinhart, 2007; Storey, 2011). Some studies even identify the role of the entrepreneur as the most important driver in terms of start-up success (Goldstein and Mayer, 1962; Gartner et al., 1999; Chorev and Anderson, 2006) and innovation (Fueglistaller et al., 2012; Kuratko and Hornsby, 2021). An overview of the analyzed definitions, which shows the multilevel nature, can be found in Supplementary Tables S3a, S3b. We anticipate that we found that no definition was used more than once in the respective articles of our first SLR loop. One of the first definitions of entrepreneurship can be traced back to Schumpeter, who said that entrepreneurship concerns the whole economy and focuses on innovation. In his view, it is about combining resources in a new combination to disrupt the current state of the market. Furthermore, he was one of the first to say that entrepreneurs do not have to be self- employed, but that there are also entrepreneurs in an organization as long as they have a certain majority of shares to exercise innovation and disruption (Schumpeter, 1934). We point out that Stevenson and Jarillo (1990) essentially define entrepreneurship by saying that entrepreneurs are the people who seize opportunities and find ways to make them happen—regardless of the resources and processes for which they are responsible. With this, they confirm that entrepreneurs may exist in start-ups and corporations (Stevenson and Jarillo, 1990). Further, Czarniawska-Joerges and Wolff (1991) refer to entrepreneurship as leadership in contexts that are novel and where leaders cannot react with routine. According to them, entrepreneurship can be defined as leadership in challenging situations (Czarniawska-Joerges and Wolff, 1991). This is why we also looked at the definitions of entrepreneurial leadership and their respective characteristics. Kuratko and Hornsby (2021) elaborated on entrepreneurial literature by defining entrepreneurs as those people who master the task of identifying and developing ideas, testing them for practicability, marketability, benefits and competition, and deploying them in accordance with the resources required and adapted to the scope. Looking at intrapreneurship SLR shows that the traits which are mentioned most in the literature include risk-taking (10), innovativeness (10), vision (8), initiative (6), creativity (6), human relations (6), and business acumen (6). Also, it is extremely difficult to confirm a causal link between psychological characteristics and entrepreneurship (Stevenson and Jarillo, 1990). Much research in this area has been conducted within a framework that has been supported by public policy and is therefore not considered entirely independent. Furthermore, much literature denies the causal effect of psychological traits and entrepreneurship. Hence, we can only speak of a correlation rather than a causal effect. Another problem with the research is that too little distinction has been made between entrepreneurs as individuals and organizations. Entrepreneurship was often equated with management, which diluted the results. In other words, personal characteristics are important, but environmental and organizational variables are important, too (Stevenson and Jarillo, 1990). The authors in our SLR emphasize the economic significance that large corporations have drawn from the conclusion that entrepreneurship may also exist in corporations (Kuratko and Hornsby, 2021). We found that interest in intrapreneurship goes back to the 1980s (Schollhammer, 1981; Pinchot, 1985, 1987) and is therefore not a new field of research, but it has not yet been scientifically fully explored as most papers mention in their conclusion. Furthermore, the state of research is very fragmented, and the scope of when to speak of intrapreneurship and when not is very diversely presented. Concerning the scope of intrapreneurship, Schumpeter already includes activities of individuals who are not merely self-employed; this paper pursues the general understanding that intrapreneurs are employees, which limits their scope of responsibility and ownership and limits innovation (Schumpeter, 1934; Wennekers, 2006). Wennekers (2006) distinguishes entrepreneurs from intrapreneurs based on the degree of employment (self-employed vs. employee; first level) and a behavioral dimension (second level). Either a person exhibits entrepreneurial behavior, which is self-initiated and aims to identify, exploit and implement opportunities. Or the person exhibits behavior that relates to the coordination and organization of resources and is, therefore, more managerial behavior. According to Wennekers, only those who are employed and who exhibit entrepreneurial behavior in this setting are intrapreneurs (Wennekers, 2006). It shows that it depends on both the degree of employment and the individual’s behavior, whether s/he is an intrapreneur in a corporation and that the degree of intrapreneurship of a corporation depends on the sum of the individuals. Organizational First of all, hierarchy levels, operational and organizational structure as well as the legal form of an organization, are one of the organizational antecedents which is mentioned the most (3) followed by leadership and management support (Urbano et al., 2022). Concerning support from within the organization, mentors, consultants, and co-founding friends/colleagues can positively affect and enable entrepreneurship but may distort decision taking and operating by emotional attitudes (Kollmann, 2007), so either direction of influence is mentioned in the articles. A third ambiguously discussed organizational antecedent is financial management and proper handling of resources: both, too little and too many resources are crucial. For instance, too much spending and thus liquidity bottlenecks or wrong spending of resources that resources are not procured, careless handling of credits can be impediments, while not investing and taking risks can be an impediment as well (Zacharakis et al., 1999; Cardon et al., 2011). Looking at intrapreneurship Moreover, Stevenson and Jarillo (1990) also focused on the behavioral aspect in their definition. They described intrapreneurship as the intrinsically motivated, innovation-oriented, and entrepreneurial actions of employees in an organization as if they were entrepreneurs. Whether intrapreneurship can exist in corporations depends to a large degree on the extent to which the employees below the management level also have an entrepreneurial attitude and (are able to) practice it (Stevenson and Jarillo, 1990). Frontiers in Sociology Environmental Enabling factors can also concern the environment (Stevenson and Jarillo, 1990; Sitkin, 1992; Atsan, 2016; Urbano et al., 2022). Our SLR confirms that it is noticeable that through the increased use of the internet and social media, people are far more keen to exchange ideas about entrepreneurship, and teach each other, and thus, the threshold for starting one’s own entrepreneurial journey is much lower (Stevenson and Jarillo, 1990; Kollmann et al., 2021). This can also be confirmed by the numerous articles excluded from the first loop of our SLR, which focused on entrepreneurial education or cultural and gender differences. It also supports the fact that entrepreneurial education is mentioned numerous times (3) as an environmental antecedent specifically for entrepreneurship. For instance, by offering a less risky scope rather than operating on the market, universities and governmental institutions increase the opportunity for entrepreneurship (Kuratko and Hornsby, 2021). Here, too, ambiguity was mentioned concerning governmental policies that may impede entrepreneurship when they do not support start-ups, which makes it difficult for them to enter the market or challenge the process of business foundation. Also, customer proximity and market forces, e.g., stronger competition and market innovations, may affect entrepreneurship (Sitkin, 1992; McGrath, 1999; Kollmann et al., 2021). frontiersin.org Entrepreneurship Our SLR shows that there is disagreement in science with regard to the personality traits that (should) exist in an entrepreneur. Our According to various studies, from the early years and recently, the entrepreneur of a start-up is considered to have an essential role 08 frontiersin.org frontiersin.org 10.3389/fsoc.2023.1267706 Knossalla and Carbon Looking at intrapreneurship Looking at intrapreneurship Environmental Much research has been carried out on organizational obstacles and conducive factors (Schollhammer, 1981; Pinchot, 1985, 1987; Hornsby et al., 1993; Pryor and Shays, 1993; Antoncic and Hisrich, 2001; Cannon and Edmondson, 2005). Our SLR indicates that resource provision (7), independence rather than bureaucracy and monitoring processes (6), as well as management commitment (6), are the most mentioned antecedents referred to intrapreneurship. Commitment is often also mentioned along with trust and integrity of the management in order to facilitate entrepreneurial work in a corporation. For instance, managers must represent and exemplify intrapreneurship, approaching new paths, being able to fail, and contributing to growth by innovation with credibility. Some articles emphasize the need for sponsors who, on the one hand, have the influence and power to make decisions and provide the dedicated resources, so being on the management/leadership level, and on the other hand, who listen to the intrapreneurs and have the time to deal with them, coach and accompany them without interfering (Pryor and Shays, 1993). When intrapreneurs are constrained within the corporation, either by management or processes, they are initially angered, may vocalize their displeasure, or leave the corporation and go to competitors or start their own corporation, thus competing with the corporation they once belonged to. This also includes independence for the intrapreneur in decision-making processes (Kanter, 1983; Pinchot, 1985, 1987; Hornsby et al., 1993; Pryor and Shays, 1993). A relevant finding concerning the context to split-offs is that further papers indicate that people cannot be appointed or convinced to become intrapreneurs, but they have to carry these characteristics within themselves. Accordingly, it is important not only to look for topics and innovations in the corporation, but also for employees who already distinguish themselves as intrapreneurs (Pryor and Shays, 1993). Also, with regard to split-offs, we found out that organizational structure is often mentioned as crucial but with different implications, for instance, differentiating between new venture entrepreneurship or strategic entrepreneurship (Hornsby et al., 1993; Neessen et al., 2019). Nevertheless, the type of organization and in-depth study of their impact has so far remained neglected in the literature (Belousova et al., 2020), and our SLR strengthens the background of the paper to examine intrapreneurship in the niche of corporate start-ups more closely as it represents a special context. Environmental The organizational structure is of unique relevance as, e.g., some antecedents of entrepreneurship are per se excluded and cannot be artificially created, e.g., the motive to create a business due to continuing family businesses or out of necessity. In organizations, the urgency is not necessarily high due to the payroll, such as in Germany, due to works constitution law and other employee-friendly measures (Stevenson and Jarillo, 1990). Since splitting-off is a decision concerning the corporation’s strategy, we would like to highlight that we found out that too much and too sudden confrontation with change can harm intrapreneurship as well as many personal changes in the management or when dismissals are carried out by lower levels of Various papers in our SLR mentioned customer proximity (5) as important as being aware of the market (5) (e.g., product addresses market needs, intrapreneur follows market trends) as an antecedent for intrapreneurship because it is the customers and the market who give quick and valuable feedback, counteracting getting tied up in internal processes and bureaucracy. Also, communities to network are not only internally relevant but also externally in order to inspire and create synergy effects (Pryor and Shays, 1993). Individual Our SLR shows that here, too, is disagreement in science with regard to the traits of an intrapreneur. We found that the traits that are mentioned most in the literature include being innovative (9), opportunity oriented (6), having entrepreneurial qualification or experience (5), and being able to create a vision (5). Concerning the development of research on individual antecedents, we found that Hornsby et al. (1993) reacted to the research gap that until then, preconditions for intrapreneurship were only examined from an organizational perspective. They supplemented the existing scientific findings with individual characteristics and stated risk-taking propensity, desire for authority, need for achievement, goal orientation, and internal locus of control as the most mentioned and most relevant individual antecedents for intrapreneurship (Hornsby et al., 1993). Our SLR showed that other characteristics were mentioned more often (see above). We also found that the prerequisites mentioned in the model by Hornsby et al. (1993) need to be supplemented by other perspectives that now exist. Especially entrepreneurial experience and qualification increases in being addressed by authors as well as connecting the dots in strategic, future-oriented thinking and seeing the impact of an opportunity are 09 frontiersin.org 10.3389/fsoc.2023.1267706 10.3389/fsoc.2023.1267706 Knossalla and Carbon management as a pawn sacrifice (Kanter, 1983). Also, like entrepreneurs, it is crucial that employees perceive recognition of intrapreneurship to intensify the sense of ownership, e.g., through intra-capital, to set positive incentives so strongly that the motivation to create, see opportunities, and implement them is increased. Successful intrapreneurs should use intra-capital for (re-)investments in other projects so they become inter-venture capitalists (Pinchot, 1987; Stevenson and Jarillo, 1990). skills that already must be present in the employees or trained when it comes to intrapreneurship in contrast to entrepreneurship (Stevenson and Jarillo, 1990). Some authors go beyond entrepreneurial skills and mention entrepreneurial motivation, meaning the desire to establish a business, the recognition of opportunities, and the sustainability with which they are realized, as a prerequisite for intrapreneurship (Naffziger et al., 1994; Shane et al., 2003). Frontiers in Sociology frontiersin.org Synthesis The findings of our SLR indicate that a comparison of both terminologies, “entrepreneurship” and “intrapreneurship” is indispensable, which is why we made the tabular comparison in Table 3. We found that both terms differ in scope, goal, focus, resource availability, dependencies, external influences, time constraints, risk, and profit. Again, this shows that we need a definition for corporate start-up entrepreneurship, which also not yet exists. Our definition of corporate start-up entrepreneurship fits the setting of corporate start-ups (see above) and is based on the detailed definition of an entrepreneur by Kuratko and Hornsby (2021), who define “entrepreneurs [as] those people who master the task of identifying and developing ideas, testing them for practicability, marketability, benefits and competition, and deploying them under the resources required and adapted to the scope; this makes them innovators and catalysts for change in the business world” (Kuratko and Hornsby, 2021, p.5). We have deliberately chosen this definition among the many available because it is detailed, includes several characteristics, distinguishes between having ideas and also implementing ideas, says that the impact of innovation affects the business world in general, and does not exclude the framework that entrepreneurship prevails only in start-ups. Moreover, it is a novel entrepreneurship definition that results from a literature review reflecting developments in recent years. It was also important to us that the definition of corporate start-up entrepreneurship should make it clear that the corporate start-up entrepreneurs are the ones who bring innovation into the parent corporation and not the divisions of the core business that are not spun off. The original definition provides a good basis for specification. Thus, we derive: Corporate Start-up Entrepreneurship means leading a decentralized part of a corporate group with the aim of innovation and growth in a new market (=corporate start-up), i.e., to identify and developing ideas initially in the frame of the parent corporation, testing them for practicability, marketability, benefits, and competition, and deploy them in accordance with the resources required provided by the parent corporation and adapted to the given scope, existing culture and prevailing politics. This makes corporate start-up 10 frontiersin.org 10.3389/fsoc.2023.1267706 Knossalla and Carbon TABLE 3 Deriving corporate start up entrepreneurship from entrepreneurship vs. intrapreneurship comparison (own representation based on a literature review). Synthesis Entrepreneurship Intrapreneurship Corporate start-up entrepreneurship Scope Self-employed and sets of own entrepreneurial ideas (Wennekers, 2006; Fueglistaller et al., 2012); mostly exploration Employed and sets of entrepreneurial rather than managerial ideas; mostly ambidextrous (Schollhammer, 1981; Pinchot, 1987; Antoncic and Hisrich, 2003; Wennekers, 2006) The corporate entrepreneur of CSE shall eventually be employed by the corporate start-up, holding a respective amount of ownership and responsibility (at least 20%). S/he may start the pre-seed and seed phase in the parent corporation but proceed with subsequent phases mandatorily in corporate start- up, therefore unavoidably not being employed by the parent corporation anymore; business concept shall be explorative rather than exploitative Ownership of concept and ideas Entrepreneur (Kuratko and Hornsby, 2021) Intrapreneur is employed; therefore, a corporation is true owner of concepts and ideas (Wennekers, 2006) Ownership shall be with the CSE, e.g., by shareholding, organizational structure as an independent corporation, etc.; there needs to be financial liability Resources Mainly provided by entrepreneur, partly by investors (Fueglistaller et al., 2012; Kuratko and Hornsby, 2021) Provided by the corporation (Pinchot, 1987; Stevenson and Jarillo, 1990; Hornsby et al., 1993; Pryor and Shays, 1993) In pre-seed phase and seed phase, CSE shall be given a certain amount of time (e.g., 20% innovation time) and resources besides day to day work to be able to work on innovation. In subsequent phases, as of start-up phase, resources shall be provided to a certain degree by the parent corporation with fixed time-frame and goal- orientation. Furthermore, being part of a split-off team/division should not mean that one is automatically allocated to the corporate start-up. Synthesis Personnel resources should result from entrepreneurial motivation and not from historical, organizational affiliations; recruiting shall include CSE checks Focus of work Entrepreneurs develop innovation through either new knowledge, new services new processes, new products, or new markets (Schumpeter, 1934; Gartner, 1988) Intrapreneurs work is not necessarily innovate sometimes rather strategic entrepreneurship, meaning restructuring; mainly entering the existing market (Hornsby et al., 1993; Neessen et al., 2019) CSE shall work on new venture and innovation rather than strategic entrepreneurship (restructuring/rejuvenation etc.) of the parent corporation in the existing market Dependencies and decisions Entrepreneur acts autonomously; create, act and decide upon own target/vision (Fritsch, 2020; Kuratko and Hornsby, 2021) Intrapreneur works within existing system (non-autonomously); act upon set goals/ vision and have restricted freedom to design (Wennekers, 2006; Maier and Pop Zenovia, 2011) CSE acts autonomously in corporate start-up External influences Entrepreneur is susceptible to outside influences, be it positive or negative (Stevenson and Jarillo, 1990; Kollmann, 2007; Kuratko and Hornsby, 2021) Intrapreneur and corporation are less susceptible to external influences (Pryor and Shays, 1993) CSE shall build up network internally (parent corporation) and externally (market) Risk Entrepreneur takes all risks (Schumpeter, 1934; Kollmann et al., 2021) Corporation takes risks (Stevenson and Jarillo, 1990) CSE and parent corporation share risks according to distribution of share Time constraints Pressure to show quick results and success (Kuratko and Hornsby, 2021) Moderate pressure to show quick results (Pryor and Shays, 1993) Defining of certain time frame in pre-seed phase among shareholders shall determine time constraints Compensation/profit The entrepreneur gets compensation according to ownership (Zacharakis et al., 1999; Cardon et al., 2011; Kollmann et al., 2021) Limited compensation for intrapreneurs (Pinchot, 1987; Stevenson and Jarillo, 1990) CSE shall not be paid fixed salary but a variable salary according to corporate start-up performance (Continued) 11 11 Frontiers in Sociology frontiersin.org 10.3389/fsoc.2023.1267706 Knossalla and Carbon Entrepreneurship Intrapreneurship Corporate start-up entrepreneurship Failures Little flexibility: there is no sanction by superiors (possibly just by investors), but there might be instant existential threats (Sternberg et al., 2021a,b) Dependent on culture of failure, a single mistake can lead to substantial consequences (e.g., withdrawing from project) but also more flexibility in staying with the corporation and balancing out smaller mistakes (Kanter, 1983; Maier and Pop Zenovia, 2011) Failures of the corporate start-up entrepreneur may lead to dissolving the corporate start-up and losing ownership; yet, the CSE shall be supported by the parent corporation to apply his entrepreneurial skills within the internal or external network/projects for a certain period of time (e.g., fallback to parent corporation no longer than 3 month) Challenges Novel contexts, market know-how and acceptance, credibility (Czarniawska- Joerges and Wolff, 1991) Corporate culture, politics, processes and regulations limit independence in operation (Kanter, 1983) Balance between co-determination among owners; setting the framework (target, budget, etc.) upfront Effect on innovation Entrepreneurship as most important driver for innovation (Schumpeter, 1934; Fueglistaller et al., 2012; Kuratko and Hornsby, 2021) Effect of intrapreneurship depends on whether organization is suppressing innovational processes, e.g., by silo- thinking, limiting risk-taking, blaming culture etc. Synthesis (Kanter, 1983; Maier and Pop Zenovia, 2011) and ownership, e.g., through intra-capital for the intrapreneur (Pinchot, 1987) Corporate start-up entrepreneurship shall drive innovation; no restructuring purposes split-offs remain on the payroll, they are not personally liable, and they do not fall into risks—at most, the compensation is bonus- related, as in the case of salespeople. The corporate entrepreneur must expect a loss of employment in case of mistakes as well as experience profit when the business model is successful. Thus, corporate start-up entrepreneurship requires that the corporate entrepreneur also experiences financially whether the business model is successful or not, for example, through a variable compensation model. In general, the intrapreneur can fall back on the group structures, find employment there again and not have to reckon with loss of employment. In addition, it was generally assumed that the managers and employees in the intrapreneurship setting also exhibit an entrepreneurial type of behavior. Suppose one goes by Wennekers’ rudimentary comparison of entrepreneurship to intrapreneurship. In that case, this shows that just because an employee is no longer self-employed, that does not make him an intrapreneur. If this employee were to exhibit managerial behavior, then s/he would be an executive manager, not an intrapreneur and still far from being an entrepreneur. Entrepreneurs act freely out of their entrepreneurial motivation. This would also be a desirable state for intrapreneurs, because people cannot be appointed to become entrepreneurs; they must already have the essential characteristics. Here, too, are significant differences, especially if you look at the framework of a corporate start-up. It has been shown that a corporate start-up is characterized by the fact that entire areas are spun off as the definition of split-off states (Jung et al., 2015). Those who are employed in the area at the time of the split-off are usually also spun off no matter what. However, this does not mean that these people demonstrate entrepreneurial behavior, motivation, or skills. Thus, for corporate start-ups, it is essential to employ employees with entrepreneurial motivation and skills (=corporate entrepreneurs). Frontiers in Sociology frontiersin.org Synthesis In general, the question arises as to what extent entrepreneurial motivation/skills exist and are promoted in corporations so that corporations can entrepreneurs the innovators for the parent corporation for the previously defined field of the corporate start-up and catalysts for change in order to continue to drive the group's competitiveness through the modified economic structure and to achieve innovation from its own resources. For instance, Table 3 shows that there are significant differences in terms of decision-making (autonomous vs. dependent), the goal (innovative vision vs. exploring existing markets), the handling of risks, liability, and personnel and financial bottlenecks. Intrapreneurship is not the same as entrepreneurship in corporate start-up settings. If intrapreneurship were simply entrepreneurship in a corporate setting, according to the literature review, leaders and employees would always have to proactively identify opportunities and work to generate growth and innovation. However, the review shows that dependencies on the corporation make it impossible to freely decide the vision, the product or the day-to-day decisions and to leverage resources so that innovation can grow. Thus, in order to leverage a corporate start-up to bring innovative business models to the market, independence from the parent corporation and ownership are required. This can be initiated by a suitable legal form for the corporate start-up, e.g., by shareholding. Furthermore, the fact that entrepreneurs always have to deal with uncertainty and that this drives their creativity and innovation is also not given in intrapreneurial settings, e.g., corporate start-ups. The sense of uncertainty and risk must also be created for corporate start-ups so that innovation-oriented behavior can take place. This can be achieved not only through the legal form, but also through financial liability, explorative business models and filling positions with corporate entrepreneurs instead of transferring employees from the parent corporation to the split-off. Intrapreneurship is also not entrepreneurship in corporate start-ups, because the compensation is mostly consistent, whether positively remuneration or negatively, when making mistakes. Executives and employees in 12 frontiersin.org 10.3389/fsoc.2023.1267706 Knossalla and Carbon 10.3389/fsoc.2023.1267706 we recommend is, in particular, the quantitative study on corporate start-up entrepreneurship conditions based on this work. Further research could also be carried out here (e.g., a case study), to what extent corporations consider the general conditions in practice for split-offs or to what extent corporate start-up entrepreneurship prevails in corporate start-ups. There may be country-specific differences in the case of split-offs from corporations. Author contributions CK: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft. C-CC: Conceptualization, Formal analysis, Methodology, Project administration, Resources, Supervision, Writing – review & editing. Funding The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article. Synthesis Further future research topics could address the relation between corporate start-up entrepreneurship and corporate (start-up) performance or in-depth studies considering the relationship between corporate start-ups and the corporate group. Also, for practitioners, it could be of future interest how corporate groups prepare their employees (e.g., via trainings, assessments) and the processes (e.g., placement) regarding corporate start-up entrepreneurship. Here the antecedents could give a guiding structure. Since there was limited literature on the environmental factors available and incorporated in our SLR, future research could deepen the impact of environmental factors on corporate start-up entrepreneurship. As with all studies, our review is subject to some limitations but also offers opportunities for further research. First of all, it is possible that we missed some high-quality papers in the field of entrepreneurship in corporate start-ups. This may be because our first loop of SLR was reduced to the database EBSCO only, and our second loop, snowball systematic analyzing the references of our first loop, focused on those indices. Additionally, we did not include papers that were not written in English or German. We deliberately chose EBSCO and took our keywords derived from theoretical considerations in order to focus our research, but the linguistic limitation was not a prerequisite but unavoidable. Nevertheless, we strongly feel that due to our two-fold approach, SLR via EBSCO and the snowball approach via EBSCO and Google Scholar, we included a sufficient number of articles in our research, especially since the niche area of corporate start-ups has not yet been researched well. Despite the mentioned limitations, our results of the SLR and especially our definitions of the research gap of corporate start-up entrepreneurship serve as a solid basis for further research in this niche area. leverage them when setting up new corporations. The differences show that the desired state in a corporate start-up cannot arise as long as intrapreneurship prevails. As a synthesis, for corporate start-ups to realize innovation for large corporations, there needs to be (1) the appropriate legal form, which ensures ownership but also the risk of the leaders, (2) an explorative focus rather than exploitation, (3) variable compensation rather than fixed and (4) corporate entrepreneurs rather than employees and managers. This can also be seen in the tabular comparison and derivative of corporate start-up entrepreneurship in Table 3. Conclusion Our research focused on the research question of how entrepreneurship may exist in the niche of corporate start-ups. The result of our work shows that entrepreneurship can exist in the form of corporate start-up entrepreneurship in split-offs, provided certain antecedents and requirements are given, and there is a uniform understanding of the framework. Through a systematic literature review we have defined both the term corporate start-up and the term corporate start-up entrepreneurship, which have not yet been defined uniformly and scientifically reliable. The definition of corporate start-up entrepreneurship includes different aspects of entrepreneurship as well as a reflection of the corporate setting. Besides the definition, we deduced antecedents and impediments for corporate start-up entrepreneurship to guide corporations when splitting off divisions to pursue growth. This review confirms the strategic relevance of corporate start-up entrepreneurship concerning corporate transformations. Our SLR contributes to current academic knowledge by showing the following: Overall, we endorse the adapted conceptual model of corporate entrepreneurship by Urbano et al. (2022) as we confirm that the organizational setting (e.g., legal form along with degree of ownership, decision-making processes, resource availability and size) has an impact on entrepreneurship. The identified key antecedents for corporate start-up entrepreneurship through our SLR, (1) appropriate legal form, (2) exploratory business, (3) variable compensation and (4) corporate entrepreneurs can be assigned to the organizational and individual antecedents of the model. The degree of innovation (how far one deviates from more experienced business models and takes the respective risks) also influences whether we speak of entrepreneurship (innovative ideas) or intrapreneurship (translating ideas of others) or pure managerial behavior (consistency and order instead of change and movement). Remuneration and motivation are also critical cornerstones that determine whether a parent corporation can enable corporate entrepreneurship (when the corporate entrepreneur owns the business and the idea) or really just management/intrapreneurship in corporate start-ups (when the management of a corporate start-up acts merely on behalf of a corporation). Also, most of the current definitions of entrepreneurship and intrapreneurship assume that there is an either-or logic. Nevertheless, we showed that entrepreneurship is rather considered a continuum. A special form, which lies somewhere between entrepreneurship and intrapreneurship but arguably closer to entrepreneurship, is called corporate start-up entrepreneurship, which we have explored in this paper. We assume that other niches also need a special consideration of entrepreneurship, which could be taken into account in future research scenarios. 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https://openalex.org/W4281689006	https://bmcpediatr.biomedcentral.com/counter/pdf/10.1186/s12887-022-03390-3	English	null	Gut microbiota and atopic dermatitis in children: a scoping review	BMC pediatrics	2,022	cc-by	7,036	© 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. Abstract Background: Gut microbiota plays an important role in the development of atopic dermatitis (AD). We aimed to elu- cidate research trends in gut microbiota and AD in children, to provide evidence and insights to the clinical preven- tion and treatment of AD in children. Methods: A scoping literature review on the studies of gut microbiota and AD were conducted. Two authors inde- pendently searched Pubmed et al. databases for studies focused on gut microbiota and AD in children up to January 15, 2022. The literatures were screened and analyzed by two reviewers. Results: A total of 44 reports were finally included and analyzed. Current researches have indicated that abnormal human microecology is closely associated with AD, and the disturbance of intestinal microbiota plays an important role in the occurrence and development of AD. Probiotics can correct the microbiota disorder, have the functions of regulating immunity, antioxidant, and help to restore the microecological homeostasis. However, there is still a lack of high-quality research reports on the efficacy and safety of probiotics in the prevention and treatment of AD in children. Conclusions: The changes of gut microbiota are essential to the development of AD in children, which may be an effective target for the prevention and treatment of AD. Future studies with larger sample size and rigorous design are needed to elucidate the effects and safety of probiotics in AD. Keywords: Gut microbiota, Atopic dermatitis, Children, Probiotics, Review, Treatment, Prevention AD children. Besides, AD children over 2 years old may have a longer course of disease and are difficult to cure. If the patient suffers from the disease for a long time and develops to the age of 20 to 30 years, the patient may even develop cataracts associated with AD [7]. There- fore, active and effective treatment and prevention of AD in children are of great significance to their growth and quality of life. Liu et al. BMC Pediatrics (2022) 22:323 https://doi.org/10.1186/s12887-022-03390-3 Liu et al. BMC Pediatrics (2022) 22:323 https://doi.org/10.1186/s12887-022-03390-3 Introduction Atopic dermatitis (AD) is a chronic, recurring, itchy skin disease with an incidence of 0.2% to 25% [1, 2]. Recent epidemiological survey data in China [3–6] have showed that the incidence of AD in children aged 3–6 years old can be as high as 18.3%, and it is increasing year by year. AD is more common in infants and young chil- dren, and AD usually reappears in adulthood period of AD is a common chronic and relapsing skin disease in children with complex pathogenesis, which is related to multiple factors such as heredity, immune dysfunction, skin barrier damage, and environmental exposure [8, 9]. In recent years, with the development of high-through- put technology, the relationship between the human micro-ecosystem and AD has been gradually revealed. Some studies [10, 11] speculate that intestinal microbiota †Yue Liu and Xiaofan Du contributed equally to this work. Correspondence: h82ip7j@163.com 1 Department of Acupuncture and Tuina Science, School of Traditional Chinese Medicine, Naval Medical University, No. 800 Xiangyin Road, Shanghai 200433, China Full list of author information is available at the end of the article †Yue Liu and Xiaofan Du contributed equally to this work. Correspondence: h82ip7j@163.com 1 Department of Acupuncture and Tuina Science, School of Traditional Chinese Medicine, Naval Medical University, No. 800 Xiangyin Road, Shanghai 200433, China Full list of author information is available at the end of the article 1 Department of Acupuncture and Tuina Science, School of Traditio Chinese Medicine, Naval Medical University, No. 800 Xiangyin Road Shanghai 200433, China Full list of author information is available at the end of the article Liu et al. BMC Pediatrics (2022) 22:323 Liu et al. BMC Pediatrics (2022) 22:323 Page 2 of 8 disturbance may be related to the pathogenesis and severity of AD. Previous studies [12, 13] have pointed out that the composition of the gut microbiota is significantly different between healthy children and children with allergic diseases. Changes of gut microbiota is associated with immune dysfunction in children [14, 15]. However, there are relatively few studies on gut microbiota and AD in children, and the current research status is unclear. Therefore, this scoping review aimed to review the stud- ies on the gut microbiota and AD in children, to provide evidence for the clinical prevention and treatment of AD. gastrointestinal ∗) AND (Ti: children ∗ OR child ∗ or pedi- atric ∗) OR (Ti: prevention ∗ OR treatment ∗). Data processing and extraction Two authors independently screened the initial papers identified through the search. The title and abstract of these papers were evaluated based on the inclusion and exclusion criteria to judge their suitability for further stages of review. The retained papers following this pro- cess were rescreened by full text to assess the eligibil- ity. A template was developed to allow extraction of the characteristics of each of the studies. This template also allowed recording of the decision made by each reviewer as to whether the paper should be included or excluded or if the decision was uncertain. The result achieved by the two reviewers was evaluated and compared. Any disagreements were resolved by further discussion and reaching consensus. Study inclusionshi The first database search yielded 465 reports for possible inclusion. Title and abstract review identified 128 articles for full-text review. After full-text review, we removed 84 additional articles for failure to meet inclusion criteria. Finally, 44 studies were included in this scoping review (Fig. 1). Methodsh This scoping review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis(PRISMA) extension for Scoping Reviews [16]. The methodological framework for scoping reviews developed by Arksey et al. was used, which comprises five stages: identifying the research question, identify- ing relevant studies, selecting studies, charting the data and collating, summarizing and reporting the results. Ethical approval was not necessary for this scoping review because a literature review did not require ethical approval based on the related research guidelines. The extracted data included the name of first author, author affiliation information, year of publication, report title, study purpose, study design, sample size, the char- acteristics of participant, outcome indicators, results and main conclusions. Data analysis W d The research question was constructed and developed according to the PICO framework (Population, Interven- tion, Comparison, Outcome). To be eligible for inclusion in this study, the reports had to meet following criteria: the study focused on the AD Children with age < 16 years old; studies focused on the preventions or treatments associated with gut microbiota; randomized controlled trials (RCTs), quasi experimental or experimental stud- ies, cohort designs; the paper was reported in English or Chinese. We excluded narrative reviews, experts’ opin- ions and papers published in other languages. We conducted thematic synthesis proposed by Thomas et al. to answer this scoping review’s research question. There are three stages in the thematic synthesis: the free line-by-line coding of the findings of primary literature; the organization of these “free codes” into related areas to construct “descriptive” themes; the development of “analytical” themes. In developing the theme of this cop- ing review, each reviewer first performed line-by-line coding and descriptive theme generation. Furthermore, two reviewers discussed and integrated the patterns that appear in those themes to generate categories as themes as interpretations beyond the original literature’s content. Introduction The key- words used were synonyms generated by the databases used and MeSH terms. Search strategy Two authors independently searched following data- bases: Pubmed, CINAHL (EBSCO), MEDLINE, Cochrane library, Cochrane central register of controlled trials, China National Knowledge Infrastructure (CNKI) and Wanfang Database, China Biomedical Literature Database (CBM) for studies focused on gut microbiota and AD in children. The search time limit was from the inception of databases to January 15, 2022. Besides, we searched Google Scholar for potentially related reports. The reference lists of included papers were also screened out to identify any additional relevant papers. The fol- lowing Boolean search strategy was used to search above databases (Ti: atopic dermatitis ∗ OR dermatitis ∗) AND (Ti: microbiota ∗ OR gut microbiota ∗ OR probiotics ∗ or Characteristics of included literature Of the included 44 reports, the authors were from 19 countries, the United States was the country with the most authors, followed by the United Kingdom and Liu et al. BMC Pediatrics (2022) 22:323 Page 3 of 8 Fig. 1 The flow diagram for literature review Fig. 1 The flow diagram for literature review of skin and intestinal microbiota in children with AD is reduced before the onset of the disease, which can lead to a shift in the immune response to the T helper 2 (Th2) and increase the possibility of AD onset [20, 21]. During the investigation of Swedish and Estonian children [22, 23], Estonian children had very few aller- gies, and their intestinal microbiota was dominated by Lactobacillus. However, the main intestinal microbiota of Swedish children was Clostridium, so there were many allergic symptoms. For non-allergic individuals, allergic infants had low levels of lactobacilli in their guts, along with a large number of aerobic bacteria. The results of in vitro and in vivo experiments [24, 25] showed that Lactobacillus could regulate the immune system and resist allergy. Elevated fecal caproic acid levels suggest altered gut microbial communities in infants with allergic symptoms [26, 27]. Experimen- tal data [28] had showed that the intestinal bifidobac- teria, gram-positive aerobic bacteria and enterococci were relatively few, but children with a high number of Clostridium and Staphylococcus aureus are prone to allergic symptoms. China. In terms of regions, Europe had the largest num- ber of researchers, followed by Asia and south America. There were 30 observation studies and 14 intervention studies. of skin and intestinal microbiota in children with AD is reduced before the onset of the disease, which can lead to a shift in the immune response to the T helper 2 (Th2) and increase the possibility of AD onset [20, 21]. h During the investigation of Swedish and Estonian children [22, 23], Estonian children had very few aller- gies, and their intestinal microbiota was dominated by Lactobacillus. However, the main intestinal microbiota of Swedish children was Clostridium, so there were many allergic symptoms. For non-allergic individuals, allergic infants had low levels of lactobacilli in their guts, along with a large number of aerobic bacteria. The results of in vitro and in vivo experiments [24, 25] showed that Lactobacillus could regulate the immune system and resist allergy. Characteristics of included literature Elevated fecal caproic acid levels suggest altered gut microbial communities in infants with allergic symptoms [26, 27]. Experimen- tal data [28] had showed that the intestinal bifidobac- teria, gram-positive aerobic bacteria and enterococci were relatively few, but children with a high number of Clostridium and Staphylococcus aureus are prone to allergic symptoms. Probiotics and AD to treat children with AD, respectively. After 12 weeks, the symptoms of AD children in the probiotic group were significantly improved, the SCORAD value was sig- nificantly lower than that in the placebo group, and there were no obvious adverse reactions. The study of Kim et al. [43] has found that oral probiotic preparations had a more significant effect in the treatment of AD children with elevated total IgE. It is speculated that the therapeu- tic effect of probiotics on AD is not only related to probi- otic strains, but also to the host’s immune status. Probiotics are live microorganisms that help to promote the balance of microbiota, have immune regulation, anti- oxidant and antibacterial effects, and have been widely used in the prevention and treatment of allergic diseases in children [29]. Th2-type inflammation is the basic fea- ture of AD [30]. On the one hand, oral probiotics can inhibit the Th2 response and make the immune response develop in the direction of Th1 [31]. On the other hand, it can induce immune tolerance and maintain the bal- ance of Th1/Th2, so as to achieve the effect of treating AD [32, 33]. Previous studies have mostly focused on oral probiotics, and topical and oral probiotics are the new hotspots in the research on the relationship between microbiota and AD in recent years. It has been found that oral probiotics can reduce the abnormal colonization of Staphylococcus aureus and other bacteria, restore the diversity of microbiota, help the recovery of intestinal barrier function, and alleviate the clinical symptoms of AD [34]. Studies [44, 45] have found that probiotic supplementa- tion by pregnant women during prenatal to postpartum lactation and postnatal fetuses can effectively reduce the risk of AD in children, and the effect of multi-strain pro- biotic mixtures is better than that of single-strain formu- lations. Wickens et al. [46] have conducted an 11-year, double-center, randomized, double-blind, placebo-con- trolled trial: pregnant women in the experimental group continued to take probiotics from 35 weeks of gestation to 6 months postpartum lactation, and infants took oral probiotics from birth to 2 years of age. Pregnant women and infants in the control group were all given placebo, and the children were followed up until the age of 11. The proportion of AD in the probiotic group was significantly lower than that in the placebo group. Baldassarre et al. [45]. Gut microbiota and AD developmenth The species and quantity of human microorganisms are abundant, and as symbiotic organisms, they form the human micro-ecosystem together with the host of human body. Under normal circumstances, the micro- biota adheres, colonizes, and reproduces in specific parts of the human body, forming a biofilm barrier, thereby inhibiting the invasion of pathogenic bacte- ria and maintaining the dynamic balance between the human body and microorganisms [17, 18]. The classic “hygiene hypothesis” holds that urbanized lifestyles, excessive hygiene, and the widespread use of antibiot- ics can lead to imbalances in human microecology and induce or aggravate allergic diseases such as AD [19]. The skin and the gut are the two main micro-ecosys- tems of the human body. Some studies have found that compared with the normal population, the diversity Liu et al. BMC Pediatrics (2022) 22:323 Liu et al. BMC Pediatrics (2022) 22:323 Page 4 of 8 Probiotics and AD conducted a clinical observational trial involving 66 pregnant women. The multi-strain probiotic mixture was orally administered to pregnant women from 4 weeks before delivery to 4 weeks after delivery. The secretion of sIgA in infant feces suggests that oral probiotics dur- ing pregnancy and childbirth contribute to the improve- ment of intestinal barrier function in the neonatal period. Intestinal microbiota disorders are closely related to the occurrence and development of AD [47]. Oral and topical probiotics can regulate the distribution of local microbi- ota and improve the immune response of the body [48]. It is expected to become one of the main methods for the prevention and adjuvant treatment of AD in children. Epidemiological surveys [35, 36] showed that the pro- portions of Clostridium, Clostridium difficile, Escherichia coli and Staphylococcus aureus in the gut microbiota of AD patients increased, while the proportions of Bifido- bacterium and Bacteroides decreased, suggesting the pathogenesis of AD is closely related to the disturbance of intestinal microbiota. In 2020, Jiang et al. [37] con- ducted a meta-analysis on the role of probiotics in the prevention and treatment of AD in children, proving that oral probiotics in pregnant women and newborns can effectively reduce the prevalence of AD in children, and children with AD take probiotics orally. It can effectively relieve its clinical symptoms and improve the quality of life. Clinical studies by Navarro-López et al. [38] has found that AD children over 1 year old responded better to oral probiotics, while the efficacy of oral probiotics in children with moderate to severe AD was better than that in children with mild AD. In addition, multi-strain prepa- rations, especially the probiotic mixture containing lacto- bacillus and bifidobacteria that has a better therapeutic effect than a single strain preparation.h Discussions The reduction of bifidobacteria in the human body and the increase of other bacteria have a certain relationship with the increase of age, but the bacteria that appear in different locations have different pathogenic properties [55]. With increasing age and gradual changes in eating habits, the intestinal microbiota in the human body will also change accordingly [56, 57]. The influence of vari- ous factors changes the changes in the microbiota in the human body, and the composition of intestinal microbes will also be changed if drugs are used for treatment [58]. will also be changed if drugs are used for treatment [58]. In terms of dermatopathology, Luo et al. [59] have treated 80 AD children with randomized, double-blind, placebo trial, and the results have showed that after pro- biotic treatment, their intestinal bacteria were signifi- cantly reduced. The group was tested, and the beneficial bacteria were significantly improved, and the allergy symptoms also improved. Probiotics can effectively pro- mote the formation of endogenous barrier mechanisms in children with atopic dermatitis, reduce intestinal inflammation, and avoid allergic symptoms. IL-10 levels increased in the children after continued treatment. Pre- vious study [60] report randomized, double-blind, pla- cebo trial to treat 90 children with moderate and severe atopic dermatitis as experimental subjects, and have used probiotics for adjuvant treatment, and scored itching after 16 weeks. The results have showed that, the degree of itching in children who received probiotic adjuvant therapy is significantly reduced, which have indicated that probiotic adjuvant therapy had a certain effect on the sensitivity of children’s skin. At present, there are not many RCTs with rigorous design that can show that pro- biotics can reduce the severity of AD, and its exact thera- peutic effects and safety remain to be further investigated [61].hl Several review articles about probiotics and gut micro- biota with AD were already published and should be con- sidered. Fang et al. [28] have focused on the the potential mechanisms of probiotics on alleviating AD via upregu- lation of epidermal barrier and regulation of immune signaling, and the possible effective substances on AD, which provide the supports for targeting gut microbiota to AD prevention and treatment. Disamantiaji et al. [69] have included a total of 5 studies, and have concluded that probiotics supplementation in the management of eczema in children does not show a clinically relevant difference vs. standard treatment in reducing eczema severity. Discussions Previous studies have reported that the gut of the fetus is sterile, and the growth and fixation of its intestinal microbiota does not begin until about 24 h after birth. These bacteria are affected by the mode of delivery, liv- ing and feeding environment. Anaerobic microorganisms start to multiply when breastfeeding begins to stimulate the baby’s intestines [49]. Bifidobacterium, Lactobacillus and Bacteroides belong to this type of bacteria, because the oligosaccharides in breast milk stimulate the baby’s stomach, so the most bacteria produced are probiotics. Its main function is to help infants absorb nutrients, min- erals, promote vitamin synthesis in their body, increase the function of the immune system [50, 51]. Interfering f The RCT of Tan-Lim et al. [39] has showed that both single-strain preparations and multi-strain mixed prepa- rations can effectively reduce the clinical symptoms of AD in children, but the multi-strain mixed preparations were more effective than single-strain preparations. Oral Lactobacillus acidophilus single-strain preparations or Lactobacillus acidophilus mixed preparations with other strains of probiotics can improve clinical symptoms in children with AD [34, 40]. The combined use of Lacto- bacillus casei and Lactobacillus salivarius can reduce IgE levels [41]. Prakoeswa et al. [42]. and Kim et al. [43] applied Lactobacillus plantarum IS-10506 and CJLP133 Liu et al. BMC Pediatrics (2022) 22:323 Liu et al. BMC Pediatrics (2022) 22:323 Page 5 of 8 Page 5 of 8 Bifidobacterium Lactobacillus triple viable bacteria tab- lets (the ingredients are Bifidobacterium longum, Lac- tobacillus bulgaricus and Streptococcus thermophilus, 4 tablets/time, 3 times a day for a total of 12 weeks) can increase the number of AD children. These attempts to improve the disease and delay the recurrence of intestinal bifidobacteria and lactobacilli microbiota have brought new hope for the management of chronic disease in children with AD. It is worth noting that the balance of human microbiota is easily affected by various fac- tors such as age, immune status, and external environ- ment, and the therapeutic effect of probiotics is closely related to the strain, timing and dose of administration [66–68]. Large-scale randomized, double-blind, placebo- controlled clinical trials are still needed to determine the effective strains, appropriate doses, and duration of treat- ment of probiotics for AD prevention and treatment. with the denaturation of enzyme activity and lowering cholesterol are the main functions of probiotics [52–54]. Discussions We focused on the relationship of gut microbi- ota and AD in the children population, with more studies included, we have found that probiotics may be an effec- tive and safe treatment option for AD. Still, the current research results have great heterogeneity on the role of probiotics for AD treatment. Therefore, the results of this review must be considered with some limitations. The most significant limitation was that the language of searching databases were limited to English and Chinese, there can be many studies on the gut microbiota and AD in children reported in other language. Besides, the clini- cal trials with rigorous design are very few, future studies with larger sample size and rigorous design are needed to elucidate the role of gut microbiota in AD development and treatment. AD is a Th2-biased inflammatory disorder caused by a combination of complex factors [62]. If the intestinal mucosal immune system is not stimulated by intestinal microorganisms in the early stage of development, the natural development of the immune system will be inhib- ited, and Treg cells will not be mature enough to regulate certain balances such as Th1/Th2 [11, 63]. By compar- ing the intestinal microbiota of infants with atopic der- matitis and healthy infants, study [64] has found that the content of Clostridium in the intestine of the former is relatively high, while the content of Bifidobacterium and Lactobacillus in the intestine of healthy infants is higher. It’s been reported that indole-3-carbaldehyde, which is a tryptophan product metabolized by gut microbiota, can alleviate skin inflammation in a mouse model of atopic dermatitis by inhibiting Th2-type cytokine secretion and IgE production. Previous study [65] has pointed out that Conclusions In conclusion, the current research evidences have showed that the gut microbiota is closely related to the occurrence and development of AD. Probiotics can help immune cells to exert anti-allergic effects on AD, including enhanced antigen degradation and pro- inflammatory immune responses. Intestinal probiotics can regulate immune cells and immune factors, inhibit Liu et al. BMC Pediatrics (2022) 22:323 Liu et al. BMC Pediatrics (2022) 22:323 Liu et al. BMC Pediatrics (2022) 22:323 Page 6 of 8 7. Nutten S. Atopic dermatitis: global epidemiology and risk factors. Ann Nutr Metab. 2015;66(Suppl 1):8–16. 7. Nutten S. Atopic dermatitis: global epidemiology and risk factors. 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Allergy. 2020;75(1):63–74. Received: 22 February 2022 Accepted: 5 May 2022 23. Voor T, Julge K, Bottcher MF, Jenmalm MC, Duchen K, Bjorksten B. Atopic sensitization and atopic dermatitis in Estonian and Swedish infants. Clin Exp Allergy. 2005;35(2):153–9. Consent for publication 18. Zhu LQ, Zhang L, Zhang J, Chang GL, Liu G, Yu DD, Yu XM, Zhao MS, Ye B. Evodiamine inhibits high-fat diet-induced colitis-associated cancer in mice through regulating the gut microbiota. J Integr Med. 2021;19(1):56–65. Competing interests The authors declare that they have no competing interests. 19. Alghamdi HA, Behieldin A, Edris S. Gut microbiome skin axis in the development of atopic dermatitis. J Pak Med Assoc. 2021;71(4):1221–7 19. Alghamdi HA, Behieldin A, Edris S. Gut microbiome skin axis in the development of atopic dermatitis. J Pak Med Assoc. 2021;71(4):1221–7. Ethics approval and consent to participate In this study, all methods were performed in accordance with the relevant guidelines and regulations. Ethical approval is necessary for this scoping review because a literature review did not require ethical approval based on the related research guidelines. 17. Yu L, Deng YH, Huang YH, Ke HJ, Guo Y, Wu JL. Comparison of gut microbiota between infants with atopic dermatitis and healthy con- trols in Guangzhou. China J Asthma Allergy. 2021;14:493–500. 17. Yu L, Deng YH, Huang YH, Ke HJ, Guo Y, Wu JL. Comparison of gut microbiota between infants with atopic dermatitis and healthy con- trols in Guangzhou. China J Asthma Allergy. 2021;14:493–500. Authors’ contributions W L designed research; Y L, X D, S Z, X T, C L conducted research; Y L, X D, S Z analyzed data; W L wrote the first draft of manuscript; Y L, X D had primary responsibility for final content. All authors read and approved the final manuscript. 12. 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https://openalex.org/W2557632639	https://europepmc.org/articles/pmc5749168?pdf=render	English	null	Text normalization for named entity recognition in Vietnamese tweets	Computational social networks	2,016	cc-by	9,016	© The Author(s) 2016. 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. Text normalization for named entity recognition in Vietnamese tweets Vu H. Nguyen1, Hien T. Nguyen1* and Vaclav Snasel2 Vu H. Nguyen1, Hien T. Nguyen1* and Vaclav Snasel2 *Correspondence: hien@tdt.edu.vn 1 Faculty of Information Technology, Ton Duc Thang University, Ho Chi Minh City, Vietnam Full list of author information is available at the end of the article Abstract Background: Named entity recognition (NER) is a task of detecting named entities in documents and categorizing them to predefined classes, such as person, location, and organization. This paper focuses on tweets posted on Twitter. Since tweets are noisy, irregular, brief, and include acronyms and spelling errors, NER in those tweets is a challenging task. Many approaches have been proposed to deal with this problem in tweets written in English, Germany, Chinese, etc., but none for Vietnamese tweets. Methods: We propose a method that normalizes a tweet before taking as an input of a learning model for NER in Vietnamese tweets. The normalization step detects spelling errors in a tweet and corrects them using an improved Dice’s coefficient or n-grams. A Support Vector Machine learning algorithm is employed to learn a classifier using six different types of features. Results and Conclusion: We train our method on a training set consisting of more than 40,000 named entities and evaluate it on a testing set consisting of 3,186 named entities. The experimental results showed that our system achieves state-of-the-art performance with F1 score of 82.13%. Results and Conclusion: We train our method on a training set consisting of more than 40,000 named entities and evaluate it on a testing set consisting of 3,186 named entities. The experimental results showed that our system achieves state-of-the-art performance with F1 score of 82.13%. Keywords: Text normalization, Named entity recognition, Spelling error detection and correction Keywords: Text normalization, Named entity recognition, Spelling error detection and correction Nguyen et al. Comput Soc Netw (2016) 3:10 DOI 10.1186/s40649-016-0032-0 1 https://blog.twitter.com/2011/numbers. Related work NER Named entity recognition has been studied extensively on formal texts, such as news and authorized web content. Several approaches have been proposed using different learning models, such as condition random fields (CRF), maximum entropy model (MEM), hid- den markov model (HMM), and support vector machines (SVM). In particular, May- field et al. [34] used SVM to estimate lattice transition probabilities for NER. McCallum and Li [35] applied a feature induction method for CRF to recognize named entities. A combination of a CRF model and latent semantics to recognize named entities was pro- posed in [18]. A method using soft-constrained inference for NER was proposed in [11]. In [8] and [54], the authors proposed a maximum entropy tagger and an HMM-based chunk tagger to recognize named entities. Unfortunately, those methods gave poor per- formance on tweets, as pointed out in [31]. Vietnamese NER In the domain of Vietnamese texts, various approaches have been proposed using vari- ous learning models, such as SVM [49], classifier voting [48] and CRF [19, 52]. Some other authors have proposed other methods for NER, such as a rule-based method [36, 38], labeled propagation [21], the use of a bootstrapping algorithm and a rule-based model [51], and combined linguistically motivated and ontological features [39]. Pham et al. [41] proposed an online learning algorithm, i.e., MIRA [7] in combination with Background In recent years, social networks have become very popular. It is easy for users to share their data using online social networks. Currently, Twitter is one of the most popular social networks. According to statistics from 2011, the number of tweets was up to 140 million per day.1 With such a huge number of tweets being posted every day, effective extraction and processing of those data will be very beneficial, especially to information extraction applications. Twitter provides an interactive environment that allows the users to create their own content through tweets. Since each tweet consists of only 140 characters, users tend to use acronyms, non-standard words, and social tokens. Therefore, the tweets contain many spelling errors, and this creates a significant challenge for named entity recog- nition (NER). Several recognition methods for named entities have been proposed for tweets in English and other languages [2, 17, 27, 31, 44]. Although there have been many Nguyen et al. Comput Soc Netw (2016) 3:10 Page 2 of 16 approaches proposed in formal texts for NER in the Vietnamese language, none is avail- able for Vietnamese tweets. Thus, in this paper, we propose a method for NER in Viet- namese tweets to fill the gap. The system consists of three steps, i.e., (1) normalization of tweets by detecting and correcting spelling errors; (2) capitalization classifier; and (3) recognition of named entities. Table 1 shows an example of NER according to these three steps. In this paper, we present the first attempt to provide NER capability in Vietnamese tweets, and this contribution has three components, i.e., (1) a method for the normal- ization of Vietnamese tweets based on dictionaries and Vietnamese vocabulary struc- tures in combination with a language model; (2) a learning model for NER in Vietnamese tweets with six different types of features; and (3) a training set of more than 40,000 named entities and a testing set of 3186 named entities to evaluate the NER system of Vietnamese tweets. The rest of this paper is organized as follows. The second section presents earlier work related to this effort. Our proposed method is presented in third section; fourth section is the experiments and their results. Our conclusions are presented in fifth section. Table 1 An example of named entity recognition Table 1 An example of named entity recognition Original tweet xe đón h`ˆo ngo. c hà gây tai na.n kinhh hoàng: se˜ khởi tố tài xế http://fb.me/2MwvznBbj Step 1: Normalization xe đón h`ˆo ngo. c hà gây tai na.n kinh hoàng: se˜ khởi tố tài xế Step 2: Capitalization Xe đón H`ˆo Ngo. c Hà gây tai na.n kinh hoàng: se˜ khởi tố tài xế Step 3: NEs recognition Xe đón <PER> H`ˆo Ngo. c Hà </PER> gây tai na.n kinh hoàng: se˜ khởi tố tài xế Table 1 An example of named entity recognition Original tweet xe đón h`ˆo ngo. c hà gây tai na.n kinhh hoàng: se˜ khởi tố tài xế http://fb.me/2MwvznBbj Step 1: Normalization xe đón h`ˆo ngo. c hà gây tai na.n kinh hoàng: se˜ khởi tố tài xế Step 2: Capitalization Xe đón H`ˆo Ngo. c Hà gây tai na.n kinh hoàng: se˜ khởi tố tài xế Step 3: NEs recognition Xe đón <PER> H`ˆo Ngo. c Hà </PER> gây tai na.n kinh hoàng: se˜ khởi tố tài xế Nguyen et al. Comput Soc Netw (2016) 3:10 Page 3 of 16 CRF and bootstrapping. Sam et al. [46] used the idea of Liao and Veeramachaneni in [28] based on CRF and expanded it by combining proper name co-references and named ambiguity heuristics with a powerful sequential learning model. Nguyen and Pham [22] proposed a feature selection approach for named entity recognition using a genetic algo- rithm. To calculate the accuracy of the recognition of the named entity, this paper used KNN and CRF. Nguyen and Pham [37] proposed a systematic approach to avoid the con- flict between rules when a new rule was added to the set of rules for NER. Le and Tran [23] proposed some strategies to reduce the running time of genetic algorithms used in a feature selection task for NER. These strategies included reducing the size of the popula- tion during the evolution process of the genetic algorithm, reducing the fitness compu- tation time of individuals in the genetic algorithm using progressive sampling for finding the (near) optimal sample size of the training data, and parallelization of individual fit- ness computation in each generation. However, there have been no approaches that focused on NER in Vietnamese tweets or (short) informal Vietnamese texts. Table 1 An example of named entity recognition To better collocate our results with other existing Vietnamese NER systems that used other techniques, we report the performances of other Vietnamese NER systems in Table 2. 2 http://www.freebase.com. NER in tweets Regarding microblog texts written in English and other languages, several approaches have been proposed for NER. Among them, Ritter et al. [44] proposed an NER system for tweets, called T-NER, which employed a CRF model for training and Labeled-LDA. Ramage et al. [43] proposed an external knowledge base, i.e., Freebase2 for NER. A hybrid approach to NER on tweets was presented in [31] in which a KNN-based classifier and a CRF model were used. A combination of heuristics and MEM was proposed in [17]. In [50], a semi-supervised learning approach that combined the CRF model with a classifier based on the co-occurrence coefficient of the feature words surrounding the proper noun was proposed for NER on Twitter. Li and Liu [26] proposed non-standard word (NSW) detection and decided a word is out of vocabulary (OOV) based on the dictionary, and then applied the normalization system of [25] to normalize OOV words. The results from NSW detection was used for NER based on the pipeline strategy or the joint decoding fashion method. In [32], a named entity was recognized using three steps, i.e., (1) each tweet is pre-labeled using a sequential labeler based on the linear conditional random fields (CRFs) model; (2) tweets are clustered to put those that have similar content into the same group; and (3) each cluster refines the labels of each tweet using an enhanced CRF model that incorporates the cluster-level information. Liu et al. [33] proposed jointly conducting NER and named entity normalization (NEN) for multiple tweets using a fac- tor graph, which leverages redundancy in tweets to make up for the dearth of information in a single tweet and allows these two tasks to inform each other. Liu et al. [30] proposed a novel method for NER consisting of three core elements, i.e., normalization of tweets, combination of a KNN classifier with a linear CRF model, and a semi-supervised learning framework. Nguyen and Moschitti [40] presented a method for incorporating global 2 http://www.freebase.com. Page 4 of 16 Nguyen et al. NER in tweets Comput Soc Netw (2016) 3:10 Table 2 Results of several previous works in Vietnamese NER PER person, ORG organization, LOC location, CUR currency, NUM number, PERC percent, TIME time, NA nationality, FA facility, RE region, MISC miscellaneous System Entity types Precision (%) Recall (%) F1 (%) [19] PER 84 82.56 83.39 [36] PER, ORG, LOC, NA, FA, RE 92 76 83 [38] PER, ORG, LOC 86.05 81.11 83.51 [46] PER, ORG, LOC 93.13 88.15 79.35 [48] PER, ORG, LOC, CUR, NUM, PERC, TIME 86.44 85.86 89.12 [49] PER, ORG, LOC, CUR, NUM, PERC, TIME 89.05 86.49 87.75 [52] PER, ORG, LOC, CUR, NUM, PERC, TIME, MISC 83.69 87.41 85.51 Table 2 Results of several previous works in Vietnamese NER PER person, ORG organization, LOC location, CUR currency, NUM number, PERC percent, TIME time, NA nationality, FA facility, RE region, MISC miscellaneous features in NER using re-ranking techniques that used two kinds of features, i.e., flat and structured features and a combination of CRF and SVM. In [55], a CRF model without being focused on Gazetteers was used for NER for Arabic social media. Recently, [1] presented the results of Shared Tasks of the 2015 Workshop on Noisy User- generated Text: Twitter Lexical Normalization and Named Entity Recognition. According to this paper, most of researchers used CRF. However, several researchers in this workshop described new methods, such as [13], which used absolutely no hand-engineered features and relied entirely on embedded words and a feed-forward, neural-network (FFNN) archi- tecture; Cherry et al. [3] developed a semi-Markov MIRA trained tagger; Yamada et al. [53] used entity-linking-based features, and other researchers used CRFs. Since some of the specific features of Vietnamese were presented in [49], one cannot apply those methods directly to Vietnamese tweets. In this paper, we propose a method for NER in Vietnamese tweets to fill the gap. Our method includes three main tasks, i.e., (1) a method for normalization of Vietnamese tweets based on dictionaries and Vietnamese vocabulary structures in combination with a language model; (2) a method for detecting and correcting suitable capital letters; and (3) a model for training and recognizing named entities in Vietnamese tweets. We also conducted experiments to evaluate our NER method focused on three entity types, i.e., PERSON, LOCATION, and ORGANIZATION. Proposed method In this section, we present our method for NER in Vietnamese tweets. This model has two main parts, i.e., one for training and another for recognizing. Figure 1 describes our model. In our model, the gazetteers are used for both training and recognizing. We will provide more detail in the following subsections. Normalization When we approached NER in Vietnamese tweets, we found that, on Twitter, they are noisy, irregular, brief, and consist of acronyms and spelling errors. Processing those tweets is more challenging than processing news or formal texts. To deal with this issue, several researchers have focused on other languages than Vietnamese. For example, Han et al. [14, 15] proposed a method to detect and handle errors based on the morpho- phonemic similarity. Choi et al. [4] detected and handled many non-standard words in online social networks using a diverse coefficient method, such as Dice, Jaccard, and Ochiai. Hassan and Menezes [16] used random walks on a contextual similarity bipartite graph constructed from n-gram sequences on large unlabeled text corpus to normalize social text. Sproat et al. [47] developed a novel method for normalizing and morpho- logically analyzing Japanese noisy text by generating both character-level and word-level normalization candidates and using discriminative methods to formulate a cost function. An approach to normalize Twitter messages in Malay based on corpus-driven analysis Nguyen et al. Comput Soc Netw (2016) 3:10 Page 5 of 16 was proposed in [45]. Cotelo et al. [6] proposed a modular approach for lexical normali- zation applied to Spanish tweets. This system is proposed by including the detection of modules and candidate for correction for each out-of-vocabulary word and ranking the candidates to select the best one. Liu et al. [29] proposed a normalization system for short message service (SMS) and Twitter data based on a broad-coverage normalization system by integrating three human perspectives, i.e., enhanced letter transformation, visual priming, and string/phonetic similarity. Recently, in the Shared Tasks of the 2015 Workshop on Noisy User-generated Text: Twitter Lexical Normalization and Named Entity Recognition, several methods were proposed for the normalization of Twitter lexical usages. According to the summary of results in [1], the common approaches were lexicon-based methods, CRF, and neural net- work-based methods. Among the constrained systems, neural networks achieved strong results even without off-the-shelf tools. In contrast, CRF and lexicon-based approaches were shown to be effective in the unconstrained category. Considering the Vietnamese language, we have not found any research work that has undertaken this task. The theoretical background Currently, there are several viewpoints on what is a Vietnamese word. However, to meet the goals of automatic error detection, normalization and classification, we followed the viewpoint in [48], i.e., “A Vietnamese word is composed of special linguistic units called Vietnamese morphosyllables.” A morphosyllable may be a morpheme, a word, or some- thing else [49]. And according to the syllable dictionary of Hoang Phe [42], we split a morphosyllable into two basic parts, consonants and syllables, as follows: Support Vector Machine learning Algorithm Trained model the Vietnamese tweet with named enes tagged a Vietnamese tweet Normalizaon Capitalizaon classiﬁer Word segmentaon and POS tagging Feature extracon Gazeeers Training set Feature extracon Named Enty Recognion Fig. 1 NER model for Vietnamese tweets Nguyen et al. Comput Soc Netw (2016) 3:10 Page 6 of 16 • • Consonants: The Vietnamese language has 27 consonants, i.e., “b,” “ch,” “c,” “d,” “đ,” “gi,” “gh,” “g,” “h,” ’‘kh,” ’‘k,” “l,” “m,” “ngh,” “ng,” “nh,” “n,” “ph,” “q,” “r,” “s,” “th,” “tr,” “t,” “v,” “x,” “p.” In those consonants, there are eight tail consonants, i.e., “c,” “ch,” “n,” “nh,” “ng,” “m,” “p,” and “t.” • • Syllables: A syllable may be a vowel, a combination of vowels, or a combination of vowels and tail consonants. According to the syllable dictionary of Hoang Phe, the Vietnamese language has 158 syllables, and the vowels in these syllables do not occur consecutively more than once, except for the syllables “ooc” and “oong.” Vowels: The Vietnamese language has 12 vowels, i.e., “a,” “ă,” “â,” “e,” “ê,” “i,” “o,” “ô,” “ơ,” “u,” “ư,” and “y.” h “ư,” and “y.” Error detection Before performing this step, the noisy contents of tweets must be removed, such as emo- tion symbols (e.g., ❤❤), hashtag symbols, link url @username and others. To detect errors, we synthesized and built a dictionary for all Vietnamese morphosyllables, and it contains more than 7300 morphosyllables. A morphosyllable in a tweet will be identified as an error if it does not appear in the morphosyllable dictionary. Normally, Vietnamese tweets include two kinds of errors, i.e., typing errors and spelling errors. Normalization Because Vietnamese tweets on Twitter are noisy, irregular, and brief and consist of acro- nyms and spelling errors. Therefore, we propose a method to normalize them before performing NER. Our normalization method has two steps, i.e., error detection and error correction. a. Similarity of two morphosyllables To measure the similarity of two morphosyllables, we used the results in the research of Dice [9] with some improvements we made. To use Dice’s research, we split all of the characters of the morphosyllables to bigrams. Assuming that we have two morphosylla- bles, i.e., “nguyen” and “nguye,” the bigrams of these morphosyllables can be represented as follows: bigramnguyn = {ng, gu, uy, yn}, and bigramnguyen= {ng, gu, uy, ye,en}. Spelling errors Spelling errors occur frequently in Vietnamese tweets. Normally, they occur due to mis- takes in pronunciation. Some examples of spelling errors are as follows: • • Error due to using the wrong mark: “quyển sách” (book) to “quyễn sách” • • Initial consonant error: “bóng chuyền” (volleyball) to “bóng truyền” • • End consonant error: “bài hát” (song) to “bài hác” • • Region error: “tìm kiếm” (find) to “tìm kím” Typing errors To handle this issue, we built a set of syllable rules with their tone-marks and a set of rules to map these syllables to their errors, as shown in the following examples: • • “án”: “asn,” “ans,” “a1n,” or “an1” • • “àn”: “afn,” “anf,” “a2n,” or “an2” • • “ản”: “arn,” “anr,” “a3n,” or “an3” • • “ãn”: “axn,” “anx,” “a4n,” or “an4” • • “ạn”: “ajn,” “anj,” “a5n,” or “an5” • • “án”: “asn,” “ans,” “a1n,” or “an1” • • “àn”: “afn,” “anf,” “a2n,” or “an2” • • “ản”: “arn,” “anr,” “a3n,” or “an3” • • “ãn”: “axn,” “anx,” “a4n,” or “an4” • • “ạn”: “ajn,” “anj,” “a5n,” or “an5” • • “án”: “asn,” “ans,” “a1n,” or “an1” • • “àn”: “afn,” “anf,” “a2n,” or “an2” • • “ản”: “arn,” “anr,” “a3n,” or “an3” • • “ãn”: “axn,” “anx,” “a4n,” or “an4” • • “ạn”: “ajn,” “anj,” “a5n,” or “an5” Error correction For the detected typing and spelling errors, first, the system uses vocabulary structures and the set of syllable rules to normalize them. Then, the system uses n-gram to normal- ize these results based on the degree of similarity between them. Typing errors Two popular typing methods are used to compose Vietnamese tweets, i.e., Telex typing and VNI typing. Each method combines letters to form Vietnamese tweets. Vietnamese characters have some extra vowels that do not exist in Latin characters, i.e., â, ă, ê, ô, ơ, one more consonant, đ; Vietnamese has five types of marks, i.e., acute accent (“á”), grave accent (“à”), hook accent (“ả”), tilde (“ã”), and heavy accent (“ạ”). The combination of vowels and marks forms the Vietnamese language its own identity. • • When using Telex typing, we have the combination of characters to form Vietnamese vowels, such as aa for â, aw for ă, ee for ê, oo for ô, ow for ơ, and uw for ư. Also we have one consonant, dd, for đ. For forming marks, we have s for acute accent, f for grave accent, r for hook accent, x for tilde, and j for heavy accent. • • Similar to Telex typing, we have the combination of characters in VNI typing, such as: a6 for â, a8 for ă, e6 for ê, o6 for ô, o7 for ơ, u7 for ư, and d9 for đ. To form marks, we have 1 for accent, 2 for grave accent, 3 for hook accent, 4 for tilde, and 5 for heavy accent. Page 7 of 16 Nguyen et al. Comput Soc Netw (2016) 3:10 Tweets are very short and prepared quickly, so the typing speed can cause errors. For example: • • With the word, “Nguyễn,” we could have typing errors such as “nguyeenx,” “nguyênx,” or “nguyeenxx” with Telex typing, and “nguye6n4,” “nguyên4,” or “nguye6n44” with VNI typing. • • With the word, “người”, we could type the following incorrect words: “ngươif,” “ngươfi,” “nguowfi,” “nguowif,” “nguofwi,” “nguofiw,” “nguoifw,” “nguoiwf,” or “nguowff” with Telex typing, and “nguwowi2,” “ngươ2i,” “nguo72i,” “nguo7i2,” “nguo27i,” “nguo2i7,” “nguoi27,” or “nguoi72” with VNI typing. Proposed method to improve the Dice coefficient As observing from the experimental data using the Dice coefficient, we found that the above method will be accurate with misspelled morphosyllables which is having the mis- spelled character at the end. When misspelled characters occur close to the last charac- ter, at least we will lose the similarity of the last two grams. For a morphosyllable that has three characters, the degree of similarity is 0. For example: Dice(“rất”, “rát”) = 0; Dice(“gân”, “gần”) = 0; From the above problem, we proposed a method to improve the Dice coefficient. The improvement of coefficient was performed by combining the first character with the last character of the two morphosyllables to form a new pair of bigrams. If the two members of this pair are different, the system will use the coefficients as shown in Eq. (1). In con- trast, we use Eq. (2) as follows: (2) iDice(wi, wj) = 2 × \| bigramwi bigramwj \| + 1 \| bigramwi \| + \| bigramwj \| + 2 (2) Let fbigramw be an additional bigram of w. Each fbigram is the pair of the first and the last character of w. We can express the formula for improving the Dice coefficient as Eq. (3): (3) f Dice(wi, wj) = Dice(wi, wj) : if fbigramwi is diﬀerent from fbigramwj iDice(wi, wj) : otherwise (3) To illustrate the improvement of the Dice coefficient, we assumed that we have two morphosyllables to measure the degree of similarity, i.e., “nguyen” and “nguyn,” as pre- sented in the previous section, thus we have \| bigramwi bigramwj \| = 3. Combining the first and the last characters of the two morphosyllables we have the new pair of bigram, which has the same result, i.e., “nn.” So, using the improvement of the Dice coefficient, we have fDice(“nguyen,” “nguyn”) = 0.727. If we use the normal coefficient of Dice, we have Dice(“nguyen,” “nguyn”) = 0.667. Table 3 shows the results of measuring the simi- larity of two morphosyllables with the Dice coefficient and the improved Dice coefficient methods. With the improved method, the similarities are obviously improved. To illustrate the improvement of the Dice coefficient, we assumed that we have two morphosyllables to measure the degree of similarity, i.e., “nguyen” and “nguyn,” as pre- sented in the previous section, thus we have \| bigramwi bigramwj \| = 3. Dice coefficienthfi The Dice coefficient, developed by Lee Raymond Dice [9], is a statistical approach for comparing the similarity of two samples. The Dice coefficient of the two morphosylla- bles, wi and wj, according to bigram can be calculated using Eq. 1: Page 8 of 16 Nguyen et al. Comput Soc Netw (2016) 3:10 where (1) Dice(wi, wj) = 2× \| bigramwi bigramwj \| \| bigramwi \| + \| bigramwj \| (1) where • • \| bigramwi \| and \| bigramwj \| are the total bigrams of wi and wj • • \| bigramwi \| and \| bigramwj \| are the total bigrams of wi and wj i j • • \| bigramwi \| \| bigramwj \| are the number of bigrams which appear in wi and wj at the same time. If two morphosyllables are the same, the Dice coefficient is 1. The higher the Dice coef- ficient, the higher the degree of similarity and vice versa. b. Similarity of two sentences b. Similarity of two sentences Assume that we need to measure the similarity of two sentences, i.e., S1 = w1 w2 . . . wn and S2 = w′ 1 w′ 2 . . . w′ n. We compare the similarity of each pair of morphosyllables according to the improved Dice coefficient. Then, we compute the similarity of the two sentences by Eq. (4): (4) Sim(S1, S2) = n i=1 fDice wi, w′ i n (4) n where wi and w′ i are the corresponding morphosyllables of S1 and S2. n is the number of morphosyllables. If two sentences are the same, their degree of similarity (Sim) is 1. The higher the Sim coefficent, the higher the degree of similarity becomes, and vice versa. Table 4 shows the results of the normalization of Vietnamese tweets that have spelling errors. Proposed method to improve the Dice coefficient Combining the first and the last characters of the two morphosyllables we have the new pair of bigram, which has the same result, i.e., “nn.” So, using the improvement of the Dice coefficient, we have fDice(“nguyen,” “nguyn”) = 0.727. If we use the normal coefficient of Dice, we have Dice(“nguyen,” “nguyn”) = 0.667. Table 3 shows the results of measuring the simi- larity of two morphosyllables with the Dice coefficient and the improved Dice coefficient methods. With the improved method, the similarities are obviously improved. Page 9 of 16 Nguyen et al. Comput Soc Netw (2016) 3:10 Table 3 The results of measuring the similarity of two morphosyllables with the Dice coef- ficient and the improved of Dice coefficient methods Error morphosyllable Correct morphosyllable Dice fDice rat rất 0 0.333 rat rác 0 0 Nguễn Nguyễn 0.667 0.727 Nguễn Nguy 0.571 0.571 tượg Tượng 0.571 0.667 tượg Tương 0.286 0.444 Table 3 The results of measuring the similarity of two morphosyllables with the Dice coef- ficient and the improved of Dice coefficient methods 4 http://mim.hus.vnu.edu.vn/phuonglh/softwares/vnTagger. Extraction of features This phase aims to convert each word to a vector of feature values. Our system uses the IOB model to annotate data in the training and classification phases. IOB is expressed as follows: • • I: current morphosyllable is inside of a named entity (NE). • • O: current morphosyllable is outside of an NE. • • B: current morphosyllable is the beginning of an NE. Table 6 shows the characteristic value of labels according to the IOB model with four classes, i.e., PER, LOC, ORG, and O. The selection of specific attributes from the train- ing set has a key role in identifying the type of entity. Since the nature of the Vietnamese language is different from English, we used the most appropriate and reasonable features to achieve optimum accuracy for the system. Our system uses the following features: • • Word position The position of words in a sentence. • • Word position The position of words in a sentence. h • • POS POS tag of the current word.i • • POS POS tag of the current word. • • Orthographic Capitalization of first character, capitalization of all letters, lowercase, punctuation, numbers. • • Gazetteer We build several gazetteer lists, such as person, location, organization, and prefixes. These gazetteer lists consist of more than 50,000 names of people, nearly 12,000 names of locations, and 7000 names of organizations. • • Prefix, Suffix The first and the second character; the last and the next to the last char- acter of the current word. • • POS Prefix, POS Suffix POS tags of two previous words and POS tags of two follow- ing words of the current word. Word segmentation and part of speech (POS) tagging To perform word segmentation and POS tagging for normalized tweets, we used vnTo- kenizer3 of [20] for word segmentation and VnTagger4 of [24] for POS tagging. Capitalization classifier Capitalization is a key orthographic feature for recognizing named entities [10, 12]. Unfortunately, in tweets, capitalization is much less reliable than edited texts. Users usu- ally compose and reply to messages quickly, and they do not care much about capitaliza- tion. According to [5], a letter is capitalized in the following cases: 1. Capitalize the first letter of the first syllable of a complete sentence, after punctuation (.), question mark (?), exclamation point (!), ellipsis (. . .) and new line. 1. Capitalize the first letter of the first syllable of a complete sentence, after punctuation ii (.), question mark (?), exclamation point (!), ellipsis (. . .) and new line. Capitalize the name of people, locations, and organizations. 2. Capitalize the name of people, locations, and organizations. 3. Other cases of capitalization include, e.g., medal name, position name, days of the week, months of the year, holidays, names of books, and names of magazines Table 4 Tweets with spelling errors and their normalization Spelling error tweets Normalized tweets xe đón hồ ngo. c hà gây tai nạn kinhh hoàng: se˜ khởi tố tài xế http://fb.me/2MwvznBbj xe đón hồ ngọc hà gây tai nạn kinh hoàng: sẽ khởi tố tài xế (the car picked up ho ngoc ha caused a terrible accident: the driver will be prosecuted) hôm nay, siinh viên ddaijj ho. c tôn dduwcss thắng được nghỉ ho. c hôm nay, sinh viên đại học tôn đức thắng được nghỉ học (today, students of ton duc thang university were allowed to absent) Table 4 Tweets with spelling errors and their normalization Nguyen et al. Comput Soc Netw (2016) 3:10 Page 10 of 16 Page 10 of 16 Because our method focuses on three types of entities, i.e., person, organization, and location, in the capitalization classifier, we take the first and the second cases into account. For the first case, we detect the structure of the sentence and correct incorrect capitalization. In the second case, we use gazetteers of persons, locations, and organiza- tions. Table 5 shows the results of the capitalization classifier of Vietnamese tweets. Evaluation Data using for normalization 3 http://mim.hus.vnu.edu.vn/phuonglh/softwares/vnTokenizer. Data using for normalization In this paper, to normalize for spelling errors that cannot be normalized by Vietnamese structure or a set of syllable rules, we used the tri-gram language model (tri-gram of Page 11 of 16 Nguyen et al. Comput Soc Netw (2016) 3:10 Table 5 Some results of capitalization classifier of Vietnamese tweets Tweets before capitalization Tweets after capitalization classifier xe đón h`ˆo ngọc hà gây tai nạn kinh hoàng: sẽ khởi tố tài xế xe đón H`ˆo Ngọc Hà gây tai nạn kinh hoàng: sẽ khởi tố tài xế(the car picked up Ho Ngoc Ha caused a terrible accident: the driver will be prosecuted) hôm nay, sinh viên đại học tôn đức thắng được nghı̉ học hôm nay, sinh viên Đại học Tôn Đức Thắng được nghı̉ học (today, students of Ton Duc Thang university were allowed to absent) Table 5 Some results of capitalization classifier of Vietnamese tweets Table 6 The characteristic value of labels according to IOB model Label Value Meaning O [1] Outside a named entity B-PER [2] Beginning morphosyllable of a NE belongs to a Person class I-PER [3] Inside morphosyllable of a NE belongs to Person class B-LOC [4] Beginning morphosyllable of a NE belongs to Location class I-LOC [5] Inside morphosyllable of a NE belongs to Location class B-ORG [6] Beginning morphosyllable of a NE belongs to Organization class I-ORG [7] Inside morphosyllable of a NE belongs to Organization class Table 6 The characteristic value of labels according to IOB model word). This model was built from SRILM5 with a huge amount of data collected from online newspapers, e.g., http://www.vnexpress.net, http://nld.com.vn/, http://dantri. com.vn/, and others. The data were collected from many fields, such as current events, world, law, education, science, business, sports, and entertainment with over 429,310 articles. The total volume of collected data was about 1045 MB. The tri-gram model that was built from SRILM was about 1460 MB. To ensure the accuracy of results, we chose all of the tri-grams from the SRILM model in which the frequency of occurrences was greater than or equal five. The volume of selected tri-grams was around 81 MB, and the number of tri-grams was around 3.75 million. 5 http://www.speech.sri.com/projects/srilm/. NER training set As seen in Fig. 1, before performing feature extraction, we perform word segmentation, POS tagging, and assigning labels in Table 6 for each word in the training set. Then, the system extracts features of the words and represents each of those words as a feature vector. A support vector machine learning algorithm was used to train the model using the training set. In particular, we assigned labels for words in the training set using a semi-automatic program, meaning that we assigned labels to those words with a program we wrote and checked in hand. In our self-written program, we considered the noun phrase obtained after the tagging step with a list of dictionary of text files to label for those words. The text files of the dictionary contain: Page 12 of 16 Nguyen et al. Comput Soc Netw (2016) 3:10 Table 7 The results of assigning labels to words of two Vietnamese tweets Tweets Tweets after assigning labels xe đón Hồ Ngọc Hà gây tai nạn kinh hoàng: sẽ khởi tố tài xế xe đón <PER> Hồ Ngọc Hà </PER> gây tai nạn kinh hoàng: sẽ khởi tố tài xề (the car picked up Ho Ngoc Ha caused a terrible accident: the driver will be prosecuted) hôm nay, sinh viên Đại học Tôn Đức Thắng được nghı̉ học hôm nay, sinh viên <ORG> Đại học Tôn Đức Thắng </ORG> được nghı̉ học (today, students of Ton Duc Thang university were allowed to absent) Table 7 The results of assigning labels to words of two Vietnamese tweets Table 8 Total number of named entities in the training set Table 8 Total number of named entities in the training set Entity type Number of named entities PER 10,842 LOC 19,037 ORG 12,311 hi • • The noun prefix for organizations such as company, firm, and corporation. hii • • The noun prefix for locations such as province, city, and district. hi • • List of dictionary for states, provinces of Vietnam, and others. Table 7 shows the results of assigning labels to words of two Vietnamese tweets. The total number of entities to which we assigned labels in this phase is presented in Table 8. After assigning the labels for words in Vietnamese tweets, we analyzed these tweets to build feature vectors for those words. NER training set The structure of a feature vector includes <label> <index1>:<value1> <index2>:<value2> <index3>:<value3> and other pairs, where • • <label>: value from 1 to 7 according to 7 labels (O, B-PER, I-PER, B-LOC, I-LOC, B-ORG, I-ORG). • • <index>:<value>: order of feature and value corresponding to feature of a word, respectively. After representing words in the training set as feature vectors, we used libSVM6 to train the model. 6 http://www.csie.ntu.edu.tw/cjlin/libsvm/#download. Experiments We conducted experiments to evaluate our method using a test set including 2,271 Viet- namese tweets and 3,186 named entities. In order to show the performance of normali- zation, we also conduct experiments to evaluate the proposed normalization method. To evaluate normalization method, we ran the test on the tri-gram model with the normal Dice coefficient (Dice) and the improved Dice coefficient (fDice) to measure the similarity of the two sentences. We used three metrics to evaluate our method, i.e., the precision, the recall, and the F-Measure methods. Page 13 of 16 Nguyen et al. Comput Soc Netw (2016) 3:10 • • Precision (P): number of correctly fixed errors divided by the total number of errors detected. • • Recall (R): number of correctly fixed errors divided by the total error. • • Balance F-measure (F1): F1 = 2∗P∗R p+R Table 9 shows the experimental results of our normalization method. As seen in this table, the combination of our improved Dice coefficient and the tri-gram model achieved better performance than the normal Dice coefficient with the tri-gram model. To evaluate the NER method and make a comparison of the impact of the normaliza- tion of the test set, we conducted two experiments, i.e., one without normalization and capitalization classifier of tweets (Case 1) and the other with normalization and capitali- Table 9 The results using fDice and Dice with tri-gram model Method Precision (%) Recall (%) F-Measure (%) Dice 83.85 82.76 83.30 fDice 89.66 88.50 89.08 Table 10 Experimental results of case 1 and case 2 Case # NEs in testing set # recognized NEs # correctly recog- nized NEs # wrong recog- nized NEs P (%) R (%) F1 (%) 1 3186 2593 2163 430 83.41 67.89 74.86 2 3186 2982 2533 449 84.94 79.50 82.13 Table 11 Comparison performance of our method with that of [49] System Precision (%) Recall (%) F1 (%) Our system 84.94 79.50 82.13 System of [49] 83.10 77.62 80.27 Table 9 The results using fDice and Dice with tri-gram model Table 11 Comparison performance of our method with that of [49] System Precision (%) Recall (%) F1 (%) Our system 84.94 79.50 82.13 System of [49] 83.10 77.62 80.27 • • Precision (P): number of correctly fixed errors divided by the total number of errors detected. • • Recall (R): number of correctly fixed errors divided by the total error. Conclusions In this paper, we present the first attempt to NER in Vietnamese tweets on Twitter. We proposed a method for the normalization of Vietnamese tweets, based on the dictionar- ies and Vietnamese vocabulary structures in combination with a language model. We also proposed a learning model to recognize named entities using six different types of features. To evaluate for our normalization method, we built a tri-gram model that had a volume of about 81 MB and the number of tri-grams was around 3.75 million. The improvement in measuring the similarity of two words based on the modified Dice coefficient outperformed the original Dice coefficient, and our normalization method achieved a high performance with F1 score of 89.08%. To evaluate the NER method, we built a training set of more than 40,000 named entities and a testing set of 3186 named entities to evaluate our system. The experimental results showed that our system achieved encouraging performance, with 82.13% F1 score. We plan to acquire a larger dataset to build and test the language model with bigram, trigram, and four-gram to improve our normalization performance. In addition, we also collected the data required to increase the number of named entities in the training set as well as to expand the Gazetteers so that we can increase the NER performance of our system. Experiments 2 P R i • • Balance F-measure (F1): F1 = 2∗P∗R p+R i • • Balance F-measure (F1): F1 = 2∗P∗R p+R Table 9 shows the experimental results of our normalization method. As seen in this table, the combination of our improved Dice coefficient and the tri-gram model achieved better performance than the normal Dice coefficient with the tri-gram model. fi To evaluate the NER method and make a comparison of the impact of the normaliza- tion of the test set, we conducted two experiments, i.e., one without normalization and capitalization classifier of tweets (Case 1) and the other with normalization and capitali- zation classifier of tweets (Case 2). Table 10 shows our experimental results. In this case, we also used three metrics to evaluate our method, i.e., the precision, the recall, and the Balance F-Measure. • • Precision (P): the number of correctly recognized named entities divided by the total number of named entities recognized by the NER system. • • Recall (R): the number of correctly recognized named entities divided by the total number of named entities in the test set. • • Balance F-Measure (F1): F1 = 2∗P∗R p+R • • Balance F-Measure (F1): F1 = 2∗P∗R p+R According to Table 10, when we applied the normalization to the test set, the preci- sion, recall and balance F-Measure of this test were higher than the case of the test set without normalization. We re-implemented the state-of-the-art method proposed in [49] and compared its performance with our method. The results of this comparison are shown in Table 11. Nguyen et al. Comput Soc Netw (2016) 3:10 Page 14 of 16 Competing interests The authors declare that they have no competing interests. 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https://openalex.org/W3202686840	https://dergipark.org.tr/tr/download/article-file/1998953	English	null	FastTrafficAnalyzer: An Efficient Method for Intrusion Detection Systems to Analyze Network Traffic	Dicle Üniversitesi Mühendislik Fakültesi mühendislik dergisi/Dicle üniversitesi mühendislik fakültesi mühendislik dergisi.	2,021	cc-by	6,418	Research Article Research Article ABSTRACT ARTICLE INFO Article history: Received 9 May 2021 Received in revised form 2 September 2021 Accepted 7 September 2021 Available online 27 September 2021 Keywords: Network intrusion detection, machine learning, feature conversion, cyber security Doi: 10.24012/dumf.1001881 * Corresponding author Article history: Received 9 May 2021 Received in revised form 2 September 2021 Accepted 7 September 2021 Available online 27 September 2021 Keywords: Network intrusion detection systems are software or devices used to detect malignant attackers in modern internet networks. The success of these systems depends on the performance of the algorithm and method used to catch attacks and the time it takes for it. Due to the continuous internet traffic, these systems are expected to detect attacks in real time. In this study, using a proposed pre-processing, internet traffic data becomes more easily processable and traffic is classified by network analysis with machine learning techniques. In this way, the traffic analysis time was significantly shortened and a high level of success was achieved. The proposed model has been tested in the CSE-CIC-IDS2018 dataset and its advantaged verified. Experimental results i) 99.0% detection rate was achieved in the ExtraTree algorithm for binary classification, while a reduction of 82.96% was achieved in the processing time per sample; ii) For multiclass (15 class) detection, 98.5% detection rate was achieved with the Random Forest algorithm, while a 64.43% shortening was achieved in the processing time per sample. As a result, similar classification rate with the studies in the literature has been achieved with much shorter test time. Network intrusion detection, machine learning, feature conversion, cyber security Doi: 10.24012/dumf.1001881 * Corresponding author * Corresponding author DUJE (Dicle University Journal of Engineering) 12:4 (2021) Page 565-572 DUJE (Dicle University Journal of Engineering) 12:4 (2021) Page 565-572 FastTrafficAnalyzer: An Efficient Method for Intrusion Detection Systems to Analyze Network Traffic Recep Sinan ARSLAN1* 1 Kayseri University, Department of Computer Engineering, sinanarslanemail@gmail.com, Orcid No: 0000-0002-3028-0416 ARTICLE INFO ABSTRACT Related Works Intrusion detection systems (IDS) are an up-to-date subject that is studies both in the field of cyber security and as an academic research. For this reason, many studies have been carried both in this area in recent years. Since the proposed model was tested with the IDS2018 dataset, which is one of the best examples of current internet traffic, in this section, studies only conducted with the same dataset are given in this section. IDS systems are basically divided into two types such as signature-based intrusion detection systems (SIDS) and anomaly-based systems (AIDS). SIDS uses patterns in its database to detect an attack, previously prepared for a known attack type. When a pattern matches with the signatures in the databases, an alarm is generated. For detection, the host’s logs and command sets are analyzed. On the other hand, AIDS is an approach that can produce solutions to many constraints of signature-based systems. In this method, the normal behavior of a computer or network is extracted with the help of machine learning. When there is a deviation between an observed suspicious and the normal behavior pattern of the systems, it is interpreted as an attack. This technique is based on the fact that malicious attackers have different behavioral patterns from normal users [8]. In [17] used the SMOTE datasets balancing algorithm to oversampling the samples of minority classes and then evaluated the results with 6 different ML algorithms (RF, DT, KNN, Adaboost, GB and LDA). The experimental results indicated that the proposed model performed better that the existing models. In the study [18] on the same dataset, a GMM-based approach is proposed to solve the sample imbalance problem between classes to increase classification performance rate. Thus, by using SMOTE and GMM together, the number of samples in classes was balanced and the results were evaluated. Accordingly, a sample CNN model was designed and demonstrated that balancing the classes improves the learning process and contributes directly to performance enhancement. Machine learning approach to attack detection has been studies by researchers for a long time [9, 30]. Large volumes of network metadata and security-related data allow ML to be applied to detect intrusion into systems. Cisco Stealthwatch [10], Darktrace [11], Vectra Cognition [12] which are modern commercial IDSs use ML as part of their security strategy. Introduction Figure 1. The number of attacks in the last 5 years (CyberEdge, 2021). 79% 77% 78% 81% 86% 2 0 1 7 2 0 1 8 2 0 1 9 2 0 2 0 2 0 2 1 Figure 1. The number of attacks in the last 5 years (CyberEdge, 2021). 79% 77% 78% 81% 86% 2 0 1 7 2 0 1 8 2 0 1 9 2 0 2 0 2 0 2 1 With the emergence and development of network structures, almost all institutions, organizations and companies have established their own large-scale LANs. Large amount of network and sensitive information are stored in these networks [13]. In addition, new device types and network structures have emerged, such as 5G, cloud computing, and the Internet of Things (IoT). With the growth of these systems and networks, ensuring their cyber security is critical [14]. According to the 2021 Cyber Threat Defense Report (CyberEdge, 2021) [4] released by the CyberEdge team and compiled from data provided by 1200 IT security experts working in 19 different sectors in 17 different countries, attacks to companies connected to the internet network have been increased (Figure-1). Figure 1. The number of attacks in the last 5 years (CyberEdge, 2021). to the M-trends 2021 report, as of September 2020, the average waiting time (dwell time), which indicated that attackers have been working on a system or network for more than one year without detection, is 416 days, while the median waiting time between October 1, 2019 and September 30, 2020 is only it has decreased to 24 days [5]. These threats in recent years contain denial of service, malicious software attacks, spyware attacks, and continues threats. Especially, advanced persistent threats are dangerous and costly. Because they are carried out by experts in the field and are mostly supported by the governments. They are long-term attacks against governments or big companies for the purpose of data stealing and sabotage of the infrastructure. According First place of defense in ensuring cyber security in computer networks consist of a security infrastructure that includes Access control, privacy management, authentication and various security policies. However, despite all this defensive structure, it can be targeted by cyber attackers for reasons such as operations problems, other zero-day DUJE (Dicle University Journal of Engineering) 12:4 (2021) Page 565-572 exploits and various systemic vulnerabilities. Introduction As the second line of defense behind the firewall, IDS systems are tasked with accurately identifying malicious network attacks, providing real-time monitoring, dynamic protection measures, and creating strategies [6]. An IDS can be placed on all independent distributed hosts in a network, or they can be located on a central dedicated server and distribute its service over the network. Network intrusion detection systems (NIDS) designed to detect attacks from a single center for all systems on a network. NIDS monitor network information such as network traffic, protocols, flow metadata, activity logs and try to detect abnormal events. The main purpose of an IDS system is to detect unauthorized computer use and malicious network traffic that is not possible when using a traditional firewall. This system makes it highly protected against malicious acts that endanger the usability, integrity or privacy of computer systems [7]. (3) Repeated experimental results under similar conditions have been shown that this proposed approach provides similar classification results with existing current studies, while achieving these results 82.96% faster than them. Thus, a model that will contribute to the real-time operation requirements of IDS systems is proposed. The remainder of the article is organized as follows. In Section 2, the current studies on CSE-CIC-IDS2018 dataset are mentioned in detail. Originally designed pre-processing model are given in Section-3. Experimental results are presented in Section-4. The study has been concluded in Section-6. System design and proposed model A model has been designed in order to observe the effect of the proposed model on the classification results in IDS systems and the time required to obtain these results. The flow-chart of designed model is shown in Figure-2. The CIC-IDS 2018 dataset was used to evaluate the study. Since the experiments are carried out for both multiple and binary classification, the dataset has been subjected to a separate process to used two classification model. Two test processes were carried out, one with the original dataset and the other with the dataset subjected to proposed pre- processing. Increasing data flow in the network can cause attacks and intrusions. In order to prevent attacks, it is necessary to quickly classify large amounts of data with little cost. In [22], unsupervised feature selection method is proposed to avoid the cost of tagging network traffic. Thus, it is aimed to achieve better classification results while reducing computational complexity. Experimental results with IDS 2018 have confirmed that the proposed method is suitable for use in LAN and mobile ad-hoc networks and other networks with variable data density. Based on the insufficiency of signature-based approaches in the detection of cyber-attacks in today’s conditions, a deep learning-based approach has been proposed [23]. It is a working model only for DoS attacks. For this reason, tests were carried out on the records for DoS among the types of attacks found in the KDD and IDS2018 dataset. As a result of the tests performed with CNN and RNN-based models, it has been shown that the CNN model is more successful. In the two test processes carried out for comparison, feature selection, under-sampling, random separation for training and testing, standardization and classification models were kept exactly the same. The purpose of this is to observe the difference and contribution of the proposed model from traditional approaches. In the feature selection, 80 features found in the original dataset were reduced to 42 features. In this way, both the processing time was saved and the noisy features that negatively affected the training of the model were removed. In addition, features that cannot be mathematically processed such as IP address are eliminated in this section. In the next step, under-sampling was applied. System Architecture The evaluation of the proposed model in dataset was prepared in Python and tested with a structure supported by pandas [1], sklearn [2] and xgboost [3] modules. In the current application, 9 supervised machine learning classifiers were used. 6 of them are tree-based, decision tree, random forest, gradient-boosted trees (gradient boost), random decision trees (extra tree) and extreme gradient boosted trees(xgboost), 1 of them is neighborhood-based, k- nearest neighbor (KNN) and 2 of them are SVM based, linear SVC and logistic regression. Related Works In the study conducted on the use of artificial intelligence techniques in IDS systems, it is aimed to classify the botnet attack, which is a serious threat to the finance and banking sectors [19]. In tests performed in IDS 2018 dataset with a designed ANN, a low FP rate of 3% was achieved. ANN have been shown to produce good results in detecting botnet attacks. In the light of the challenges and requirements given above, a machine learning-based model is proposed for the classification of suspicious internet traffic. The model was evaluated with CSE-CIC-IDS2018 dataset with both multiple and binary classification scenarios. In [20], a design has been made in which deep neural network and deep autoencoders are included in the learning process together. A model that uses grid search and random search algorithms together is proposed to determine optimum model parameters. Deep autoencoder, autoencoder and stack autoencoder are used for feature extraction. As a result, more successful results have been obtained in multiclass classification than previous approaches. (1) A new pre-processing model is proposed, which provides significant gains in the training and testing time of the classification models. Thus, it was possible to achieve high classification results in a much shorter time compared to similar studies. (2) Tests have been carried out with 9 different ML techniques in order to observe the effect of this proposed new pre-processing step on both the reduction rate in the processing time and the classification results, and the results are given comparatively. An artificial neural network-based model has been proposed [21]. It is aimed at ensuring security in software defined networks, in which packet analysis is separated from the traffic routing processes. It has been shown in the 566 DUJE (Dicle University Journal of Engineering) 12:4 (2021) Page 565-572 tests that it is more beneficial to used much more complex and multilayer neural network to increase classification performance. It has been stated that LSTM-based models produce more successful results for real life usable system designs. tagged inputs is required. Thus, the system that emerged after completing the model training, can be deployed to detect new attacks. System design and proposed model The reason for this is that there are over 18M data in dataset and it is very difficult to transform and process such large amounts of data into vectors for KNN or SVM. For this reason, the number of data has been reduced with under-sampling and a subset that best represents the original dataset is obtained. Then, this data was separated randomly to be used in the training and testing as 70%-30%. Finally, after a standardization of the data, both feature vectors emerging are given to the classification model. The results were compared in terms of both the classification performance rate and the processing time. Apart from the ones given in this section, many survey articles covering all IDS related studies from past to present have been prepared [24-26], and they have been evaluated comparatively regardless of the data set. The NIDS problem CSE-CIC-IDS2018 Proposed Pre- Processing Feature Selection Feature Selection Under Sampling Under Sampling Training Set Test Set Standard Scaler Standard Scaler Classification Model Training Set Test Set Standard Scaler Standard Scaler Classification Model Before and After Pre-processing Comparison Result for Classification Rate and Time Complexity Figure 2. System overview of proposed model. F CSE-CIC-IDS2018 Proposed Pre- Processing Feature Selection Feature Selection Under Sampling Under Sampling Training Set Test Set Standard Scaler Standard Scaler Classification Model Training Set Test Set Standard Scaler Standard Scaler Classification Model Before and After Pre-processing Comparison Result for Classification Rate and Time Complexity Figure 2. System overview of proposed model. Proposed Pre- Processing Calculate sum of values for each feature in dataset Find average value for each feature All dataset feature values converted to 0 or 1 (not 12000215.45 or 12.25415252) L7_PROTO,IN_BYTES,IN_BYT ES 92.0, 375.12524, 21.00000, ... 5126.0 , 67190, 125421524.0, ... ..... L7_PROTO,IN_BYTES,IN_B YTES 1, 0, 1, ... 0 ,0, 1, ... ..... Figure 3. Details of proposed pre-processing model. Proposed Pre- Processing CSE-CIC-IDS2018 Proposed Pre- Processing Calculate sum of values for each feature in dataset Feature Selection Under Sampling Test Set Test Set Classification Model All dataset feature values converted to 0 or 1 (not 12000215.45 or 12.25415252) Model Before and After Pre-processing Comparison Result for Classification Rate and Time Complexity Figure 3. Details of proposed pre-processing model. Figure 2. System overview of proposed model. Regarding the proposed model, first of all, the average value in the whole dataset was calculated separately for each feature. According to this average, the value of each feature is changes to 1 if it is greater than the average value and 0 if it is less than the average value. Thus, the average value calculated on a feature basis is used as a threshold. Thus, instead of working with decimal numbers of internet traffic, it is possible to work with feature vectors with values of 0 and 1. As a result, it is aimed to produce feature vectors that will be easier and faster to work with in both training and testing processes of classification algorithms. Experimental Design and Results In this study, it is tried to show that contribution of new pre- processing method in the detection of attacks in IDS systems. Experimental environment, dataset details and test results, which are performed in multiclass and binary, are given in this section and the results are discussed with similar studies. Experimental design The test process was carried out on Intel Xeon a 3.5 GHz 8 core processor with 32 GB Ram and Ubuntu 18.04 operating system. It was developed in Python programming language by using sklearn library and tensorflow for machine learning. Imbalanced-learning library is used for under-sampling. As the data is changes with the approach shown in Figure- 3, it is necessary to observe whether the emerging new feature vector eliminates meaningful data. If significant value is lost, classification performance will decrease. In this case, even if you have a faster classification and testing process, performance will be lost. In order to observe this situation, the experiments, the details of which are given in the next section, have been carried out. The NIDS problem When the dataset is examined, it is seen that both the complete parts and the fraction parts of the values for each feature increases up to 10 digits (12.00215875 or 8214251652.0012). Working with high-digit numbers for 40 different properties can cause problems, especially in terms of processing time. This required processing time is quite long for most real-time IDS systems, and it is not directly possible to use the developed models in real life. In order to find a solution to this problem, all data were evaluated according to the approach shown in Figure-3 and all values were changes to 0 or 1. The main purpose of NIDS is to follow the system operating process and behavior. In this way, they determine when the attacks occur and create warning messages when necessary. Thus, it is to ensure that field experts can mitigate the bad consequences that will arise. As an input to an IDS system, traffic statistics in a computer network, packet headers and information to be obtained from packet content and process behavior, system call traces, application logs, file system change information coming from host computers can be given. And its output can be like a tagging for each input, attack scoring or categorization. Tags can be both benign or malicious, as well as multi-class attacks such as brute-force, infiltration, SQL injection. Using machine learning algorithms, this problem can be modelled as a classification or anomaly-based detection problem. In the training of the model to be prepared for anomaly detection, a data set consisting of information obtained from the network and hosts will be sufficient. On the other hand, for classification, 567 DUJE (Dicle University Journal of Engineering) 12:4 (2021) Page 565-572 CSE-CIC-IDS2018 Proposed Pre- Processing Feature Selection Feature Selection Under Sampling Under Sampling Training Set Test Set Standard Scaler Standard Scaler Classification Model Training Set Test Set Standard Scaler Standard Scaler Classification Model Before and After Pre-processing Comparison Result for Classification Rate and Time Complexity Figure 2. System overview of proposed model. Proposed Pre- Processing Calculate sum of values for each feature in dataset Find average value for each feature All dataset feature values converted to 0 or 1 (not 12000215.45 or 12.25415252) L7_PROTO,IN_BYTES,IN_BYT ES 92.0, 375.12524, 21.00000, ... 5126.0 , 67190, 125421524.0, ... ..... L7_PROTO,IN_BYTES,IN_B YTES 1, 0, 1, ... 0 ,0, 1, ... ..... Figure 3. Details of proposed pre-processing model. CSE-CIC-IDS2018 dataset CSE-CIC-IDS2018 was prepared by The Communications Security Establishment (CSE) and the Canadian Institute for Cybersecurity (CIC) for use in attack detection and prevention. It is a dataset representing the traffic flow and attack types of real life network structure and is distributed as open source [15]. It consists of 80 features, including tags. Statistical information about internet traffic constitutes most of the features and obtained with the CICFlowMeter and outputted and based on the flow of network traffic. The details of the scenario and the process of extracting traffic characteristics are explained in [15,16]. Almost all of the features in the dataset consist of continuous features such as dst_ports, protocols, 568 DUJE (Dicle University Journal of Engineering) 12:4 (2021) Page 565-572 timestamps, labels. It includes 14 types of attacks in 6 different scenarios including Brute-Force, Botnet, Web attacks, DDos and infiltration. The total number of records is 18893270. Of these, 16635579 records (%83.07%) represent benign traffic, while the remainder represent malicious. In this study 9.73% is used for training and 4.17% for testing. The amount of data to be used in the training and testing phase of the proposed model is shown in Table-1. It is aimed to prove the objectivity of the proposed model and that is a viable method in real systems by performing the tests with CSE-CIC dataset which is quite large, contains different attack types and internet traffic. classification problems. It shows the classification distribution of a model and helps determine the situation in which one class is constantly classified as incorrectly in another class. For example, a DDos attack can be classified as a continuous exfiltration. The researchers can then examine the possible causes of the problem. These situations regarding models in many different metrics cannot be easily observed. Using this table, important metrics such as acc, precision, recall and f1-measure are calculated. Thus, it is possible to evaluate the classification performance from different angles. For multi class problems, these metrics should be calculated separately on a class basis. If it desired to obtain a general value regarding the classification performance, it can be done by using following methods. Table 1. CSE-CIC-2018 data distribution (before and after random under sampling with majority). Table 1. CSE-CIC-2018 data distribution (before and after random under sampling with majority). random under sampling with majority). CSE-CIC-IDS2018 dataset Category Attack Types Size - % Train – Test (%70-%30) (After Under Sampling) Benign 166355 79 88.050 442 190 0.028% DDoS HOIC 108085 8 5.721 756601 324257 47.852% LOIC-http 307300 1.627 215110 92190 13.605% LOIC-UDP 2112 0.011 1478 634 0.094% Botnet Bot 143097 0.757 100168 42929 6.335% Brute Force FTP 25933 0.137 18153 7780 1.148% SSH 94979 0.503 66485 28494 4.205% Infilteration Infilteration 116361 0.616 81453 34908 5.152% Dos Hulk 432648 2.290 302854 129794 19.154% SlowHTTPT est 14116 0.075 9881 4235 0.625% GoldenEye 27723 0.147 19406 8317 1.227% Slowloris 9512 0.050 6658 2854 0.421% Web Attacks Web 2143 0.011 1500 643 0.095% XSS 927 0.005 649 278 0.041% SQL Injection 432 0.002 302 130 0.019% Total 188932 70 100 1581141 677632 100% • Micro average: Calculates the final value using the sum of the classification metrics for each class (TP = TPClass1 + TPClass2 + …) • Macro average: Metrics per class are calculated and averaged. It refers to the process of adding them all and dividing them by the sum of the number of classes. • Weighted macro average: It is a similar method to calculate macro average, but when obtaining the overall metric value of the model, the number of samples in the classes is also taken into account in the average calculation. All classes are not included in the overall average with equal weight. Multi class classification IDS 2018 dataset contains a total of 15 classes, 14 of which are malicious and 1 benign. The first stage of the tests of the model proposed in this study was conducted for the classification of these data. The test results are given in Table-3 in detail. Table 3. Multiclass classification rate (before and after proposed pre-processing model) Table 3. Multiclass classification rate (before and after proposed pre-processing model) Evaluation metrics Various measurement metrics and tools are used to evaluate the results in problems where ML models are used. In this way, it is possible to evaluate the performance and deficiencies of the IDS systems. Explanations of the metrics are given in this section. proposed pre processing model) MULTICLASS (15 CLASS) CLASSIFICATION RATE COMPARISON Before Proposed Pre- processing After Proposed Pre-processing Acc Prec. Recall F score Acc Prec. Recall F score Logistic Reg (LR) 0.949 0.937 0.949 0.940 0.989 0.989 0.989 0.989 RF 0.998 0.998 0.998 0.998 0.990 0.990 0.990 0.989 DT 0.998 0.998 0.998 0.998 0.990 0.990 0.990 0.989 LDA 0.965 0.972 0.965 0.967 0.959 0.981 0.959 0.968 ExtraTree 0.998 0.998 0.998 0.998 0.990 0.990 0.990 0.989 XGB 0.996 0.996 0.995 0.997 0.955 0.987 0.955 0.965 GB 0.997 0.997 0.997 0.997 0.955 0.987 0.955 0.965 SVC 0.950 0.947 0.952 0.940 0.990 0.990 0.990 0.989 KNN 0.949 0.937 0.949 0.940 0.989 0.989 0.990 0.990 MULTICLASS (15 CLASS) CLASSIFICATION RATE COMPARISON Table 2. Confusion matrix of binary classification Predicted Class Attack Benign Actual Class Attack TP FN Benign FP TN TP = True positives, TN = True Negatives, FP = False positives, FN = False Negatives Table 2. Confusion matrix of binary classification TP = True positives, TN = True Negatives, FP = False positives, FN = False Negatives The confusion matrix is a classification table based on actual class (exact reference) in the rows and the class projected on it in the columns as shown in Table-2. This table can be produced in both multiclass and binary 569 DUJE (Dicle University Journal of Engineering) 12:4 (2021) Page 565-572 Table 5. Binay classification rate (before and after proposed pre-processing model) BINARY (BENIGN- MALICIOUS) CLASSIFICATION RATE COMPARISON Before Proposed Pre- processing (original data) After Proposed Pre- processing (original data) Acc Prec. Recall F- score Acc Prec. Recall F score Logistic Reg (LR) 0.877 0.917 0.829 0.871 0.963 0.983 0.94 0.962 RF 0.975 0.978 0.971 0.975 0.975 0.996 0.95 0.974 DT 0.971 0.970 0.972 0.971 0.975 0.996 0.95 0.974 GNB 0.895 0.965 0.820 0.886 0.905 0.946 0.86 0.901 LDA 0.942 0.960 0.922 0.940 0.946 0.968 0.92 0.944 AdaB 0.976 0.994 0.958 0.976 0.957 0.973 0.94 0.956 ExtraTree 0.974 0.977 0.971 0.974 0.985 0.996 0.96 0.984 GB 0.980 0.999 0.961 0.980 0.968 0.992 0.94 0.967 XGB 0.975 0.991 0.948 0.977 0.975 0.996 0.95 0.974 Table 5. Evaluation metrics Considering that IDS systems monitor internet traffic in real time and there is an average of 30 thousand or 50 thousand packages per second, the test time of each sample is very important. Thanks to the model proposed in this study, the test time per sample is considerably shortened, and this makes it more possible to use in real- time analysis tools. It this study, it is aimed to make the model with very high classification rate with a shorter processing time. The results support this goal. Figure 4. Binary classification time comparison (before and after pre-processing). If a classification process is divided into steps as shown in Figure-4, the processing times were compared for each processing step. As a result, it is expected that the proposed model needs shorter processing times in all steps. Accordingly, as can be seen in the graph, after the proposed models, considerably shorter processing times were needed than traditional models. Evaluation metrics Binay classification rate (before and after proposed pre-processing model) With the datasets of 15 classes, the tests were tested with 9 different machine learning techniques. When the tests performed with original dataset, a very successful classification performance of 99%. However, high classification rates were obtained in similar studies on this subject. Therefore, the purpose of conducting these tests is to observe the effect of the proposed model on the classification performance. When the results were evaluated, although there was a loss of data during proposed pre-processing, the results were found to be better for some classifiers and similar values for some others. The required processing times in the experiments are shown separately for each process step in Tablo-4. Table 4. Multiclass classification time comparison (before and after pre-processing) MULTI CLASS (15 CLASS) TIME COMPARISON (seconds) Extra Tree Algorithm Before Proposed Pre-processing (original data) (sec.) After Proposed Pre- processing (sec.) Loading Data 699.6770 63.0300 Label Encoding 5.4992 0.0035 Under Sampling 743.1130 148.8480 Data Split for Training and Test 9.4730 0.7700 Training time 422.7824 330.1324 Testing time 27.2800 16.5900 Testing time for each sample 0.00004 0.00002 MULTI CLASS (15 CLASS) TIME COMPARISON (seconds) In this study, a new pre-processing method is proposed to shorten the processing time for classification. It is expected that the shortening of the processing time will not reduce the classification performance. For this purpose, the tests were repeated in the same environments. When the test results related to binary classification are examined, it is seen that generally successful results are obtained as in multi class classification. It has been observed that the pre- processing does not cause any loss in performance. The advantage provided in terms of processing time in the classification shown graphically in Figure-4. Figure 4. Binary classification time comparison (before and after pre-processing). 0,0000 500,0000 1.000,0000 1.500,0000 2.000,0000 2.500,0000Loading DataLabel EncodingUnder SamplingData SplitingTraining TimeTesting Time After Proposed Pre-processing Before Proposed Pre-processing (original data) 0,0000 500,0000 1.000,0000 1.500,0000 2.000,0000 2.500,0000Loading DataLabel EncodingUnder SamplingData SplitingTraining TimeTesting Time In Table-4, separate time periods are calculated for the process steps that may be needed in a sample machine learning model design. A much faster process is carried out thanks to the proposed pre-processing model in all steps such as reading data, label encoding, under sampling, and classifying data. In addition, more importantly, an average of 64.43% shortening is achieved in the test time per sample. Binary classification In some IDS architectures, traffic differs only as malicious or benign. Malicious traffic is not expected to be categorized separately. In order to simulate this problem, the tests were repeated as binary classification. The obtained results are shown in Table-5. In general, the results show that the direct equivalents of the values obtained from the flow of traffic in IDS systems do not have any meaning in the classification models, there are sharp distinctions between the classes in terms of traffic 570 DUJE (Dicle University Journal of Engineering) 12:4 (2021) Page 565-572 characteristics, therefore, it is understood that using the value in a simplified form makes a significant contribution to the processing time. and application details are explained. Tests were carried out with 9 different machine learning algorithms in the type of supervised. Necessary optimization processes were applied in the parameter selection of algorithms. In general, it was determined that tree-based classifiers performed best. Classifiers based on decision trees achieved a certain success value in all classes. For this reason, they have become practically applicable. The most increases were provided for the ExtraTree and SVC classifiers. Conflict of interest statement There is no need to obtain permission from the ethics committee for the article prepared. There is no conflict of interest with any person / institution in the article prepared. Comparison with similar works and discussion The classification results obtained in the studies with the same dataset used in this work are shown comparatively in Table-6. In the test results for multiclass classification were at the same level with other studies. Classification success was achieved with 99%. In todays, internet traffic is increasing and diversifying due to the increase in internet usage. For this reason, there is a need for tools/analyzers that perform much faster and accurate traffic classification and this need is increasing day by day. With the proposed model in this study, it has been shown that complex traffic can be made much simpler and used in classification. In the future studies, performing analyzes using fewer features will be beneficial in terms of speed. In addition, using artificial neural network structures will allow successful results. It is possible to get more accurate results with more complex learning structures. Finally, unsupervised learning methods can be used that can adapt itself to new attack types (zero day attacks). So that it will always be possible to create up-to-date models. In complex and heavy network traffic flows, it is very important to be fast as well as accuracy in classification of it. In the tests performed with proposed model, similar classification rated were achieved with other studies using mush shorter processing time. Thus, the classification was possible both quickly and with high accuracy. Table 6. Comparison with similar works using CSE CIC IDS 2018 dataset Table 6. Comparison with similar works using CSE CIC IDS 2018 dataset Paper - Year Dataset Model Details Results Yu et al. [27] - 2021 CIC- IDS 2018, CIC- IDS 2017 Hierarchical packet byte- based CNN (PBCNN) Acc: 0.999 Precision: 0.982 Recall: 0.983 F1-score: 0.983 Vinayakumar et al. [28]- 2019 CIC- IDS 2018, CIC- IDS 2017 ANN 5 hidden layer (1024, 768, 512, 256, 128 node), Relu Activation Function Acc: 0.962 Precision: 0.962 Recall: 0.965 F1-score: 0.957 Ferrag et al. [29]-2020 CIC- IDS 2018 DNN, RBM, DBFN, CNN, DBM, Deep autoencoder Acc: 0.9728 Proposed model with original dataset CIC- IDS 2018 RF, DT, ExtraTree Acc: 0.998 Precision: 0.998 Recall: 0.998 Proposed model with pre-processed data CIC- IDS 2018 RF, DT, ExtraTree Acc: 0.990 Precision: 0.990 Recall: 0.990 F1-score: 0.989 Acknowledgement We special thank to Iman Sharafaldin, Arash Habibi Lashkari, and Ali A. Ghorbani for providing CSE-CIC- IDS2018 Dataset publicly. [6] Liao H-J, Richard Lin C-H, Lin Y-C, Tung K. “Intrusion detection system: A comprehensive review”, Journal of Network and Computer Applications, 36(1), 16-24, 2013. References [1] McKinney Wes, “Data structures for statistical computing in Python”, Proceedings of the 9th python in science conference, 1-6, 2010. [2] Pedregosa F, Varoquaux G., Gramfort A., Michel V., Thirion B., Grisel O, et al., “Scikit-learn: machine learning in Python”, Journal of Machine Learning Research 12, 2825-2830, 2011. [3] Chen T., Guestrin C., “Xgboost: a scalable tree boosting system”, Proceedings of the 22nd ACM SIGKDD International conference on Knowledge Discovery and Data Mining, 785-794, August, 2016. [4] CyberEdge, 2021. 2021 Cyberthreat Defense Report. https://cyber-edge.com/cdr/ [5] FireEye, 2021. M-trends 2021Cyber Security Report. 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FireEye, https://www.fireeye.com/blog/threat- research/2021/04/m-trends-2021-a-view-from-the-front- lines.html Conclusion and Future Work Conclusion and Future Work [6] Liao H-J, Richard Lin C-H, Lin Y-C, Tung K. “Intrusion detection system: A comprehensive review”, Journal of Network and Computer Applications, 36(1), 16-24, 2013. In this paper, detailed experimental results of CSE-CIC- IDS2018, which is a modern dataset for the application of ML to network intrusion systems, are shown. Model design 571 DUJE (Dicle University Journal of Engineering) 12:4 (2021) Page 565-572 IDS2018 using cloud computing”, ICT Express, 5, 211-214, 2019. IDS2018 using cloud computing”, ICT Express, 5, 211-214, 2019. [7] Sunanda Gamage, Jagath Samarabandu, “Deep learning methods in network intrusion detection: a survey and an objective comparison”, Journal of Network and Computer Applications, 169, 1-21, 2020. [20] Yesi Novaria Kunang, Siti Nurmaini, Deris Stiawan, Bhakti Yudho Suprapto, “Attack clasification of an instrusion detection system using deep learning and hyperparameter optimization”, Journal of Information Security and Applications, 58, 1-15, 2021. [8] Ansam Khraisat, Ammar Alazab, “A critical review of intrusion detection systems in the internet of things: techniques, deployment strategy, validation strategy, attacks, public datasets and challenges”, Cybersecurity, 4(18), 1-27, 2021. [21] S. S. Volkov, I I Kurochkin, “Network attacks classification using Long Short-term memory based neural networks in Software Defined Networks”, 9th International Young Scientist Conference on Computational Science, 178, 394-403, 2020. [9] C Kalimuthan, J. Arokia Renjit, “Review on instrusion detection using feature selection with machine learning techniques”, Materials Todat: Proceddings, 33(7), 3794- 3802, 2020. [22] Mahendra Prasad, Sachin Tripathi, Keshav Dahal, “Unsupervised feature selection and cluster center initialization based arbitrary shaped clusters for intrusion detection”, Computers & Security, 99, 1-19, 2020. [10] Cisco, 2021. Cisco Security Analytics Whitepaper. https://www.cisco.com/c/dam/en/us/products/collateral/sec urity/stealthwatch/sw-siem-optimization-wp.pdf. [11] Darktrace, 2021, Preparing for AI-enabled Cyberattacks, Whitepaper, https://www.darktrace.com/en/mit-preparing-for- cyberattacks/. [23] Jiyeon Kim, Jiwon Kim, Hyunjung Kim, Minsun Shim, Eunjung Choi, “CNN-based network intrusion detection against Denial-of-Service Attacks”, Mdpi electronics, 1-21, 2020. [12] Vectra Cognito, 2020. How to Augment Security Operations Center with Artificial Intellignece, Whitepaper, https://content.vectra.ai/rs/748-MCE- 447/images/WhitePaper_AugmentSOCwithAI.pdf. [24] Mokhtar Mohammadi, Tarik A. Rashid, Sarkhel H.Taher Karim, Adil Hussain Mohammed Aldalwie, Quan Thanh Tho, Moazam Bidaki, Amir Masoud Rahmani, Mehdi Hosseinzadeh, “A comprehensive survey and taxonomy of the SVM-based intrusion detection systems”, Journal of Network and Computer Applications,178, 1-23, 2021. 447/images/WhitePaper_AugmentSOCwithAI.pdf. [13] Chencheng MA, XueHui Du, Lifeng Cao, “Analysis of Multi-Types of Flow Features Based on Hybrid Neural Network for Improving Network Anomaly Detection”, IEEE Access, 7, 1-18, 2019. [25] T. Daniya, K. Suresh Kumar, B. Conclusion and Future Work Santhosh Kumar, Chandra Sekhar Kolli, “A survey on anomaly-based intrusion detection system”, Materials Today: Proceedings, 1-4, 2021. [14] Lan Liu, PengCheng Wang, Jun Lin, LangZhou Liu, “Intrusion Detection of Imbalanced Network Traffic based on Machine Learning and Deep Learning”, IEEE Access, 9, 1-14, 2021. [26] Arwa Aldweesh, Abdelouahid Derhab, Ahmed Z. Emam, “Deep learning approaches for anomaly-based intrusion detection systems: A survey, taxonomy, and open issues”, Knowledge-Based Systems, 189,1-19, 2020. [15] University of New Brunswick (UNB). A realistic cyber defense dataset (CSE-CIC-IDS2018), https://www.unb.ca/cic/datasets/ids-2018.html. [27] Lian Yu, Jingtao Dong, Lihao Chen, Mengyuan Li, Bingfeng Xu, Zhao Li, Lin Qiao, Lijun Liu, Bei Zhao, Chen Zhang, “PBCNN: Packet Bytes-based Convolutional Neural Network for Network Intrusion Detection”, Computer Networks, 1-24, 2021. [16] Iman Sharafaldin, Arash Habibi Lashkari, Ali A. Ghorbani, “Toward Generating a New Intrusion Detection Dataset and Intrusion Traffic Characterization”, 4th International Conference on Information Systems Security and Privacy, 1-9, 2018. [28] R. Vinayakumar, M. Alazab, K. P. Soman, P. Poornachandran, A. Al-Nemrat and S. Venkatraman, "Deep Learning Approach for Intelligent Intrusion Detection System," in IEEE Access, vol. 7, pp. 41525-41550, 2019, doi: 10.1109/ACCESS.2019.2895334. [17] G. Karatas , O. Demir , O.K. Sahingoz , Increasing the performance of machine learn- ing-based IDSs on an imbalanced and up-to-date dataset, IEEE Access 8 (2020) 32150–32162 . [18] Hongpro Zhang, Lulu Huang, Chase Q. Wu, Zhanbo Li, “An effective convolutional neural network based on SMOTE and Gaussian mixture model for intrusion detection in imbalanced dataset”, Computer Networks, 177, 1-10, 2020. [29] Mohamed Amine Ferrag, Leandros Maglaras, Sotiris Moschoyiannis, Helge Janicke, “Deep learning for cyber security intrusion detection: Approaches, datasets, and comparative study”, Journal of Information Security and Applications, 50, 1-19, 2020. [30] Mesut Uğurlu, İbrahim Alper Doğru, Recep Sinan ARSLAN, “A new classification method for encrypted internet traffic using machine learning”, Turkish Journal of Electrical Engineering and Computer Sciences, Accepted. 2021 [19] V. Kanimozhi, T. Prem Jabob, “Artificial Intelligence based Network Intrusion Detection with hyper-parameter optimization tuning on the realistic cyber dataset CSE-CIC- 572
https://openalex.org/W4386746402	https://www.qeios.com/read/WXB7QK/pdf	English	null	Review of: "Digital Persona: Reflection on the Power of Generative AI for Customer Profiling in Social Media Marketing"	null	2,023	cc-by	118	Qeios, CC-BY 4.0 · Review, September 14, 2023 Review of: "Digital Persona: Reflection on the Power of Generative AI for Customer Profiling in Social Media Marketing" Asad Butt1 1 University of Central Punjab Potential competing interests: No potential competing interests to declare. It looked more of an article than a research paper. Qeios ID: WXB7QK · https://doi.org/10.32388/WXB7QK It looked more of an article than a research paper. Data analysis are weak. Need more authentication of references. Elaborate methodology further. How it was collected and from whom. Qualitative survey needs more elaboration. Segments and target markets and samplze size require more information. More references can be added. More tabular form and figures can be added to the article. 1/1
https://openalex.org/W3200515920	https://knepublishing.com/index.php/espoch/article/download/9659/16086	es		Probiotics and Reduction of the Inflammatory Response for Prevention of Preeclampsia	ESPOCH congresses	2,021	cc-by	4,637	ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M. Volume 1, Issue no. 6, DOI 10.18502/espoch.v1i6.9659 Production and Hosting by Knowledge E Conference Paper Probiotics and Reduction of the Inflammatory Response for Prevention of Preeclampsia Probióticos y Reducción de la Respuesta Inflamatoria para la Prevención de la Preeclampsia Iván Enrique Naranjo Logroño1 , Anthony Alfonso Naranjo Coronel2 , Cynthia Johana Navarro Rivadeneira1 , and Erika Alejandra Romero Minaya1,3 1 XVIII International Seminar on Health, Food and Human Nutrition Corresponding Author: Iván Enrique Naranjo Logroño naranjometropolitana@hotmail.com Published: 9 September 2021 Production and Hosting by Knowledge E Iván Enrique Naranjo Logroño et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Carrera de Medicina, Facultad de Salud Pública, ESPOCH, Riobamba, Chimborazo, Ecuador Médico Cirujano, COLPOMED Centro, Hospital del Día, Riobamba, Chimborazo, Ecuador 3 Hospital Provincial General Docente de Riobamba, Riobamba, Ecuador 2 Abstract Introduction: In the field of medicine and obstetrics, the most common hypertensive disorders are preeclampsia and it constitutes a great question that has not yet been completely resolved. Objective: To identify the effectiveness of the use of probiotics and the reduction of the inflammatory response in the prevention of pre-eclampsia. Method: This search took place in large databases both globally and locally, being Elsevier, La Reference, Scielo, Pubmed, Google Scholar, Redalyc, Scopus, among others, where probiotic and pre-eclampsia terms were found. Results: 37 articles were found which were preselected, after correct appreciation 32 articles were selected and 5 articles were discarded, the selected ones met the inclusion and exclusion criteria, therefore they are related to the topic and objective of the present study review. Discussion: The epithelial cells of the intestine produce TNF-α, once mixtures of different strains of probiotics are made, causing the immune response to be generated, especially if consumed before pregnancy or in early pregnancy and continued throughout of the same. the administration is safe and has a protective role in pre-eclampsia, gestational diabetes, vaginal infections, maternal, infant, and subsequent weight gain. Conclusion: The preventive effect occurs as long as probiotics are administered mainly in the second and third trimesters of pregnancy, although the consumption of probiotics before these results in a much greater benefit, and is even more potent if probiotic mixtures are made. Keywords: probiotics, preeclampsia, hypertensive disorder. Resumen Introducción: En el campo de la medicina y obstetricia, los trastornos hipertensivos más comunes son preeclampsia y compone una gran interrogante que aún no está resuelta completamente. Objetivo: Identificar la efectividad del uso de probióticos y la reducción de la respuesta inflamatoria en la prevención de la preeclampsia. Método: Esta búsqueda tuvo lugar en grandes bases de datos tanto a nivel mundial como local, siendo Elsevier, La Referencia, Scielo, Pubmed, Google Académico, Redalyc, Scopus, entre otras, donde se encontraron términos de probióticos y preeclampsia. Resultados: Se encontró 37 artículos los cuales fueron preseleccionados, luego de la correcta apreciación se seleccionaron 32 artículos y se descartaron 5 artículos, los seleccionados cumplían con los criterios inclusión y de exclusión por lo tanto van en relación con el tema y objetivo del presente estudio de revisión. Discusión: Las células epiteliales del intestino producen TNF-α, una vez que se realicen mezclas de How to cite this article: Logroño IEN, Coronel AAN, Rivadeneira CJN, Minaya EAR (2021). Probiotics and Reduction of the Inflammatory Response for Prevention of Preeclampsia. ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M., 1(6), 230–239. DOI 10.18502/espoch.v1i6.9659 ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M. distintas cepas de probióticos, haciendo que se genere la respuesta inmunitaria, en especial si se consume antes del embarazo o en el embarazo temprano y continua a lo largo del mismo. La administración es segura y tiene un papel protector en la preeclampsia, diabetes gestacional, infecciones vaginales, aumento de peso materno, infantil, y posteriores. Conclusión: El efecto preventivo se da siempre y cuando se administre probióticos principalmente en el segundo y tercer trimestre de embarazo, aunque el consumo de probióticos con anterioridad a estos resulta de mucho mayor beneficio, y se potencia aún más si se realizan mezclas de probióticos. Palabras Clave: probióticos, preeclampsia, trastorno hipertensivo. 1. Introducción El embarazo es un estado que implica muchas adaptaciones fisiológicas que permitirán albergar al nuevo ser en formación, dicha etapa no está exenta de complicaciones. Reportes de la OMS, en todo el mundo, a diario mueren aproximadamente 830 mujeres embarazadas por complicaciones relacionadas con el mismo embarazo o con el parto [1]. Los trastornos hipertensivos constituyen uno de los problemas más intrigantes, y aún no resuelto en el campo de la medicina y obstetricia, de acuerdo con las estadísticas disponibles hoy en día se calcula que de un 2 a un 8% los embarazos a nivel mundial son complicados y con ello más de un 20% de muertes maternas en todo el mundo se dan debido a la preeclampsia [2, 3]. A pesar de que la preeclampsia representa un riesgo potencial de muerte para la madre, también repercute en el desarrollo normal del feto, puesto que se disminuye el suministro de sangre, mecanismo por el cual el feto recibe nutrientes, con lo cual no existirá este último, a esta condición se le conoce como restricción del crecimiento intrauterino (RCIU). Alrededor del mundo se estima que cada año nacen 30 millones de bebés con RCIU, de ellos el 15% (4,5 millones) son repercusión de preeclampsia [4]. Este trastorno es más frecuente en aquellos países que están aún en vías de desarrollo, Ecuador es uno de ellos y en el 2010 la preeclampsia-eclampsia fue una de las principales causas de morbimortalidad materna y perinatal [5]. La fisiopatología de la preeclampsia se da por la implantación placentaria anómala lo que conlleva a un estado de hipoxia, y por ende se produce la respuesta inflamatoria materna, sin embargo esta causa aún no está del todo clara, en estudios ya realizados aluden que poseen características inmunológicas, puesto que en el sistema inmune existe una gran variabilidad de genes, estos tienen la función de codificar las moléculas que forman el complejo de histocompatibilidad por lo tanto al sufrir esta modificación los receptores de las células llamadas asesinas naturales, modifican la placentación generando cambios en la misma [6]. Además de esto el incremento de costos de un parto con riesgos maternos neonatales son mayores, que aquellos partos que suceden si eventualidades. El impacto de los trastornos hipertensivos en el embarazo es muy grave puesto que afecta social y económicamente en el desarrollo de un país [7]. El tratamiento de este trastorno en DOI 10.18502/espoch.v1i6.9659 Page 231 ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M. la actualidad es el nacimiento del bebe y la expulsión de la placenta, puesto que la medicación utilizada solo nos permite aumentar las posibilidades de supervivencia del recién nacido y con el uso de anticonvulsivantes para la madre, se pretende evitar efectos adversos, por lo tanto el diagnostico oportuno y la prevención son los mejores pilares, por lo cual surge la pregunta ¿tienen los probióticos mecanismos para reducir la respuesta inflamatoria y con ello evitar la preeclampsia? [8]. Con el presente estudio se pretende: analizar las diversas investigaciones sobre el efecto de los probióticos en la prevención de la preeclampsia y de esa manera contribuir con la literatura médica de Latinoamérica y del mundo que se enfocan en la alimentación materna como base para que la mujer durante la etapa de embarazo tenga un sistema inmunológico sano; el consumo de probióticos, que son microorganismos productores de ácido láctico con lo cual disminuyen la permeabilidad intestinal a bacterias patógenas, incrementa la producción de los linfocitos T helper, y de inmunoglobulina A secretora, así influyen en las respuestas inmunitarias, mediadas por el tejido linfoide asociado al intestino [9]. 2. Metodología 2.1. Diseño: En el siguiente artículo de revisión, los criterios meta analíticos no forman parte del mismo Estrategia de búsqueda: Para tomar en cuenta los criterios de inclusión, las investigaciones que enfocaron la atención en probióticos y reducción de la respuesta inflamatoria para la prevención de la preeclampsia han sido aceptadas; los estudios publicados desde el año 2005, han servido de base; posteriormente, se incluyeron solo artículos originales. Al momento de realizar la exclusión se toma en cuenta los artículos que contengan más variables fenomenológicas o búsquedas sin resultados en su contenido y artículos sin texto selecto. Cuando se realizó la preselección, los artículos, fueron sometidos a un segundo proceso de indagación, sobresaliendo los que contenían información acerca de: probióticos, respuesta inflamatoria, prevención de preeclampsia, logrando conseguir artículos originales de relevancia, que, en su gran mayoría fueron experimentales. Esta búsqueda tuvo lugar en grandes bases de datos tanto a nivel mundial como local, siendo Elsevier, La Referencia, Scielo, Pubmed, Google Académico, Redalyc, Scopus, entre otras, donde se encontraron términos de probióticos y preeclampsia. Entre diciembre 2019–febrero 2020, se investigó toda la bibliografía seleccionada. Se utilizó palabras clave en todas las bases ya mencionadas, siendo estas: Probióticos, preeclampsia, además de utilizar términos boleanos ‘and’ y ‘or’, descriptores en singular o plural e incluso buscando términos en inglés como español. DOI 10.18502/espoch.v1i6.9659 Page 232 ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M. 3. Resultados Selección de estudios: Al disponer de distintas bases de datos se emplearon diversos métodos para realizar la búsqueda. Como se recopiló 37 artículos, y se eliminó 5, se trabajó con los 32 artículos con la mejor información y el cumplimiento de los criterios, pues los descartados no se ajustaban del todo con el objetivo de la revisión. 4. Discusión Probióticos: Son microorganismos vivos que están destinados a mantener o mejorar el microbiota normal, a diferencia de los prebióticos, que son alimentos que sirven de nutrientes para la microbiota. Se encuentra principalmente en alimentos como el yogurt y el chucrut (col blanca fermentada con vinagre) [10]. Preeclampsia: Es una complicación asociada al embarazo y relativamente común, caracteriza por la presencia de cierto signos y síntomas como la hipertensión arterial, proteínas en la orina y daño a órganos, siendo más frecuente el hígado y riñones. Ocurre a partir de la semana 20 de embarazo tras tener una presión arterial hasta ese entonces, normal. Existen diferentes factores de riesgo para que aparezca, como, ser madre primeriza, embarazo previo cursado con esta patología, embarazos múltiples, edad materna menor de 20 o mayor de 40 años, obesidad o antecedentes de enfermedad renal, entre otros. Además, se clasifica en leve y severa, dependiendo la sintomatología [11]. La etiología de la preeclampsia se cree que se debe a un fallo en la placentación. Al inicio del desarrollo embrionario la nutrición está dada por secreciones de las glándulas endometriales, conocido como nutrición histiotrófica (nutrición de sustratos disponibles en útero), posteriormente el trofoblasto invade los tejidos maternos y se forman lagunas que después se unen para crear el espacio intervelloso, la apertura de las arterias espirales en dicho espacio permite el cambio de nutrición de tipo histiotrófica a hematotrófica. Cuando el proceso de placentación no es el óptimo, se produce una oxigenación inadecuada, lo que conlleva a un estado de estrés oxidativo causado por una deficiente perfusión del trofoblasto. Inmunológicamente en el embarazo normal el perfil Th (T-helper) 2 es dominante, mientras que en la preeclampsia domina el Th1. Cuando se activa el perfil Th1 (productores de Inter Leucina-2, Factor de necrosis tumoral interferón), las citoquinas activan las células T citotóxicas y las células NK, ampliando aún más su efecto citotóxico, causando un estado inflamatorio generalizado [12]. Al realizar cierto estudio se pudo observar que, si se realizan mezclas de cepas de probióticos, estas pueden inducir en las células epiteliales intestinales la producción de factor necrótico tumoral alfa (TNF-α), quien reacciona frente a la presencia de alguna respuesta inmunitaria, actuando de manera más eficiente que al no consumir probióticos. La producción de inmunoglobulinas A y M, la producción de citoquinas cuando existe modulación de respuesta, la liberación de las quemoquinas, el desarrollo de los linfocitos T reguladores y la activación de las células asesinas naturales, son activados por los probióticos al encontrarse frente a procesos inmunológicos [13]. DOI 10.18502/espoch.v1i6.9659 Page 233 ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M. Lactobacillus casei es una bacteria anaerobia Gram positiva que habita en el intestino y en la boca de los humanos y es utilizado en la elaboración de alimentos probióticos, en un estudio realizado por T Ogawa, Y Asai se demostró que en conjunción con dextrano, elevó significativamente las actividades de las células asesinas naturales (NK) en células mononucleares de bazo de ratones BALB/c en comparación con el Lactobacillus casei ssp. casei solo o con otras especies de Lactobacillus con o sin dextrano [14]. Un estudio de cohorte prospectivo mostró recientemente que la administración de una leche suplementada con probióticos durante el embarazo redujo la preeclampsia y el riesgo de parto prematuro [15]. Nordqvist M, Jacobsson B, Brantsæter A-L, mencionan que los probióticos son microorganismos gran positivos fermentadores de carbohidratos y productores de ácido láctico. Su importancia fundamental desde el punto de vista bioterapéutico se da porque facilitan la digestión de la lactosa y equilibran el sistema inmunitario encontramos que protegen de enfermedades del aparato gastrointestinales. Se realizó también un estudio en 37.050 mujeres nulíparas y se encontró que 1851 presentaban preeclampsia leve y 550 preeclampsia grave. La ingesta de leche con probióticos durante el embarazo tardío evidenció un menor beneficio en la reducción de riesgo para la preeclampsia en comparación con las mujeres que consumieron la leche con prebióticos antes del embarazo y en el embarazo temprano, con lo cual podemos entender el efecto beneficioso tangible que tiene para el desarrollo del embarazo. Los probióticos tienen el potencial de afectar los procesos fisiopatológicos involucrados en la hipertensión, la inflamación, la función renal y la diabetes [16]. Según Garrote y Bonet, mencionan que los probióticos, mantiene la integridad del epitelio intestinal, previene los daños que pueden efectuarse por acción de microorganismos patógenos alergenos, citoquinas proinflamatorias o bien facilitan la reparación, mejorando la respuesta inflamatoria intestinal. Además, ayuda al sistema inmunológico, al activar los macrófagos locales, quienes participan en la presentación de antígenos a los linfocitos B y producen inmunoglobulina a secretora local y sistémica [17]. Pradenas Marcelo, explica en su artículo que la mayoría de probióticos contienen lactobacillus, lo cuales al estar en mayor cantidad, brindan un ambiente sano, al producir ácido láctico y al estar presentes en la flora vaginal, mantienen un ambiente inhóspito para muchas bacterias, estas especies también producen peróxido de hidrogeno que son radicales hidroxilados tóxicos y que impiden el crecimiento de bacterias patógenas, lo que resulta muy favorable para la embarazada que esta propensa a sufrir infecciones por sus cambios anatómicos [18]. Entre las bacterias que con más frecuencia se hallan en el tracto urogenital se encuentra la Chlamydia trachomatis, Trichomonas vaginalis, Escherichia coli, Enterococcus faecalis, Gardnerella vaginalis, Staphylococcus saprophyticus, Neisseria gonorrhoeae, Candida albicans y Klebsiella pneumoniae [19]. Otro artículo publicado por Espinoza, Vidaeff, Pettker y Simhan sobre el consumo de probiótico de leche durante el embarazo y su ayuda en la prevención de preeclampsia, señalan la importancia de una alimentación materna preconcepcional rica en vegetales favorecida con un equilibrado ambiente creado por dichos microrganismos probióticos [20]. DOI 10.18502/espoch.v1i6.9659 Page 234 ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M. En la Guía de práctica clínica del Ministerio de Salud Pública sobre los trastornos hipertensivos del embarazo destaca que la preeclampsia y eclampsia son las primeras causas de muerte materna desde el año 2006 a 2014, y representan el 27,53% de todas las muertes maternas, por lo que representa una patología de alto riesgo en nuestro medio, además se relaciona con muchas complicaciones tanto maternas como perinatales [21]. Existe una respuesta inflamatoria materna que es muy característica en la fisiopatología de esta afección. La preeclamsia comprende la activación del sistema de coagulación. Se piensa que la disfunción endotelial se relaciona con una respuesta inflamatoria y esta desempeña una característica principal en el inicio temprano y grave de preeclamsia. Se plantea que la dieta materna tiene mucha influencia en el desarrollo de un trastorno hipertensivo en el embarazo, por ejemplo, una dieta caracterizada por un consumo de verduras, frutas y aceites vegetales se relacionan con un riesgo reducido de preeclampsia [22]. Menon y Fortunato manifiestan en su revisión que los probióticos son microorganismos vivos que cuando son administrados en cantidades correctas dan un beneficio adicional para la salud del huésped. Los prebióticos cuando son ingeridos por vía oral tienen una mayor probabilidad de colonizar la vagina y normalizar la flora bacteriana en el tracto genital inferior, los probióticos pueden tener un efecto antinflamatorio de los liposacaridos en las células del trofoblasto placentario humano, un estudio dio a conocer que las madres que se les administro leche que contenían probióticos durante la primera mitad del embarazo presentaban un riesgo reducido de preeclampsia [23]. Halkjaer y sus colaboradores en su artículo de revisión afirman que el consumo de probióticos puede aminorar el riesgo de preeclampsia, modulando la presión arterial. Esto serviría como hallazgo potencial al tratar de explicar y entender la asociación que existe entre el consumo de probióticos y la preeclampsia, especialmente cuando los probióticos se consumen al final del embarazo o si la ingesta durante el embarazo temprano fue certera, dará como resultado un menor riesgo de parto prematuro [5, 24]. Según Sohn uno de los papeles fundamentales que proporciona la administración de los probióticos durante el embarazo es la de protección frente a la preeclampsia, diabetes gestacional, infecciones vaginales, aumento de peso materno, infantil, y posteriores. Adicionalmente, de su seguridad, se ha demostrado también que su uso reduce la proteína C reactiva, que es un reactante de fase aguda, que se asocia con efectos adversos cuando ocurren afecciones maternas como es la preeclampsia y diabetes gestacional. Si se consume una mezcla de probióticos en las etapas finales del embarazo también ocurre la modulación en las citoquinas antiinflamatorias en especial la interleucina (IL)-4 e IL-10 disminuyendo por ende en gran medida estas [25]. En otro estudio se destaca que la ingesta de la leche con probióticos fue más frecuente en un grupo de mujeres mayores, primíparas que se encontraban en un rango de IMC < 25, las cuales no fumaban, usaban suplementos dietéticos, consumían yogurt y presentaban un nivel de educación alto acompañado con los ingresos familiares. Entre ellas se estimó un promedio de 1,5 a 2 tazas leche con probióticos durante la etapa de embarazo especialmente en el tercer trimestre lo cual proporcionó un menor riesgo de padecer preeclampsia y en aquellas que cursaban con un embarazo tardío esto DOI 10.18502/espoch.v1i6.9659 Page 235 ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M. tuvo una repercusión muy importante al disminuir la probabilidad de tener un parto prematuro ya sea espontaneo y parto prematuro iatrogénico [26]. Para Karen y sus colaboradores, los probióticos tienen un efecto sobre la cascada inflamatoria cuando sucede una posible infección, por este motivo podría ocurrir un parto prematuro; el efecto de la leche probiótica sobre la preeclamsia y parto prematuro también tuvo mucha relación con la FFQ (Frecuencia de Consumo de Alimentos) a mediados del embarazo, teniendo siempre en cuenta factores ambientales, infecciones y consumo de medicamentos que también pueden influir [19]. Se debe tener en cuenta que las mujeres al estar embarazas tienden a cambiar las conductas alimenticias y se debería aprovechar aumentando el consumo de los probióticos de mejor manera si se lo hace en el primer trimestre del embarazo, sin embargo algunas mujeres disminuyen el consumo de comida durante el segundo y tercer trimestre del embarazo, cuando presentan los síntomas más fuertes como náuseas y vómitos, esto hace pensar que el consumo de probióticos tiene mucha relación cuando se consume durante diferentes puntos de tiempo en el embarazo; de manera profiláctica resulta de mayor beneficio el consumo de leche con probióticos y se recomienda ingerir también antes del embarazo logrando la disminución del índice de infecciones y el aumento de la inmunidad materna ya que tienen el potencial de impactar el estado fisiopatológico como los procesos implicados en la hipertensión, inflamación, funciones renales y diabetes [27, 28]. Otro estudio que duro aproximadamente 9 semanas, en un total de 70 mujeres, se midió el estrés oxidativo y el aumento de los niveles de GR (Glutatión Reductasa) después del consumo de yogurt con probiótico, fue positivo con lo cual se corrobora de nuevo la importancia de reducir el estrés oxidativo, hay que recordar el hecho de que la placenta es rica en mitocondrias y se requiere mayor cantidad de oxígeno durante el tercer trimestre, por consiguiente, esta etapa del embarazo es la que se encuentra relacionada con niveles altos de estrés [29]. Entre el estrés oxidativo y disfunción endotelial ocurre una asociación que cuando se generaliza, se cree que puede ser la causa del aparecimiento de la preeclamsia, peso neonatal bajo, parto prematuro, restricción del crecimiento fetal, eclampsia, infecciones, desnutrición materna y diabetes. Los probióticos al ser bacterias vivas y al consumir con frecuencia mejoran el estrés oxidativo al reducirlo y a la vez aumentar los niveles de glutatión. Los rangos de edad en el estudio realizado cursaron entre 24 y 25 años y con un rango de peso de 68 a 71 kg correspondiendo a un IMC de 28 y 29, en ellas se encontró que al haber consumido yogurt con probióticos en especial los que contienen L. acidophilus LA5 y B. lactis BB12 se generó un aumento de los niveles de GR (Glutatión Reductasa), lo cual fue beneficioso puesto que esta enzima juega un papel importante en la defensa de diferentes tejidos y órganos en la fisiopatología de varias enfermedades; este estudio también se encontró la relación con la proteína C reactiva como biomarcador de alta sensibilidad que como otras investigaciones afirman que puede disminuir los niveles [30, 31]. DOI 10.18502/espoch.v1i6.9659 Page 236 ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M. 5. Conclusiones La finalidad de los probióticos es mantener o mejorar el microbiota normal del individuo, actuales estudios muestran evidencia que emplear alimentos que contengan probióticos, ayuda a la reducción de la respuesta inflamatoria por lo cual disminuye la probabilidad de padecer preeclampsia, que es la complicación más común en una mujer embarazada después de la semana 20 de gestación, posterior a una insuficiente oxigenación que genera un estado de estrés oxidativo por la deficiencia de perfusión del trofoblasto. Para que el mecanismo realice su efecto preventivo, es necesario que se administre principalmente en el segundo y tercer trimestre de embarazo, aunque el consumo de probióticos con anterioridad a estos, resulta de mucho mayor beneficio, en especial si se consume en combinación de varias cepas de probióticos. Su mecanismo de acción consiste en mantener la integridad del epitelio particularmente intestinal o bien facilitando la reparación; ayuda al sistema inmunológico, al activar los macrófagos locales que presentan antígenos a los linfocitos B y producen inmunoglobulina A secretora local y sistémica, y a la vez disminuye la proteína C reactiva. Además de prevenir la preeclampsia puede reducir el riesgo a infecciones vaginales, ya que producen ácido láctico, el mismo que crea un ambiente inhóspito para bacterias. Agradecimientos Los autores agradecemos formalmente a la Dirección de Bibliotecas y Recursos para el Aprendizaje y la Investigación, por la asesoría brindada para la búsqueda técnica en las bases de datos. Conflicto de Intereses Los autores del siguiente artículo, expresan no poseer ningún conflicto de interés en la realización del trabajo de investigación. Limitación de Responsabilidad Toda la información proporcionada en la siguiente revisión bibliográfica son absoluta responsabilidad de los autores legítimos. Fuentes de Apoyo Propias del grupo de autores de este artículo. References [1] Alkema L, Chou D, Hogan D, Zhang S, Moller ABGA. Global, regional, and national levels and trends in maternal mortality between 1990 and 2015, with scenario-based projections to 2030: A systematic DOI 10.18502/espoch.v1i6.9659 Page 237 ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M. analysis by the UN Maternal Mortality Estimation Inter-Agency Group. Lancet. 2016;17(287):462–74. [2] Ronsmans CGW. Maternal survival series steering group, “maternal mortality; who, when, where and why.” Lancet. 2006;1(23–3). [3] Villar J, Say L, Gulmezoglu AM, et al. L. Eclampsia and pre-eclampsia: A health problem for 2000 years. Lancet. 2003;3:189–207. [4] Godoy TGM, Mabel ZJ. Restricción de crecimiento intrauterino: Causas, características clínicas, y evaluación de factores asociados a policitemia sintomática. Rev. chil. pediatr. 2010;81(4):366-367. [5] Organizacion Panamericana de la Salud. Recomendaciones de la OMS en la prevencion y tratamiento de preclampsia y eclampsia. 2019. Available from: https://www.paho.org/clap/index.php?option=com_ content&view=article&id=570:dia-de-concientizacion-sobre-la-preeclampsia-22-de-mayo&Itemid= 215&lang=es [6] Martín L, Carbajal G. Actualización en la fisiopatología de la preeclampsia. Scielo Rev chil Obs ginecol. 2014;60(2304–5132):6. [7] HVMV, AGA, EMAM. Preeclampsia un problema de salud pública mundial. Scielo Rev chil Obs ginecol. 2012;77(6):471–6. [8] Organización Panamericana de la Salud. Guias de Manejo de las Complicaciones obstetricas. Minist Salud Panama; 2009. [9] Carnicé RT. Probióticos. Concepto y mecanismos de acción. An Pediatr. 2006;4(1):30–41. [10] Mayo Clinic. ¿Qué son los probióticos y los Prebióticos? Mayo Foundation for Medical Education and Research; 2019. [11] Acton A. Preeclampsia: Nex insights for the healthcare professional. ScholarlyBrief. 2011. Available from: https://books.google.com.ec/books?id=b-o6rDwR7qsC&printsec=frontcover&dq=preeclampsia&hl= es-419&sa=X&ved=0ahUKEwj4tZCS-OHnAhWhmOAKHYnpDhQQ6AEIKjAA#v=onepage&q&f=false [12] Herna AS. Celulas colaboradoras artritis reumatoide. Reumatologia Clin. 2009;5:1–5. [13] Marteau P, Vaerman JP, Dehennin JP, Bord S, Brassart D PP. Effects of intrajejunal perfusion and chronic ingestion of Lactobacillus johnsonii strain La1 on serum concentrations and jejunal secretions of immunoglobulins and serum protein in healthy humans. Gastroenterol Clin Biol. 1997;4(293):8. [14] Ogawa T, Asai Y, Tamai R, et al. Natural killer cell activities of synbiotic Lactobacillus casei ssp. casei in conjunction with dextran. Clin Exp Immunol. 2005;103–9. [15] Myhre R., Brantsæter AL, Myking S, et al. La ingesta de alimentos probióticos y el riesgo de parto prematuro espontáneo. Clin Nutr. 2011;93. [16] Nordqvist M, Jacobsson B BA-L. Timing of probiotic milk consumption during pregnancy and effects on the incidence of preeclampsia and preterm delivery: a prospective observational cohort study in Norway. BMJ Open. 2018. [17] Garrote A, Bonet YR. Probioticos. Elsevier. 2017;31:13–6. [18] Pradenas DM. Infecciones cérvico vaginales y. Genit Infect pregnancy. 2014;25(6):925–35. [19] Vizcaíno R, Macias-Tomei C, Márquez SJC, Morales ATN. Usos clínicos de los probióticos. Scielo Rev chil Obs ginecol. 2016;1:029–040. [20] Escobar L. Anestesia Local y locorregional en cirugia menor. Reveure. 2006;32(2):73–98. [21] Ministerio de Salud Pública. Trastornos hipertensivos del embarazo. Guía de Practica Clínica (GPC). 2016;2. [22] Alexandra L, Villamil T, Carolina A, et al. Cuidado nutricional en la prevención de la preeclampsia: Una revisión sistemática Introducción Material y métodos. 2018;24(2). [23] Aguiló IP. Las bacterias contraatacan. Not Segur. 2017;6(2):1–27. [24] Halkjaer S, Nilas L, Carlsen E, Cortes D, Halldórsson T, Pedersen AKKT. Effects of probiotics (Vivomixx®) in obese pregnant women and their newborn: study protocol for a randomized controlled trial. Pubmed. 2016;17:491. [25] Sohn K, Underwood MA. Prenatal and postnatal administration of prebiotics and probiotics. Seminars in Fetal & Neonatal Medicine. 2017. [26] Zatollah A, Samimi M, Tabasi Z, Talebian P, Azarbad Z, Esmaillzadeh A. Effect of daily consumption of probiotic yoghurt on lipid profiles in pregnant women: a randomized controlled clinical trial. [Internet]. 2018;25(9):1552- 1556. [27] Lindsay KL, Walsh CA, Brennan L, McAuliffe FM. Probiotics in pregnancy and maternal outcomes: A systematic review. The Journal of Maternal-Fetal & Neonatal Medicine. 2013;26(8):772-778. [28] Doege K, Garjecki D, Zyriax B-C, et al. Impact of maternal supplementation with probiotics during pregnancy on atopic eczema in childhood – a meta-analysis. Br J Nutr [Internet]. 2012;107:1–6. [29] Badehnoosh B, Karamali M, Zarrati M, et al. The effects of probiotic supplementation on biomarkers of inflammation, oxidative stress and pregnancy outcomes in gestational diabetes. The Journal of Maternal-Fetal & Neonatal Medicine. 2017. DOI 10.18502/espoch.v1i6.9659 Page 238 ESPOCH Congresses: The Ecuadorian Journal of S.T.E.A.M. [30] Jamilian M, Bahmani F, Vahedpoor Z, et al. Effects of probiotic supplementation on metabolic status in pregnant women: A randomized, double-blind, placebo-controlled trial. Arch Iran Med. 2016;19:687-2. [31] Olveira G, González-molero I. Endocrinología y nutrición actualización de probióticos, prebióticos y simbióticos. Elsevier. 2016;63(9):482–94. [32] Pagnini C, Saeed R, Bamias G, Arseneau KO, Pizarro TT C. 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W4390549714.txt	https://ejournal.uinsalatiga.ac.id/index.php/register/article/download/368/175	en		Intertextuality in Pre-service Teachers’ Argumentative Essay in Raising AI: Practices and Beliefs	Register journal/Register	2,023	cc-by-sa	7,217	REGISTER JOURNAL – Vol 16, No 2 (2023), pp. 186-206 DOI: 10.18326/rgt.v16i2.186-206 p-ISSN: 1979-8903; e-ISSN: 2503-040X Intertextuality in Pre-service Teachers' Argumentative Essay in Raising AI: Practices and Beliefs Martono1, Nur Arifah Drajati2, Dewi Rochsantiningsih3, Surya Agung Wijaya4 1,2,3,4 Department of English Education, Faculty of Teacher Training and Education, Universitas Sebelas Maret, Surakarta, Indonesia ) Corresponding Author Email: martono.bs@staff.uns.ac.id DOI: 10.18326/rgt.v16i2.186-206 Submission Track: Received: 25-09-2023 Final Revision: 19-12-2023 Available Online: 31-12-2023 Copyright © 2023 Authors This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Abstract English as a Foreign Language (EFL) pre-service teachers arguably face more challenges regarding rhetorical moves in argumentative essays, and one of them is intertextuality because EFL pre-service teachers' arguments require sufficient and high-quality support and evidence from other scholars. Intertextuality was mainly studied, grounding in texts without external tools, such as, Artificial Intelligence (AI). In a rising AI era, the objective of this study is to investigate Indonesian EFL pre-service teachers' intertextuality in argumentative essays assisted by AI. Ten EFL pre-service teachers who attended sixteen courses in Academic Writing, with neither teaching nor writing experience, were recruited as participants. We employed a case study design to portray the nature of the phenomena, and the data were collected through documents (academic essays) to portray the practices, and interviews to represent teachers' beliefs on explicit and implicit intertextuality beyond their argumentative essays in facing AI. We employed content analysis from academic essays and interviews. The findings show that 1) EFL pre-service teachers mostly used reporting phrases and iconic references, but they were oriented to local references that targeted local audiences, so international references should be more practiced; and 2) EFL pre-service teachers' beliefs that assistance using AI while writing argumentative essays was limited to writing accuracy, but it helped them to focus on Martono, Nur Arifah Drajati, Dewi Rochsantiningsih, Surya Agung Wijaya intertextuality. Although they were enough to give sufficient intertextuality references in practice, they could not present their voices to tailor their arguments. Moreover, the intertextuality praxis and policy implications will be discussed in EFL pre-service teachers' argumentative essays assisted by AI. Keywords: academic intertextuality essays, artificial intelligence, EFL pre-service teachers, INTRODUCTION To help EFL pre-service teachers become skilled writers, having only knowledge and teaching experiences in argumentative teaching strategies is insufficient. However, a broader understanding and practice of argumentative essays is necessary for success (Valdivia & Martínez, 2018). Moreover, writing plays an essential role in connecting EFL pre-service teachers to academia and teacher community practices based on expressing their creative teaching of ideas, relating to teachers' community, preserving cultural and social relevance, and achieving professional requirements (Latham, 2020; Yoo, 2018). More specifically, argumentative essays can elevate EFL pre-service teachers' scientific thinking by integrating causal claims with supporting evidence in the writing process (Valdivia & Martínez, 2018). Moreover, one of the essential elements in the argumentative essays is intertextuality, so it requires knowledge of citations. Therefore, pre-service teachers should be capable of critically navigating in a body of literature, critical reading, and critical writing to argue and give sufficient evidence to support their arguments. Kristeva (1996) acknowledges all texts are interconnected through references, so pre-service teachers could distinguish various voices in their augmentative essay between their own and experts to avoid plagiarism. Furthermore, the current body literature on intertextuality mainly discussed intertextuality and plagiarism awareness (Hu & Shen, 2021), literature review on the thesis (Badenhorst, 2017), writing e-mail (Bremner & Costley, 2018), writing social arguments (Olsen et al., 2018), and online writing (Strickland, 2019). Furthermore, intertextuality was reported to be the most challenging part for preservice teachers in writing argumentative essays (Valdivia & Martínez, 2018). With the rise of AI, there is scant evidence for intertextuality study in argumentative essays assisted by AI. Therefore, this study explored EFL pre-service teachers' intertextuality practices and beliefs in argumentative essays assisted by AI. 187 REGISTER JOURNAL – Vol 16, No 2 (2023) Intertextuality in Pre-service Teachers' Argumentative Essay in Raising AI: ... Pre-service Teachers on Intertextuality in Argumentative Essays Argumentative essays are essential for pre-service teachers during academic years in universities because numerous subjects require them to develop argumentative skills and critical thinking. These skills were developed through various writing projects, including classroom reports, mini-research projects, graduation papers, and similar assignments. In writing scientific argumentative essays, pre-service teachers must have sufficient skill to justify whether they accept or reject other ideas based on scientific principles or knowledge. Furthermore, justification of pre-service teachers' arguments can be found in three operations (Jorba et al., 2000): 1. Producing arguments or claims; 2. Establishing relationships that modify the epistemic value of arguments according to available knowledge; 3. Examining the validity of arguments according to available scientific knowledge. Pre-service teachers use their prior knowledge to construct scientific arguments to convey perspectives and values to their readers. Scientific arguments go beyond simply organizing theories and empirical studies in order, but teachers involve critical reflection and evaluation. Prior studies on argumentation within educational settings have primarily centered on pre-service teachers' argumentative essay training (Fajaryani et al., 2021; Valdivia & Martínez, 2018), pre-service teachers' collaborative writing (Rosales et al., 2020), and where scholars also employed various technologies AI chatbot (Guo et al., 2022) and mind mapping (Barzilai et al., 2021). However, those studies did not explicitly discuss pre-service teachers' intertextuality of argumentative essays. Moreover, Castelló et al. (2011) point out that the quality of scientific argumentative essays can be assessed according to their structure and the argumentative nature of academic texts, including three criteria, and one of them is intertextuality: 1. Intertextuality refers to dialogue established with other texts and authors used as an explicit reference. It includes: (i) sufficiency, clarity and relevance of statements; (ii) evaluative comments on statements, use of other texts or voices with that purpose; (iii) convergence with other accepted theories, laws or models. REGISTER JOURNAL – Vol 16, No 2 (2023) 188 Martono, Nur Arifah Drajati, Dewi Rochsantiningsih, Surya Agung Wijaya 2. Critical approach is characterized by the writer's stance and the use of discursive resources for: (i) making personal attitudes and choices explicit according to assumptions and evidence; and (ii) achieving coherence between arguments and ideas to convince a given audience. 3. Formal aspects represent of texts that follow specific rules on formal discourse elements. Such characteristics include command of technical language, and grammatical and spelling correctness. Moreover, among quality of argumentative essays (e.g., intertextuality, critical approach, and formal aspects), intertextuality was found to be an area that requires room for improvement because pre-service teachers lack the ability to argue and explain the relationship within intertextuality (Valdivia & Martínez, 2018). Therefore, this study focused on the intertextuality aspect, so we expect that preservice teachers have wider understanding to differentiate various voices (e.g., experts studied theory, methodology, and empiric; and their own voices as writers to interweave and critically reflect). The concept of intertextuality, influenced by Kristeva (1996), proposes that all texts are interconnected. Moreover, intertextuality can be defined as how writers incorporate existing texts and audiences to generate a fresh text and audience. It embodies the post-modern idea that every new text element has a background and can be linked to its origins. Essentially, intertextuality involves portraying other sources within one's writing (Badenhorst, 2017). Furthermore, Groom (2000) acknowledges as a "given point whose "voice" is "speaking" (p. 15). Therefore, the origin of the idea can be traceable. When writers do not quote or cite, readers will be expected to take responsibility for the statement and credit. However, Bazerman (2004) acknowledges two types of intertextuality, including, explicit (evident through direct source citation) and implicit (only be discerned by knowledgeable individuals within a discourse community). Furthermore, explicit and implicit intertextuality can connect a text or statement to previous, current, or possible future texts (Bazerman, 2004). Moreover, implicit intertextuality relies on commonly circulated beliefs, issues, ideas, and statements, often considered common knowledge. However, recognizing and understanding the underlying connections and the voices behind the text may require additional or background knowledge, especially for individuals not part of the specific community or context in which the text originated. 189 REGISTER JOURNAL – Vol 16, No 2 (2023) Intertextuality in Pre-service Teachers' Argumentative Essay in Raising AI: ... Arguably as the most challenging part of writing, Farrelly's (2020) work notes that Fairclough (1992) operationalized Kristeva's idea about intertextuality to 'make the concept of intertextuality somewhat more concrete by using it to analyze texts' and to 'set out rather more systematically the potential of the concept for discourse analysis' (p. 101). Fairclough (1992) divided intertextuality into six forms (e.g., discourse representation: direct discourse, discourse representation: indirect discourse, presupposition, negation, meta-discourse, and irony). However, Fairclough (1992) also faced criticisms by Farrelly (2020) in terms of the fact that 1) presupposition and negation are problematically viewed by manifestation of intertextuality; 2) presupposition and negation should be included in assumption; and 3) meta-discourse should be excluded because it represents the text itself, not other texts. Therefore, Fairclough (2003) narrowed down his idea about intertextuality by referring to 'the presence of actual elements of other texts within a text – quotations' (p. 39). EFL Pre-service Teachers' Academic Essay Challenges on Raising AI Many EFL pre-service teachers have complained because they had inadequate English language proficiencies, so their manuscripts were difficult to follow (Vintzileos et al., 2023). In the case of pre-service teachers, some needed to make a graduation paper, although they did not need to publish it. They were tested by internal examiners from their campus. Some of them gave up during the writing of the graduation paper and did not graduate from university. However, writing has become easier for pre-service teachers because they can get 24-hour-assisted language from Artificial Intelligence (AI). AI is not new in education, but it has become more advanced. Since the 1950s, Artificial Intelligence (AI) has been in multidisciplinary and interdisciplinary growth discussions among scholars, from conceptual to practical, by mimicking human intelligence, for instance, in language skills (Chowdhary, 2020; Haenlein & Kaplan, 2019; Lund et al., 2023). Moreover, generative AI is different from AI tools. Generative AI offers a "shortcut" to the writing process, and it becomes a concern to scholars to ethically educate pre-service teachers on what they could do and what they could not do with AI for writing argumentative essays. Moreover, the emerging integration between generative AI and various writing tools (e.g., Google Docs, Grammarly, and Turnitin) was used to improve writing quality in terms of accuracy and decrease plagiarism (Ebadi & Rahimi, 2019; Li & Li, 2018; Liao, 2016). REGISTER JOURNAL – Vol 16, No 2 (2023) 190 Martono, Nur Arifah Drajati, Dewi Rochsantiningsih, Surya Agung Wijaya Therefore, pre-service teachers' skills in intertextuality were challenged and they should have the ability to distinguish and articulate various voices of writers and prior scholars. Moreover, employing writing tools powered by AI that could offer collaborative writing, so they did not limited to shortcut use to produce writing product, but writing process in peer review could be done to maintain accuracy and fluency that were missed by AI's feedback (Ebadi & Rahimi, 2018, 2019; Li & Li, 2018). Therefore, the roles of AI and peer reviewers could strengthen feedback generated by AI and peer reviewers. Moreover, pre-service teachers can take benefit from focusing more attention on the quality of argumentation because writing accuracy can be assisted by AI so that pre-service teachers can focus on other areas of writing process (Cotos et al., 2020; Knight et al., 2020; Palermo & Wilson, 2020; Shermis & Hamner, 2013). In November 2022, more advanced AI - ChatGPT or AI chatbot - was massively used by various users until it reached over 100 million users (Meyer et al., 2023; Vintzileos et al., 2023). Moreover, this AI could also improve "several aspects of language, such as vocabulary, spelling, punctuation, and grammar" (Vintzileos et al., 2023, p. 89), where Grammarly was already offered. Although this AI chatbot could have similar roles like Grammarly, Quillbot, RewriteGuru, etc., this chatbot can elevate by generating arguments. AI not only generates sentences but also creates paragraphs. However, the validity and reliability of the result have been criticized by scholars. Although ChatGPT’s use in language classrooms has been debated, EFL teachers have also shown a positive attitude toward implementing ChatGPT to enhance traditional language classrooms (Mohamed, 2023; Ulla et al., 2023). Ulla et al. (2023) point out that teachers' role and technology knowledge should be empowered to implement ChatGPT in the classroom where the role of ChatGPT could help teachers to assist students in problem-solving activities during their studies. In comparison, traditional classrooms mostly position teachers as the main sources to provide feedback. Moreover, in the EFL context, students indicated high affective filters while learning English in the classroom, and ChatGPT helps to reduce students' affective filters because they could get assistance before performing English (Mohamed, 2023). However, when it comes to writing, pre-service teachers have faced teaching dilemmas in terms of three main issues using AI in academic essays, including 1) 191 REGISTER JOURNAL – Vol 16, No 2 (2023) Intertextuality in Pre-service Teachers' Argumentative Essay in Raising AI: ... authorship, 2) copyright, and 3) plagiarism (Lund et al., 2023). Moreover, authorship issues related to the intertextuality of the text became ambiguous when the AI produced the arguments because they might produce augments based on the data and without directly citing the authors. Moreover, the practice of citation was essential for early career researchers to be introduced. Furthermore, it raises the question, "Is a graduation paper still reliable as one of the graduation requirements?". However, this also reminded policymakers and faculty members of a question: if they did not practice from their undergraduate years to write academic essays, how do they survive when they pursue master or doctoral degree or work in a research field because they need to publish journal articles as a graduation requirement? Today, academic essays are challenged by generated AI. AI can develop argumentation for academic essays, which is painful to scholars who spend many hours navigating, reading, and writing. However, scholars and educators cannot ultimately hinder academic writing classes, but AI literacy should be introduced to pre-service teachers so they can use AI wisely. Regarding management, beliefs, and practices, Indonesian education in the context of this study reform should be carefully reviewed. Although the existence of AI has tremendously impacted various aspects, it must be noted that AI cannot replace pre-service teachers' critical reading and writing because AI does not comprehend the same way as professional writers do. We speculate that AI can assist English language quality, but it still requires critical review from the writers. Therefore, this study attempted to unfold the status quo of AI in argumentative essays from intertextuality because educators need clear borders to use AI in the classroom, especially in academic essays referring to previous works. We generated two research questions. 1) What are the characteristics of pre-service teachers' intertextuality in argumentative essays facing AI? 2) How do pre-service teachers' beliefs on argumentative essays with AI contribute to intertextuality selections? REGISTER JOURNAL – Vol 16, No 2 (2023) 192 Martono, Nur Arifah Drajati, Dewi Rochsantiningsih, Surya Agung Wijaya RESEARCH METHOD To portray the nature of the data, we employed case study research (Yin, 2018), where we investigated ten EFL pre-service teachers who practiced writing argumentative essays in one of the university courses in Indonesia. This program employed writing tools powered by AI called Scribo. It is capable in terms of 1) classroom management, grouping students in some classes and groups, 2) seeking feedback (e.g., self-feedback, peer-feedback, and AI-feedback), and 3) providing initial scores in each progress and detailed language proficiency progress. During this study, they were in the stage of beginning practice, so it was only focused on the introduction of argumentative essays. Therefore, EFL pre-service teachers' introductions were based on create-a-research-space (or CARS) model (Swales, 2014), including: establishing a research territory, establishing a niche, and occupying the niche. There were sixteen meetings during this study. This program allowed preservice teachers to review each other's argumentative essays. During the first and third meetings, students were educated about AI literacy in argumentative essays and how to use Scribo for argumentative essay purposes. Further meetings were started by introducing Swales's (2014) concept of create-a-research-space (or CARS) model, and pre-service teachers practiced writing and reviewing. Moreover, we provided a consent form and got permission to use their argumentative essay as the primary data source. The course's main argumentative essay topics focused on integrating technology into teaching. To collect the data, we collected documents, argumentative essays, to be analyzed through content analysis. Moreover, we also used interviews (e.g., 1) What types of references do you use to support your argumentative essay?; 2) How far does AI assist you in writing your argumentative essay?; 3) What challenges do you face in using references to support your argumentative essay? And How do you face it?; 4) Do you use any AI from third-party apps instead of built-in AI in our learning management system?) with stimulus recall; the purpose was to investigate and clarify the nature of the teachers' text claims and the underlying value system underlying the attributions made about argumentative essays assisted by AI. In the data analysis process, we employed Bengtsson's (2016) content analysis. First is "decontextualization;" we familiarized ourselves with the data by 193 REGISTER JOURNAL – Vol 16, No 2 (2023) Intertextuality in Pre-service Teachers' Argumentative Essay in Raising AI: ... reading the argumentative essays to know what was happening in the practices and reading the interview results to understand what was happening with preservice teachers' beliefs. During the decontextualization, we labeled the data with code to start open coding (e.g., reporting phrases, named text whole text, iconic references, etc.). Second is "recontextualization;" we reread all data and highlighted to distinguish each meaning-making from the data. During this process, we compared highlighted data with research questions and aims of this study and excluded out-of-topic data. Third is "categorization;" we categorized selected data into practices and beliefs of intertextuality. Then, we grouped the selected data into sub-themes under practices and beliefs. Fourth, "compilation", we analyzed grouped data under Farrelly's (2020) intertextual reference types as references. To check the consistency of our analysis, we referred back to the original data. To strengthen the validity of the data, we employed intercoder reliability. The first author played as the main coder, and the other authors were the co-coders. We worked under Bengtsson's (2016) content analysis in different places and met to see similarities and differences among our coding data. Then, we excluded some of the data that were differently interpreted and did not meet the agreement of the interpretations. FINDING AND DISCUSSION Pre-service teachers' intertextuality practices in argumentative essays with AI At the macro analysis, this study grouped introductions based on Swales's (2014) create-a-research-space (or CARS) model. We found that all teachers already fulfilled the criteria in general, but they lacked coherence and cohesion. They mostly quoted previous studies and Indonesian policies without considering connector sentences or ideas. Theoretically, scientific arguments must be built based on sufficient justifications and teachers' skills in determining or locating their works, whether they agree or disagree with the prior studies (Castelló et al., 2011; Jorba et al., 2000). We added that the teacher-teacher reviewing process during the academic writing course was insufficient to build the skills in the short term. We argued it required a long-term commitment to make pre-service teachers engage with the lecturer in the same discourse. REGISTER JOURNAL – Vol 16, No 2 (2023) 194 Martono, Nur Arifah Drajati, Dewi Rochsantiningsih, Surya Agung Wijaya Intertextuality is the most challenging part of argumentative writing (Valdivia & Martínez, 2018), and we found that pre-service teachers required sufficient navigating skills as a foundation. Therefore, pre-service teachers seemingly overclaimed; for instance, teacher 3 stated "However, none of the previous studies showed the influence and effectiveness of augmented reality on the writing abilities of students. (Step 1B: Indicating gap)". However, this statement was not sufficiently supported by prior studies, such as studies on literature review, systematic review, critical review, etc., because they did not put more effort into navigating or reading the literature, although they were already trained. In line with Valdivia and Martínez's (2018) work, their participants had challenges developing argumentative thesis and generating intertextual dialogue. Moreover, this study added that pre-service teachers' introductions are less focused on some key areas or variables in the introduction; for example, teacher 3 neglected descriptive texts, writing skills, and the context of the research with senior high school students. Teacher 3 only focused on one of the media that was not commonly used in Indonesia and without any specific regulation for education. Similar to prior studies reports, they found EFL pre-service teachers sufficient in terms of micro-skills (e.g., grammar and vocabulary), macro-skills (e.g., coherence and cohesion) in writing (Fajaryani et al., 2021; Valdivia & Martínez, 2018) although we found from our mental process with pre-service teachers that they tend to more skills on cognitive, meta-cognitive, and social strategies. Therefore, we argued that the complex system of the writing process and sufficient writing skills must be built from pre-service teachers' awareness of intertextuality when they have adequate cognitive, meta-cognitive, and social strategies. Figure 1. Pre-service Teachers' Intertextuality 195 REGISTER JOURNAL – Vol 16, No 2 (2023) Intertextuality in Pre-service Teachers' Argumentative Essay in Raising AI: ... Our content analysis shows that pre-service teachers mostly used reporting phrases (n= 32), named text whole text (n= 9), iconic references (n=10), and generic communicative act type (n= 4) from the introduction; quotation parts of the text (n= 2), generic text-type (n= 1), and ambiguity were used (n= 7). In Figure 2, pre-service teachers use many reporting phrases, seemingly forgetting that argumentative writing is not limited to reporting previous studies to support the argumentation. Furthermore, pre-service teachers' intertextuality is displayed (See Table 3) as reprepresentingachers' works. Table 1. Examples of Pre-service Teachers' Intertextuality Types Examples Quotation Parts of the text Language program evaluation enables "a variety of evidence-based decisions and actions, from designing programs and implementing practices to judge effectiveness and improving outcomes" (Norris, 2016, p.169-189). (Teacher 9) Reporting phrases This previous study reported that the students in night grades' reading motivation are strongly influenced by their school-related reading practices (Tegmark et al., 2022). (Teacher 2) In relation to the effectiveness of learning English as Foreign Language (EFL), the previous research stated that the interaction relationship between teachers and students is also one of the factors supporting success (Vattøy & Gamlem, 2020). This research was conducted in two junior high schools with the aim of knowing the quality of interaction between teachers and students and provide feedback in teaching English as a foreign language (EFL). This research was conducted through the analysis of video recording data from 13 classrooms conducted 65 English learning. The result revealed that interaction between teacher and students and providing feedback is a regulatory process needed to achieve the learning objectives. This research found that English teachers in those two schools were still struggling to provide positive feedback so that it could influence students' learning to be more effective. (Teacher 9) Named Text Whole text Information and Communication Technology (ICT), in addition to pedagogy integration, refers to three key factors (Setyawan & Istiawan 2021): pedagogy, technology, including social interaction. (Teacher 4) Oakley (2011) stated that reading is a skill of combining background knowledge with reading texts. (Teacher 6) REGISTER JOURNAL – Vol 16, No 2 (2023) 196 Martono, Nur Arifah Drajati, Dewi Rochsantiningsih, Surya Agung Wijaya Types Iconic Reference Examples According to RI Law No. 20 of 2003 about the National Education System, early childhood education is a coaching effort directed at children from birth until the age of six. (Teacher 10) Another piece of research about reading from PISA (Program for International Student Assessment) shows that Indonesia's literacy rate is low compared to other countries in the world. This is the result of a study of 72 countries. Indonesia is ranked 62 out of 70 countries surveyed. (Teacher 2) The government's policy of allowing elementary schools in Indonesia to teach English starting from the fourth grade (Depdikbud, 1994) provides early readiness for L2 students in Indonesia to master English as a second language. (Teacher 8) Generic Texttype They said that according to the Program for International Student Assessment (PISA) report, Indonesia has a low literacy mastery problem. (Teacher 7) Generic Communicative Act The Indonesian government has created a curriculum, called curriculum 2013. In this curriculum, the process of English class requires the use of a scientific approach in the learning process, where the learning is more focused on students' activity rather than teachers' activity. (Teacher 7) In the case of COVID-19 in Indonesia, the Indonesian government decided to suspend all school-related activities in March 2020 to keep up against the virus. The Ministry of Education in Indonesia recommended schools establish remote teaching arrangements and provide online education possibilities for children. (Teacher 5) The government through the ministry of education made various adjustments to learning activities during the pandemic. One of them is the implementation of an online class system. (Teacher 1) Ambiguity There are many studies being conducted and compared to evaluate the effectiveness of written asynchronous computer mediated communication (WACMC) and oral face to face interaction (OF2F) that is used to give feedback in writing class. (Teacher 1) Despite the rise in popularity of digital games as pastimes and research demonstrating the affordability of digital game-based language learning (DGBLL) for English as a Foreign Language (EFL), DGBLL is not widely used in Indonesia. (Teacher 4) 197 REGISTER JOURNAL – Vol 16, No 2 (2023) Intertextuality in Pre-service Teachers' Argumentative Essay in Raising AI: ... As novice writers, pre-service teachers showed a low level of intertextuality because they were in an ongoing process of developing reading experience to recognize theoretical, conceptual, and empirical studies to layer their complexity of arguments (Badenhorst, 2017; Hu & Shen, 2021; Valdivia & Martínez, 2018). Valdivia and Martínez (2018) found that the most challenging part is that novice teachers tend to use direct citations. In contrast, our analysis showed why more experienced teachers used indirect citation in the form of "reporting phrases" without considering their voices, so they only reported (see Table 1 "reporting phrases" from Teacher 9). Teacher 9 only summarized what prior studies were conducted and found without critical analysis of other studies or relationships to Teacher 9's study. Teachers argued by providing various sources that could make their augmentative text because they developed based on facts. We found that many pre-service teachers did the same things because they forgot to interweave what prior studies had already seen and their argumentation. Furthermore, complexity of represented citations draws pre-service teachers' capacity on intertextuality, and our study showed teachers cited on the level of empirical studies and left behind the theoretical or conceptual framework. In the introduction of argumentative writing, pre-service teachers providing clear definitions become valuable for readers because readers might need help understanding some discourse used in some studies. Some pre-service teachers tended to give descriptions or parameters of their key terms in the introduction by using "named text whole text." However, some teachers also used "ambiguity, referring to some knowledge or terms from previous studies without clearly mentioning who is speaking in the text. Pre-service teachers' beliefs on intertextuality in argumentative essay with AI Using a writing management system powered by AI made pre-service teachers believe they could focus on their arguments. However, pre-service teachers seemingly followed Indonesian writers in local journals where they wanted to show their knowledge and expertise about government policies related to education. Sezen-Barrie et al. (2017) also found a similar movement of teachers to use government resources as references. Still, their study showed that teachers supported government policy and that rebuttal was presented. Our participants tended to play safe and hold their norms; rebuttal was stereotyped as an impolite movement. Furthermore, international readers might see this act as uninterested REGISTER JOURNAL – Vol 16, No 2 (2023) 198 Martono, Nur Arifah Drajati, Dewi Rochsantiningsih, Surya Agung Wijaya reports because pre-service teachers only reported and did not critically elaborate on previous regulations or other countries' similar policies (see Table 1 "Iconic Reference and Generic Communicative Act"). Before I thought my research and writing did not need to be perfect, my audiences are not experts, and I do not need to publish. Therefore, I used local resources to build a context of the study in my introduction. However, when I wrote my academic writing, the AI gave me scores that motivated me to write better. I did not want scores under 70. Although it was very difficult for me as a novice writer to get higher scores, I was zeal to write the topic because it was based on my research interest. (Teacher 1) I learned that I need to be careful in selecting resources as references, for example, when my participants are in junior high school, I need to find the same participants on my topics to compare the result with the literature. But, I believe in my introduction that I need to put what is going on in my country to give updates to local readers. (Teacher 10) Putting more arguments and facts based on local reports or government policies, it is more valuable for other Indonesian readers because they might replicate my idea in their classroom and be considered by local context and published in a local journal because I am still new so local journal probably is good step to enter academia. (Teacher 3) Pre-service teachers' beliefs seem to be reproducing knowledge rather than seeking new knowledge from the current body of literature (Badenhorst, 2017). Pre-service teachers argued their academic writing as a practice to conduct classroom research to help students for local audiences, so they did not need to achieve novelty in their work. Moreover, AI made them keep on revising because it provided initial scores of their arguments. Therefore, pre-service teachers could reflect on the scores and target higher scores from AI. Although in self-assessment, AI scores did not finally score pre-service teachers, it is considered external feedback for internal feedback seeking. They built awareness of what they could seek or not from AI for the writing process (Guo et al., 2022). 199 REGISTER JOURNAL – Vol 16, No 2 (2023) Intertextuality in Pre-service Teachers' Argumentative Essay in Raising AI: ... Moreover, pre-service teachers' intertextuality capacity was not magically changed because we found their capacity to use intertextuality was still developing and insufficient, and they needed more time to practice. Furthermore, we found that pre-service teachers only reported and forgot to argue, so their voices were not represented in their works. It indicated a lack of pre-service teachers' reading and writing strategies. I find it challenging to synthesize by creating a group of similar studies' results into one argument. Although my class gave me the idea to use mindmaping, it is not an essay to write my work's result. Although I did not get lost in my writing, tailoring and summarizing the idea was difficult. (Teacher 7) The AI is quite helpful in checking my vocabulary use and grammar, so I do not worry about my summary or paraphrasing from other scholars. However, if the AI could not differentiate between my claim and my citation, it could benefit someone who learned academic writing. I still relied on my friends' or lecturer's feedback about my citations. Because this was my first time learning to cite and write a paragraph, I needed to combine many resources. AI was beneficial for me to avoid plagiarism because it provided me with feedback about paraphrasing or summarising. (Teacher 4) After attending this class, I realized that I needed to read the journal articles I cited because I tried to compare them with the results of ChatGPT, but it was different. I felt embarrassed if I misinterpreted by following the AI about paraphrasing. Then, I strategized using AI to give me more options about vocabulary when I paraphrased. My lecturer showed that if I directly copied and pasted from ChatGPT, Turnitin could also detect my plagiarism with AI from my citation or summary. This class made me aware that I could use AI, but I need to use it wisely. (Teacher 2) Those strategies need to be supported by pre-service teachers viewing intertextuality as a way to critically interact with body literature rather than anchored conventions (Vardi, 2012). Learning intertextuality, this community contributed to giving fundamental skills and aspects for pre-service teachers as preparing for graduation paper by acknowledging pre-service teachers about 1) navigating and connecting literature review, 2) promoting various methodologies REGISTER JOURNAL – Vol 16, No 2 (2023) 200 Martono, Nur Arifah Drajati, Dewi Rochsantiningsih, Surya Agung Wijaya (Badenhorst, 2017), 3) using direct and indirect quotations, and 4) reporting previous studies to give credit and evidence (Guo et al., 2022; Hu & Shen, 2021). Moreover, this study added teacher-mentor and teacher-teacher interactions essential to building intertextuality because prior studies suggested three areas of teachers' development: intertextuality, engagement with various sources, and contextual mediation (Badenhorst, 2017; Fajaryani et al., 2021; Valdivia & Martínez, 2018). CONCLUSION This study reports on pre-service teachers' practices and beliefs. In practice, it shows that pre-service teachers need more time to engage with academic essay discourse which is repeated from higher use of reporting phrases (n= 32) to provide evidence of their argumentative essay. However, less use of iconic references (n=10) shows that some pre-service teachers find it challenging to offer their expertise to local or national knowledge to tailor to international issues. Moreover, to support their reporting phrases, pre-service teachers tended to use whole text (n= 9), generic communicative act type (n= 4), quotation parts of the text (n= 2), and generic text type (n= 1). To express their expertise in one of the research areas, they used ambiguity (n= 7), but it was low. Although their beliefs showed that AI feedback facilitated their focus on their arguments and quotations, leading to decreased worry about writing errors on accuracy, using various intertextuality in argumentative writing could not be magically achieved with AI support. We argue that writing argumentative essays cannot be achieved using only generative AI; it requires high-quality feedback to elevate students' intertextuality. Moreover, this study shows that students build initial awareness of AI literacy during this study, although it requires in-depth investigation. The praxis implication of this study contributes to Farrelly's (2020) intertextual reference-type implementation that can be fostered by integrating writing classes assisted by AI, so pre-service teachers can focus on critical engagement with literature review. To connect students with various discourses in a body of literature, our study recommends 1) building critical reading, 2) familiarizing them with mind mapping of body literature, and 3) practicing summarizing, paraphrasing, synthesizing, and arguing. Therefore, they not only report similarly to "reporters," but they also know how to voice their arguments 201 REGISTER JOURNAL – Vol 16, No 2 (2023) Intertextuality in Pre-service Teachers' Argumentative Essay in Raising AI: ... and to locate their study in the body of literature. Therefore, at the end of the day, we expect that pre-service teachers will build their capacity to criticize or rebut based on the existing literature or policy. Regarding the policy implication of this study in higher education, although this study's early responses to the Indonesian government's policy of making "graduation paper and publication" optional, this study showed that the regulation of academic writing for publication classes needs to be reformed in the higher education curriculum. This study indicated pre-service teachers' insufficient intertextuality skills or early stages to engage with academic discourses, requiring more time to develop AI literacy in responding to the AI-raising era. This study suggests policymakers and faculty members should carefully design academic writing for publication classes with a single national framework. Therefore, although pre-service teachers did not need to write graduation papers or choose other optional requirements, they still engaged with academic writing or argumentative essays as the language of academia, and AI was adequately utilized and ethically regulated. 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https://openalex.org/W4296036007	https://www.eurosurveillance.org/deliver/fulltext/eurosurveillance/27/37/eurosurv-27-37-3.pdf?itemId=%2Fcontent%2F10.2807%2F1560-7917.ES.2022.27.37.2101028&mimeType=pdf&containerItemId=content/eurosurveillance	English	null	Seroconversion rate and socio-economic and ethnic risk factors for SARS-CoV-2 infection in children in a population-based cohort, Germany, June 2020 to February 2021	Euro surveillance/Eurosurveillance	2,022	cc-by	4,692	What are the implications of your findings for public health? Seroconversion rates among children in all age groups increased 3–4- fold from June 2020 to February 2021. Only 24 of 58 (41%) seropositive children reported symptoms. In 51% of infected children, at least one parent was also What are the implications of your findings for public health? Populations at risk for infection should be the focus of public health interventions to improve infection control and other preventative measures (e.g. vaccine uptake). SARS-CoV-2-positive. Low level of parental educa- tion (OR = 3.13; 95% CI: 0.72–13.69) non-significantly increased the risk of infection. Of the total cohort, 38.5% had a migration background, 9% of Turkish and 5% of Middle Eastern origin, and had the high- est risk for SARS-CoV-2 infections (OR = 6.24; 95% CI: 1.38–28.12 and OR = 6.44 (95% CI: 1.14–36.45) after adjustment for other risk factors. Conclusion: In the second half of 2020, seroprevalence for SARS-CoV-2 in children increased especially in families with lower- socioeconomic status. Culture-sensitive approaches are essential to limit transmission and could serve as a blueprint for vaccination strategies. Introduction: Socio-economic and ethnic background have been discussed as possible risk factors for SARS- CoV-2 infections in children. Improved knowledge could lead to tailored prevention strategies and help improve infection control. Aim: We aimed to identify risk factors for SARS-CoV-2 infections in children in the first and second wave of the pandemic. Methods: We performed an observational population-based cohort study in children (6 months–18 years) sched- uled for legally required preventive examination and their parents in a metropolitan region in Germany. Primary endpoint was the SARS-CoV-2 seroconversion rate during the study period. Risk factors assessed included age, pre-existing medical conditions, socio- economic factors and ethnicity. Results: We included 2,124 children and their parents. Seroconversion rates among children in all age groups increased 3–4- fold from June 2020 to February 2021. Only 24 of 58 (41%) seropositive children reported symptoms. In 51% of infected children, at least one parent was also Introduction: Socio-economic and ethnic background have been discussed as possible risk factors for SARS- CoV-2 infections in children. Improved knowledge could lead to tailored prevention strategies and help improve infection control. Aim: We aimed to identify risk factors for SARS-CoV-2 infections in children in the first and second wave of the pandemic. Methods: We performed an observational population-based cohort study in children (6 months–18 years) sched- uled for legally required preventive examination and their parents in a metropolitan region in Germany. Primary endpoint was the SARS-CoV-2 seroconversion rate during the study period. Risk factors assessed included age, pre-existing medical conditions, socio- economic factors and ethnicity. Results: We included 2,124 children and their parents. What did you want to address in this study? This study was initiated to estimate seropositivity for SARS-CoV-2 during the first year of the pandemic in an unselected cohort of children and adolescents and identify risk factors for infection. Citation style for this article: Brinkmann Folke, Diebner Hans H, Matenar Chantal, Schlegtendal Anne, Eitner Lynn, Timmesfeld Nina, Maier Christoph, Lücke Thomas. Seroconversion rate and socio-economic and ethnic risk factors for SARS-CoV-2 infection in children in a population-based cohort, Germany, June 2020 to February 2021. Euro Surveill. 2022;27(37):pii=2101028. https://doi.org/10.2807/1560-7917.ES.2022.27.37.2101028 What have we learnt from this study? What have we learnt from this study? Neither age nor sex were risk factors for SARS-CoV-2 infection, whereas contact to infected family members and, independent of other findings, a Middle Eastern or Turkish migration background increased the risk for infection. www.eurosurveillance.org Research Research Seroconversion rate and socio-economic and ethnic risk factors for SARS-CoV-2 infection in children in a population-based cohort, Germany, June 2020 to February 2021 Folke Brinkmann1 , Hans H Diebner2 , Chantal Matenar1 , Anne Schlegtendal1 , Lynn Eitner1 , Nina Timmesfeld2 , Christoph Maier1 , Th Lü k 1 lke Brinkmann1 , Hans H Diebner2 , Chantal Matenar1 , Anne Schlegtendal1 , Lynn Eitner1 , Nina Timmesfeld2 omas Lücke1 bner2 , Chantal Matenar1 , Anne Schlegtendal1 , Lynn Eitner1 , Nina Timmesfeld2 , Christoph Maier1 , Folke Brinkmann1 , Hans H Diebner2 , Chantal Matenar1 , Anne Schlegtendal1 , Lynn Eitner1 , Nina Timmesfeld2 , Christoph Maier1 , Thomas Lücke1 omas Lücke University Children’s Hospital, Ruhr University Bochum, Bochum, Germany D t t f M di l I f ti Bi t d E id i l R h U i i 1. University Children’s Hospital, Ruhr University Bochum, Bochum, Germany 2. Department of Medical Informatics, Biometry and Epidemiology, Ruhr Univer 1. University Children’s Hospital, Ruhr University Bochum, Bochum, Germany 2. Department of Medical Informatics, Biometry and Epidemiology, Ruhr University Bochum, Bochum, Germany y p a , u y u , u , a y 2. Department of Medical Informatics, Biometry and Epidemiology, Ruhr University Bochum, Bochum, Ger Correspondence: Folke Brinkmann (folke.brinkmann@rub.de) Citation style for this article: Citation style for this article: Brinkmann Folke, Diebner Hans H, Matenar Chantal, Schlegtendal Anne, Eitner Lynn, Timmesfeld Nina, Maier Christoph, Lücke Thomas. Seroconversion rate and socio-economic and ethnic risk factors for SARS-CoV-2 infection in children in a population-based cohort, Germany, June 2020 to February 2021. Euro Surveill. 2022;27(37):pii=2101028. https://doi.org/10.2807/1560-7917.ES.2022.27.37.2101028 Article submitted on 22 Oct 2021 / accepted on 02 Jun 2022 / published on 15 Sep 2022 Introduction The rate of infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in children was discussed controversially during the first months of the pandemic. Children are frequently oligo- or 1 Table 1 Demographic and clinical data of study participants, SARS-CoV-2 infection in children, Germany, June 2020–February 2021 (n = 2,124) Demographic and clinical data of study participants, SARS-CoV-2 infection in children, Germany, June 2020–February 2021 (n = 2,124) Demographic and clinical data of study participants, SARS-CoV-2 infection in children, Germany, June 2020–February 2021 ( 2 124) All SARS-CoV-2-positive SARS-CoV-2-negative OR (SARS-CoV-2 yes/no) n % n % n % 95% CI Total 2,124 58 2,066 NA Female 1,020 48.0 27 46.6 993 48.1 0.94 (0.56–1.59) Male 1,104 52.0 31 53.4 1,073 52.9 0,92 (0,45–1,65) ≤ 3 yearsa 429 20.2 13 22.4 416 20.1 1.15 (0.61–2.14) 4–6 years (preschool) 817 38.5 22 37.9 795 38.5 0.98 (0.57–1.67) 7–10 years (primary school) 290 13.7 8 13.8 282 13.7 1.01 (0.47–2.16) 11–18 years (secondary school) 588 27.7 15 25.9 573 27.7 0.91 (0.5–1.65) Underweight (< 3 percentile) 159 7.5 3 5.2 156 7.6 0.67 (0.21–2.16) Obesity (> 97% percentile) 156 7.3 4 6.9 152 7.4 0.93 (0.33–2.61) Any chronic medical conditionb 389 18.3 8 13.8 381 18.4 0.71 (0.33–1.5) Chronic airway disease 211 9.9 4 6.9 207 10.0 0.67 (0.24–1.86) CI: confidence interval; NA: not applicable; OR: odds ratio; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2. a In our cohort during the study period, only 71 children (16.5%) and only 1/13 of the children who tested positive for SARS-CoV-2 attended day care regularly. b All medical conditions requiring constant medical attendance and/or medication. adolescents from 6 months to 18 years of age who attended outpatient paediatric practices in three regions of Germany for scheduled mandatory routine examinations were invited to participate in the study (Corkid study, ‘Corona in Kids’) [3]. Participants and their parents were asked to fill in an electronic ques- tionnaire (pre-installed on a tablet) with 14 questions (see Supplementary material) available in five dif- ferent languages (German, English, Turkish, Arabic and Russian). It also asked about former SARS-CoV-2 infections (either symptomatic or asymptomatic). Recalled symptoms were categorised as, among oth- ers, fever, cough, loss of smell and/or taste, influ- enza-like illness and diarrhoea. Introduction Medical history as well as information on socio-economic and migration background (Germany/Western Europe, Eastern and Southern Europe, Turkey, Middle East, Commonwealth of Independent States (CIS states) America and Asia) of parents and grandparents was collected. Western European background included Austria, Belgium, France, Liechtenstein, Luxembourg, Monaco, the Netherlands and Switzerland; for this study, we did not classify individuals from these countries as ‘migrants’ because of comprehensive similarities in educa- tion system, standard of living and healthcare sys- tem. Southern Europe comprised Greece, Italy, Malta, Portugal and Spain. Eastern Europe included Albania, Bulgaria, Croatia, Hungary, North Macedonia, Poland, Romania, Slovakia and Slovenia. asymptomatic and therefore undertested and underdi- agnosed. Seropositivity studies, however, reveal that the rate of SARS-CoV-2 infections in adults and chil- dren is similar [1-3]. Most transmissions probably take place in households, where contacts are most intense [2,4]. Crowded living conditions and poverty have been associated with a higher rate of SARS-CoV-2 infection both in adults and children as well as increased morbidity and mortality in Europe and in the United States (US) [5,6]. Ethnic origin has also been described as a risk factor for hos- pitalisation for coronavirus disease (COVID-19) in chil- dren in the US [7,8]. Moreover, socio-economic factors have been identified as risk factors for SARS-CoV-2 infection [5,9]. This study took place during the first phase of the pan- demic and before vaccinations became available. At that time most infections were caused by the wild type SARS-CoV-2 while the Alpha variant started spreading in Germany from December 2020 and became domi- nant within the following 3 months. It was performed in an unselected COVID-19-asymptomatic paediatric population in a Western German metropolitan region where ca 40% of families have migration background. The study had been set up to identify risk factors for SARS-CoV-2 infection in children and adolescents with the aim to identify populations at risk in Germany and to introduce preventive measures for the affected chil- dren and their families. Education level was defined as the highest level of education in terms of graduation of one of the par- ents/guardians. High level of education referred to high school/grammar school (Fachhochschule/Abitur), medium-level education to general secondary school (Realschule) and low-level education included edu- cation up to primary school/basic secondary school Methods Study design and participants In a standardised approach from 1 June 2020 to 1 February 2021, all asymptomatic children and www.eurosurveillance.org Table 2 Socio-economic parameters and migration background of study participants, SARS-CoV-2 infection in children, Germany, June 2020–February 2021 (n = 2,073) Socio-economic parameters and migration background of study participants, SARS-CoV-2 infection in children, Germany, June 2020–February 2021 (n = 2,073) All SARS-CoV-2-positive SARS-CoV-2-negative n % n % n % Total 2,073 56 2,017 Parent SARS-CoV-2-positivea 59 3.0 27 50.0 32 1.7 SARS-CoV-2 contacta 268 14.5 33 64.7 235 13.1 Parents in high-risk occupations for SARS-CoV-2a 1,024 53.3 28 56.0 996 53.2 Parents‘ highest educational degree High school/grammar school 1,408 67.9 32 57.1 1,376 68.2 General secondary school 463 22.3 12 21.4 451 22.4 Basic secondary/primary school 132 6.4 9 16.1 123 6.1 No educational degree 70 3.4 3 5.4 67 3.3 Any migration background in any of parents/ grandparents 816 39.4 36 64.3 780 38.7 Turkey 180 8.7 13 23.2 167 8.3 Middle East 108 5.2 8 14.3 100 5.0 Asia 28 1.4 3 5.4 25 1.2 Commonwealth of Independent States (CIS states) 127 6.1 3 5.4 124 6.1 Eastern Europe 288 13.8 7 12.5 281 13.9 Other 85 4.1 2 3.6 83 4.1 Germany/Western Europe 1,257 60.6 20 35.7 1,237 61.3 Members in the householda 1–3 564 27.2 13 23.2 551 27.5 4–5 1,337 64.8 36 64.3 1,301 64.8 > 5 162 7.9 7 12.5 155 7.7 Rooms in the householda 1–3 522 25.4 24 42.9 498 24.9 4–5 1,029 50.0 23 41.1 1,006 50.3 > 5 505 24.6 9 16.1 496 24.8 SARS-CoV-2: severe acute respiratory syndrome coronavirus 2. a Missing value. Denominator for the percentages excludes study participants with missing information. (Grundschule/Hauptschule). No educational degree (school education not completed, i.e. without final graduation) formed a fourth group. In addition, we col- lected information on the parent’s working background and included high-risk working conditions with sev- eral hours of contact with many people per day (public transport, sales assistance, medical or nursing profes- sion, teaching or childcare). We documented both the number of persons and the number of available rooms per household (rooms per person). History of chronic diseases and medication of the children was obtained from parents and verified by matching with medical charts. Accompanying parents were also invited to par- ticipate in the study, fill in the questionnaire and have their SARS-CoV-2 antibodies measured. seropositivity or by previous positive PCR test results reported by the family; however, PCR testing was not performed as part of the study. Table 2 We aimed at compar- ing demographical data (age, sex), underlying medical conditions, socio-economic parameters and migration background between children and adolescents with and without evidence of SARS-CoV-2 infection. www.eurosurveillance.org Study design and participants y g p p In a standardised approach from 1 June 2020 to 1 February 2021, all asymptomatic children and 2 Table 3 3 Random effect logistic regression and univariate analysis, SARS-CoV-2 infection in children, Germany, June 2020–February 2021 ( 2 052) 3 Random effect logistic regression and univariate analysis, SARS-CoV-2 infection in children, Germany, June 2020–February 2021 (n = 2,052) Predictors OR (univariate) p value OR (logistic regression) 95% CI p value Background Germany/Western Europe Reference 1.0 Reference Turkey 4.81 < 0.001 6.24 1.38–28.13 0.017 Middle East 4.95 < 0.001 6.44 1.14- 36.45 0.035 Eastern Europe 1.54 > 0,05 1.56 0.42–5.78 0.503 Asia 7.42 0.002 16.36 1.07–250.36 0.045 Living conditions Rooms per person 0.25 0.001 0.35 0.08–1.5 0.158 Level of education High level Reference 1.0 Reference Medium level 1.08 > 0,05 1.02 0.37–2.81 0.972 Low level 3.15 0.003 3.13 0.72–13.69 0.129 No educational degree 1.6 > 0,05 1.63 0.23–11.69 0.628 CI: confidence interval; OR: odds ratio; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2. confidence interval; OR: odds ratio; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2. background had evidence of SARS-CoV-2 infection, only 1.6% (20/1,257 of children with Western European background had evidence of infection (OR = 2.78; 95% CI: 1.63–4.74). Details about the socio-economic and migration background of the participants are shown in Table 2. background had evidence of SARS-CoV-2 infection, only 1.6% (20/1,257 of children with Western European background had evidence of infection (OR = 2.78; 95% CI: 1.63–4.74). Details about the socio-economic and migration background of the participants are shown in Table 2. effect term in our model given by a family identifica- tion number. Insignificance of other factors was shown by a likelihood ratio-based model reduction process. Consequently, irrelevant factors were dropped in order to avoid overfitting. For the random effect analysis, we used the lme4 software package within statistical programming language R [10]. We considered p values under 0.05 as statistically significant. A random effect logistic regression yielded the results listed in Table 3. Due to missing records for the num- bers of rooms in the household and for family mem- bers, the regression was based on 2,052 instead of 2,073 observations. Specifically, the highest risks for infection were detected in children of Turkish (OR = 6.24; 95% CI: 1.38–28.13; p = 0.017) and Middle Eastern (OR = 6.44; 95% CI: 1.14–36.45; p = 0.035) origin. In the Asian population, seroprevalence was also high (OR = 16.6; 95% CI: 1.07–250), but because only 1.4% of the cohort originated from this region this result must be interpreted with caution. Results Demographics and clinical characteristics In this cohort, 2,848 children and adolescents were asked initially to participate and 25% refused, mainly because of fear of drawing blood. Of the remaining 2,124 (1,019 female; 48%) with a median age of 7.1 years (range: 0.6–18 years), 58 (2.7%) had evidence of SARS-CoV-2 infection (33 by SARS-CoV-2 antibody test only, six by PCR only and 19 by both methods). Our cohort exhibited an increasing seroprevalence rate from 0.5% in mid-2020 to almost 6% at the beginning of 2021 [3]. There were no clini- cal differences regarding sex, age or weight of positive cases compared with the SARS-CoV-2-negative chil- dren. Likewise, the number of children with pre-exist- ing conditions (8/58 vs 381/2,066; 14 vs 18%) did not differ much between the groups. Twenty-four of the 58 infected children did not recall any infection during the preceding 3 months. Clinical characteristics of the par- ticipants by group are displayed in Table 1. Other risk factors relevant for risk adjustment, although below significance, included low level education of the parents (OR = 3.13; 95% CI: 0.72–13.69) and crowded living conditions, inversely expressed by rooms per person (OR = 0.35; 95% CI: 0.72–13.69). Of note, these two factors had statistically significant effects in uni- variate analyses (Table 3). Discussion This population-based study identified seropositiv- ity rates and risk factors for SARS-CoV-2 infections in asymptomatic children and adolescents who attended legally required preventive examinations. Our key find- ings were: (i) Less than half of the seropositive partici- pants of all age groups recalled symptoms of infection within the 3 months preceding the study, suggest- ing that more than half of SARS-CoV-2 infections go unnoticed [3]. (ii) The cumulative incidence reflected by prevalence of antibodies in serum of SARS-CoV-2 Statistical analysis hi d li Demographic and clinical parameters between groups are presented with univariate odds ratios (OR) and 95% confidence intervals (CI). The impact of migration back- ground was analysed by means of an adjusted random effects logistic regression. Thereby, we adjusted for the ratio of number of available rooms vs the num- ber of persons living in the same household as well as for the highest educational level in the family. The ratio of rooms per person was introduced following the rationale that the available space per person is the essential surrogate for crowded living conditions and, therefore, a potential risk factor for transmission. Some families participated with more than one child in the Corkid study. Therefore, we included a random Blood samples were taken from all participating chil- dren on the occasion of their mandatory medical appointment. Serum samples were analysed for SARS- CoV-2 IgM and IgG nucleocapsid (N) antibodies (Elecsys Anti-SARS-CoV-2, Roche, sensitivity 99.5%, specificity 99.8%) using the manufacturer’s cut-off. The target variable was SARS-CoV-2 infection, either given by www.eurosurveillance.org 3 Table 3 Random effect logistic regression and univariate analysis, SARS-CoV-2 infection in children, Germany, June 2020–February 2021 (n = 2,052) www.eurosurveillance.org Socio-economic and migration background Because information on migration background was not documented for 51 participants, the following analysis is based on the remaining 2,073 patients. Within the remaining study cohort, 816 (39%) children and adoles- cents had a migration background (Table 2). While 4.4 (36/816) of children and adolescents with a migration 4 our results can help identify opportunities for timely targeted interventions. infections was significantly higher in families of Turkish or Middle Eastern descent irrespective of other risk factors, i.e. after adjustment for other socio-economic confounders. Our study had limitations: Paediatric patients were recruited only from three regions in Germany, and not all paediatric practices in the area participated. In addi- tion, initially low seroprevalence rates could have intro- duced a bias. The proportion of children from Turkish families, in contrast to those of other origins, was 9%, lower than expected seeing as 20% of the families in the regions studied are of Turkish origin [19]. However, this rather indicates an underestimation of the true infection rate in this group. The migration status was not differentiated with regards to legal status or num- ber of years lived in Germany. In some of the calcula- tions regarding infection risk in children with migration background, 95% CI were wide and the findings have to be interpreted with caution. Another limitation might be the use of surrogate parameters for the assessment of living conditions and income, such as the number of rooms etc. Different educational backgrounds not matching the German system (participants who had part of their education in other countries) might have posed difficulties in answering questions about educa- tional status and might have also led to an underesti- mation of the corresponding risk factor. We attempted to reduce a possible bias through semi structured inter- views to validate the answers drawn from the Internet- based questionnaire. In addition to this, we included children and adolescents from a wide range of ages (0–18 years). Different mechanism of transmission may have played a role in different age groups. As in other seroprevalence data from German or Swiss cohorts, we observed an increase in seroprevalence from 0.5 to 6% during the study period [2,11,12]. The dynamics of this increase in the seroprevalence of SARS-CoV-2 infections match well with national data of acute SARS-CoV-2 infection in the same geographical region [4,13]. Infection rates were comparable across all age groups in keeping with the findings of other studies [1,4,8]. Conclusions Thi d h The most relevant risk factor for SARS-CoV-2 infec- tion in our study population was a Turkish or Middle Eastern migration background. Although socio-eco- nomic factors confound the factor of migration history to some extent, our findings result from an adjusted regression model and are therefore independent of educational standard and housing conditions. Reasons for this observation could include different family and social structures favouring closer contacts and there- fore increasing the risk of transmission as described by Gaskell et al. for a tight Jewish Orthodox community in the US [18]. Apart from that, language as well as cul- tural barriers might play a role [9] as well as travelling habits. Visiting friends and relatives in countries with higher incidence of infection might further increase the risk. Scepticism regarding politics and health authori- ties are an additional issue, especially in prevention and vaccination programmes [1]. To approach these families, tailored, culture-sensitive strategies need to be developed. This study shows that Turkish or Middle Eastern migra- tion background even after correcting for poor socio- economic status, were risk factors for SARS-CoV-2 infection in a population-based cohort of children and adolescents in Germany. Targeted interventions and tailored prevention strategies in these groups are necessary to improve infection control and to protect these vulnerable populations from excess morbid- ity and mortality. They could serve as a blueprint for vaccination programmes, and there is a need to shift the focus of politics towards more culture-sensitive interventions. Socio-economic and migration background It is not surprising that known exposure to SARS-CoV- 2-positive individuals doubled the risk for seroconver- sion. In most families, at least one parent also had evidence of SARS-CoV-2 infection, which is slightly more than described in other cohorts [14,15]. However, half of these SARS-CoV-2-exposed children did not develop evidence of infection (seronegative). Recent studies from the US state that socio-econom- ically disadvantaged children, especially those from ethnic minorities, are at higher risk of infection [5,7,8]. Data from the United Kingdom (UK) and the US also show increased morbidity and mortality from SARS- CoV-2 in adults from ethnic minorities and with poor socio-economic status [6,9]. Limited access to health- care systems and migration background might also play a role in spreading the disease [16,17]. www.eurosurveillance.org License, supplementary material and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made. 5. Goyal MK, Simpson JN, Boyle MD, Badolato GM, Delaney M, McCarter R, et al. Racial and/or ethnic and socioeconomic disparities of SARS-CoV-2 infection among children. Pediatrics. 2020;146(4):e2020009951. https://doi.org/10.1542/ peds.2020-009951 PMID: 32759379 Any supplementary material referenced in the article can be found in the online version. 6. Smith AR, DeVies J, Caruso E, Radhakrishnan L, Sheppard M, Stein Z, et al. Emergency department visits for COVID-19 by race and ethnicity - 13 States, October-December 2020. MMWR Morb Mortal Wkly Rep. 2021;70(15):566-9. https://doi. org/10.15585/mmwr.mm7015e3 PMID: 33857062 This article is copyright of the authors or their affiliated in- stitutions, 2022. 7. Moreira A, Chorath K, Rajasekaran K, Burmeister F, Ahmed M, Moreira A. Demographic predictors of hospitalization and mortality in US children with COVID-19. Eur J Pediatr. 2021;180(5):1659-63. https://doi.org/10.1007/s00431-021- 03955-x PMID: 33474580 8. Bailey LC, Razzaghi H, Burrows EK, Bunnell HT, Camacho PEF, Christakis DA, et al. Assessment of 135 794 pediatric patients tested for severe acute respiratory syndrome coronavirus 2 across the United States. JAMA Pediatr. 2021;175(2):176-84. https://doi.org/10.1001/jamapediatrics.2020.5052 PMID: 33226415 9. Mathur R, Rentsch CT, Morton CE, Hulme WJ, Schultze A, MacKenna B, et al. , OpenSAFELY Collaborative. 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Clustering and longitudinal change in SARS-CoV-2 seroprevalence in school children in the canton of Zurich, Switzerland: prospective cohort study of 55 schools. BMJ. 2021;372(616):n616. https://doi.org/10.1136/bmj.n616 PMID: 33731327 13. Robert Koch Institute (RKI). Coronavirus Fallzahlen. [Coronavirus case numbers]. Berlin: RKI. [Accessed: 21 Oct 2021]. German. Available from: https://www.rki.de/DE/ Content/InfAZ/N/Neuartiges_Coronavirus/Fallzahlen.html 14. Ethical statement The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Ruhr-University Bochum (04/2020, No 20/6027). 5 Funding statement BMBF funding registration 01KI20173 (Corkid). Conflict of interest None declared. In keeping with data from other studies on seropositiv- ity in children and families [1,2,11,12] and with observa- tions in other populations with a migration background e.g. in the UK or the US [5-8], we here provide data from a region of Germany with a high percentage of families with a migration background. We assume that Funding statement BMBF funding registration 01KI20173 (Corkid). Conflict of interest None declared. Funding statement BMBF funding registration 01KI20173 (Corkid). www.eurosurveillance.org 5 Dataset: preliminary findings from a novel passive surveillance system of COVID-19. Int J Epidemiol. 2021;50(3):743-52. https://doi.org/10.1093/ije/dyab057 PMID: 33837417 References 1. Hippich M, Sifft P, Zapardiel-Gonzalo J, Böhmer MM, Lampasona V, Bonifacio E, et al. A public health antibody screening indicates a marked increase of SARS-CoV-2 exposure rate in children during the second wave. 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Brinkmann F, Diebner HH, Matenar C, Schlegtendal A, Spiecker J, Eitner L, et al. Longitudinal rise in seroprevalence of SARS- CoV-2 infections in children in western Germany-a blind spot in epidemiology? Infect Dis Rep. 2021;13(4):957-64. https://doi. org/10.3390/idr13040088 PMID: 34842714 4. Brinkmann F, Schlegtendal A, Hoffmann A, Theile K, Hippert F, Strodka R, et al. SARS-CoV-2 infections among children and adolescents with acute infections in the Ruhr region. Dtsch Arztebl Int. 2021;118(21):363-4. https://doi.org/10.3238/ arztebl.m2021.0227 PMID: 34247701 Authors’ contributions Conceptualisation F.B. C.M., T.L.; methodology H.D., N.T; validation H.D., N.T.; formal analysis, C.M., H.D.; investi- gation, F.B., C.M., A.S, L.E. ; resources, T.L.; data curation C.M., C.M.; writing—original draft preparation, F.B. ; writ- ing—review and editing, H.D., A.S. L.E. C.M. N.T C.M., T.L.; supervision T.L. N.T.; project administration F.B., C.M.; fund- ing acquisition, T.L. All authors have read and agreed to the published version of the manuscript. 15. Galow L, Haag L, Kahre E, Blankenburg J, Dalpke AH, Lück C, et al. Lower household transmission rates of SARS-CoV-2 from children compared to adults. J Infect. 2021;83(1):e34-6. https://doi.org/10.1016/j.jinf.2021.04.022 PMID: 33930468 16. Lee EH, Kepler KL, Geevarughese A, Paneth-Pollak R, Dorsinville MS, Ngai S, et al. Race/ethnicity among children with COVID-19-associated multisystem inflammatory syndrome. JAMA Netw Open. 2020;3(11):e2030280. https:// doi.org/10.1001/jamanetworkopen.2020.30280 PMID: 33252688 17. Saatci D, Ranger TA, Garriga C, Clift AK, Zaccardi F, Tan PS, et al. Association between race and COVID-19 outcomes among 2.6 million children in England. JAMA Pediatr. 2021;175(9):928- 38. https://doi.org/10.1001/jamapediatrics.2021.1685 PMID: 34152371 License, supplementary material and copyright Hall JA, Harris RJ, Zaidi A, Woodhall SC, Dabrera G, Dunbar JK. HOSTED-England’s Household Transmission Evaluation 6 www.eurosurveillance.org
https://openalex.org/W2553381844	https://link.springer.com/content/pdf/10.1007%2Fs00018-016-2421-9.pdf	English	null	A threshold of transmembrane potential is required for mitochondrial dynamic balance mediated by DRP1 and OMA1	Cellular and molecular life sciences	2,016	cc-by	13,874	& Robert Gilkerson robert.gilkerson@utrgv.edu 1 Department of Biology, The University of Texas Rio Grande Valley, 1201 West University Drive, Edinburg, TX 78539-2999, USA E. Jones and N. Gaytan contributed equally. Edith Jones1 • Norma Gaytan1 • Iraselia Garcia1 • Alan Herrera1 • Manuel Ramos1 • Divya Agarwala1 • Maahrose Rana1 • Wendy Innis-Whitehouse2 • Erin Schuenzel1 • Robert Gilkerson1,3 Edith Jones1 • Norma Gaytan1 • Iraselia Garcia1 • Alan Herrera1 • Manuel Ramos1 • Divya Agarwala1 • Maahrose Rana1 • Wendy Innis-Whitehouse2 • Erin Schuenzel1 • Robert Gilkerson1,3 Received: 1 April 2016 / Revised: 6 November 2016 / Accepted: 14 November 2016 / Published online: 17 November 2016 The Author(s) 2016. This article is published with open access at Springerlink.com Abstract As an organellar network, mitochondria dynamically regulate their organization via opposing fusion and ﬁssion pathways to maintain bioenergetic homeostasis and contribute to key cellular pathways. This dynamic balance is directly linked to bioenergetic function: loss of transmembrane potential across the inner membrane (Dwm) disrupts mitochondrial ﬁssion/fusion balance, causing fragmentation of the network. However, the level of Dwm required for mitochondrial dynamic balance, as well as the relative contributions of ﬁssion and fusion pathways, have remained unclear. To explore this, mito- chondrial morphology and Dwm were examined via confocal imaging and tetramethyl rhodamine ester (TMRE) ﬂow cytometry, respectively, in cultured 143B osteosar- coma cells. When normalized to the TMRE value of untreated 143B cells as 100%, both genetic (mtDNA-de- pleted q0) and pharmacological [carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated] cell models below 34% TMRE ﬂuorescence were unable to maintain mitochondrial interconnection, correlating with loss of fusion-active long OPA1 isoforms (L-OPA1). Mechanisti- cally, this threshold is maintained by mechanistic coordination of DRP1-mediated ﬁssion and OPA1-medi- ated fusion: cells lacking either DRP1 or the OMA1 metalloprotease were insensitive to loss of Dwm, instead maintaining an obligately fused morphology. Collectively, these ﬁndings demonstrate a mitochondrial ‘tipping point’ threshold mediated by the interaction of Dwm with both DRP1 and OMA1; moreover, DRP1 appears to be required for effective OPA1 maintenance and processing, consistent with growing evidence for direct interaction of ﬁssion and fusion pathways. These results suggest that Dwm below threshold coordinately activates both DRP1-mediated ﬁs- sion and OMA1 cleavage of OPA1, collapsing mitochondrial dynamic balance, with major implications for a range of signaling pathways and cellular life/death events. Keywords Oxidative phosphorylation mtDNA S-OPA1 Protonophore Proteolytic cleavage E. Jones and N. Gaytan contributed equally. Electronic supplementary material The online version of this article (doi:10.1007/s00018-016-2421-9) contains supplementary material, which is available to authorized users. 3 Department of Clinical Laboratory Sciences, The University of Texas Rio Grande Valley, 1201 West University Drive, Edinburg, TX 78539-2999, USA Cell. Mol. Life Sci. (2017) 74:1347–1363 DOI 10.1007/s00018-016-2421-9 Cell. Mol. Life Sci. (2017) 74:1347–1363 DOI 10.1007/s00018-016-2421-9 Cellular and Molecular Life Sciences ORIGINAL ARTICLE ORIGINAL ARTICLE 2 Department of Biomedical Sciences, The University of Texas Rio Grande Valley, Edinburg, TX 78539-2999, USA Introduction Mitochondria have emerged as a highly responsive organellar network that dynamically balances between two states: a collection of individual spherical organelles, or an elaborately interconnected reticular network [1, 2], thus adapting its organization to directly integrate into crucial cellular processes including metabolism, apoptosis, autop- hagy, and proliferation [3]. Moreover, mitochondrial dynamics are directly linked with bioenergetic function in an integrated structure/function relationship: loss of trans- membrane potential (Dwm), which is critical to oxidative phosphorylation (OxPhos), causes collapse of structural homeostasis, leading to complete fragmentation of the mitochondrial network [4]. Despite this, it has remained unclear what level of bioenergetic function is required for mitochondrial dynamic balance, as well as the contributing mechanisms necessary. Here, our results reveal a ‘tipping point’ threshold of mitochondrial structure/function. w p y Fission/fusion dynamics are integral to mitochondrial participation in vital cellular processes: ﬁssion is required for stemness [22], mitosis [23, 24], apoptosis [25], and autophagy [26, 27], while fusion is a necessary adaptation to nutrient starvation and increased metabolic demand [3], allowing transmission of Dwm along inter- connected mitochondria [1]. Moreover, disruption of mitochondrial dynamics and bioenergetics are emerging in prevalent diseases such as heart failure and neurode- generative disorders [28, 29]. As such, the interaction of Dwm and ﬁssion/fusion balance is likely to have major impact as an underlying mechanism of prevalent human disease. Mitochondrial dynamics employ an elegant balance of fusion and ﬁssion pathways, with each process mediated by a distinct set of interacting factors. Fusion is carried out by optic atrophy-1 (OPA1), a dynamin-related GTPase [5] that mediates fusion of the inner membrane [6, 7], while fusion of the outer mitochondrial membrane is accomplished by mitofusins 1 (MFN1) and 2 (MFN2). Fusion of the mito- chondrial outer and inner membranes allows individual organelles to exchange components [4] and permits com- plementation between functional and dysfunctional organelles [8]. The opposing process, ﬁssion, is mediated by dynamin-related protein-1 (DRP1) [9], which is recruited to the mitochondrial outer membrane by inter- acting proteins FIS1 [10, 11] and MFF1 [12] where it forms an oligomeric ring, dividing mitochondria at discrete sites. As these processes are carried out by different sets of interacting factors, inhibition of one pathway causes an increase in the other: inhibition of DRP1-mediated ﬁssion causes unopposed mitochondrial fusion [13, 14]. Edith Jones1 • Norma Gaytan1 • Iraselia Garcia1 • Alan Herrera1 • Manuel Ramos1 • Divya Agarwala1 • Maahrose Rana1 • Wendy Innis-Whitehouse2 • Erin Schuenzel1 • Robert Gilkerson1,3 Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrazine DAPI Diaminophenylindole DMEM Dulbecco’s Modiﬁed Eagle’s medium DMSO Dimethylsulfoxide EtBr Ethidium bromide FBS Fetal bovine serum MEF Mouse embryonic ﬁbroblast mtDNA Mitochondrial DNA OxPhos Oxidative phosphorylation PBS Phosphate-buffered saline TMRE Tetramethyl rhodamine ester Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrazine DAPI Diaminophenylindole DMEM Dulbecco’s Modiﬁed Eagle’s medium DMSO Dimethylsulfoxide EtBr Ethidium bromide FBS Fetal bovine serum MEF Mouse embryonic ﬁbroblast mtDNA Mitochondrial DNA OxPhos Oxidative phosphorylation PBS Phosphate-buffered saline TMRE Tetramethyl rhodamine ester 2 Department of Biomedical Sciences, The University of Texas Rio Grande Valley, Edinburg, TX 78539-2999, USA 2 Department of Biomedical Sciences, The University of Texas Rio Grande Valley, Edinburg, TX 78539-2999, USA 3 Department of Clinical Laboratory Sciences, The University of Texas Rio Grande Valley, 1201 West University Drive, Edinburg, TX 78539-2999, USA 12 3 1348 E. Jones et al. on both chromosomal and mitochondrial DNA (mtDNA). Strikingly, cells with partial or complete loss of Dwm are unable to maintain an interconnected, reticular mitochon- drial morphology: cells depleted of mtDNA (q0 cells), as well as cells carrying mtDNA mutations affecting Dwm, show completely fragmented mitochondrial ultrastructure [16–19], while pharmacological dissipation of Dwm via the protonophore CCCP and the ionophore valinomycin also cause fragmentation of the mitochondrial network [4, 20]. While the mitochondria of mtDNA-depleted q0 cells show a weak ability to fuse and exchange contents via ‘kiss-and- run’ events [21], both q0 and CCCP-treated cells are unable to maintain ﬁssion/fusion balance, with their mitochondria existing instead as a fragmented population of organelles [4, 21]. Despite the importance of Dwm to mitochondrial structure/function homeostasis, however, it is unclear i) what level of Dwm is required for maintenance of mito- chondrial ﬁssion/fusion balance, and ii) the relative contribution of the opposing fusion and ﬁssion processes to Dwm-dependent mitochondrial dynamics in the cell. 123 Cells with low Dwm lack reticular mitochondrial organization Previous studies have found that cells with OxPhos defects have complete fragmentation of the mitochondrial network [4, 16, 21]. To conﬁrm these ﬁndings, we examined both genetic and pharmacological cell models of Dwm loss in a 143B osteosarcoma cell background. 143B206q0 cells have been depleted of all mtDNA through treatment with ethidium bromide (EtBr) [36], resulting in loss of mtDNA- encoded polypeptides, Dwm, and OxPhos function [16, 19]. The protonophore CCCP allows for complete dissipation of the proton gradient across the mitochondrial inner mem- brane, providing a pharmacological method to eliminate Dwm. To examine mitochondrial morphology in WT, q0, and CCCP-treated WT lines, cells were immunolabeled for the TOM20 protein, which is located in the mitochondrial outer membrane, providing imaging of the mitochondrial network in these cells. Mitochondrial localization was conﬁrmed by colocalization with MitoTracker (not shown). WT cells visualized by confocal ﬂuorescence microscopy revealed a balance of both fusion and ﬁssion, with highly interconnected, reticular mitochondria existing in the same cell with fragmented individual mitochondria. Conversely, q0 cells displayed fragmented, somewhat swollen, mito- chondria, while WT cells treated with 10 lM CCCP for 1 h also showed complete fragmentation of the mitochondrial network (Fig. 1a). MtDNA content was veriﬁed by PCR from total DNA of indicated cell lines. Ampliﬁcation of mtDNA nt7130-8113 revealed a strong band in both WT and CCCP-treated WT cells, while q0 cells showed no mtDNA present (Fig. 1b). Western blot analysis showed that the mtDNA-encoded cytochrome c oxidase II Intriguingly, q0 cells maintain a modest, intermediate TMRE signal, statistically greater than that of WT cells treated with 10 lM CCCP [1197 ± 179 versus 385 ± 26 a.u., respectively (Fig. 1e)], likely due to reversal of the F1F0 ATP synthase [37]. Despite this, both have com- pletely fragmented mitochondrial morphology (Fig. 1a). This suggests that a minimum level of Dwm, greater than that seen in either q0 or CCCP-treated WT cells, is required for reticular mitochondrial organization. To explore this, WT cells were incubated with a range of concentrations of CCCP for 1 h, and mitochondrial morphology visualized by anti-TOM20 immunolabeling, as above (Fig. 2a). At 0, 0.1, and 1 lM CCCP, cells displayed full ability to main- tain mitochondrial interconnection, as reticular mitochondria were clearly evident. At 10 lM CCCP, however, no cells were observed with reticular mitochon- dria, as all cells had fragmented mitochondria (Fig. Introduction In a unique organellar structure/function relationship, ﬁs- sion/fusion dynamics are increasingly linked to mitochondrial bioenergetics via the transmembrane potential across the inner membrane (Dwm) [15]. In the classical mitochondrial genetic threshold effect, cells can withstand a high mtDNA mutation load (often up to 80–90% mutant mtDNA) and maintain full OxPhos function, but when the overall proportion of mutant mtDNA exceeds a critical threshold, mitochondrial bioen- ergetic function collapses, leading to a variety of systemic and tissue-speciﬁc pathologies [30, 31]. Previously, we showed that heteroplasmic cells (carrying both WT and mutant mtDNAs) above 80% mutation load have com- pletely fragmented mitochondria, while cells below 80% mutation load have effective ﬁssion/fusion balance [17], suggesting that a functional threshold of Dwm is required to maintain effective ﬁssion/fusion balance. Intriguingly, the OPA1 fusion factor undergoes cleavage and inactivation by the OMA1 metalloprotease in response to dissipation of Dwm, causing mitochondrial fragmentation [32–34]; how- ever, the level of Dwm required and the necessity of coordinating OPA1 cleavage with other ﬁssion/fusion factors in balancing mitochondrial dynamics and function remains unclear. Mitochondrial ATP production is accomplished by the ﬁve OxPhos complexes in the mitochondrial inner mem- brane. Complexes I–IV utilize electron transfer to generate the proton-motive Dwm that is used by Complex V, the F1F0 ATP synthase, to create ATP from ADP and Pi. These complexes are composed of polypeptide subunits encoded 123 1349 A threshold of transmembrane potential is required for mitochondrial dynamic balance… To explore this, we employed genetic and pharmaco- logical models of decreased Dwm to determine the functional and mechanistic requirements for effective mitochondrial ﬁssion/fusion dynamic balance in human cells. Cultured cells grown in high-glucose media obtain ATP primarily via glycolysis, allowing the growth and study of cells with genetic or pharmacological defects in bioenergetics [35]. Using TMRE ﬂow cytometry to moni- tor Dwm, our data indicate that cells below 34% of untreated wild-type (WT) TMRE values cannot maintain mitochondrial dynamic balance, with concomitant loss of fusion-active OPA1: this threshold is mediated by DRP1 and OMA1, reﬂecting a crucial breakpoint of Dwm as a determinant of mitochondrial homeostasis, with severe impacts on cell viability and broad relevance to human disease. (MTCO2) protein was present in WT and CCCP-treated WT cells, but absent in q0 cells, as expected (Fig. 1c). Consistent with this, immunolabeling microscopy showed strong MTCO2 signal in WT and CCCP-treated WT cells, but not in q0 cells (not shown). Cells below a threshold of 34% TMRE ﬂuorescence cannot maintain mitochondrial interconnection Cells with low Dwm lack reticular mitochondrial organization Introduction To determine Dwm, cells were given media containing the Dwm-dependent dye tetramethyl rhodamine ester (TMRE) and assayed by ﬂow cytometry (representative histograms, Fig. 1d). This assay is a novel adaptation of our previous method [19]. WT cells show a robust average ﬂuorescence of 4172 ± 172 arbi- trary units (a.u.), while q0s and CCCP-treated WT cells maintain signiﬁcantly lower values of 1197 ± 179 a.u and 385 ± 26 a.u., respectively (Fig. 1e). These values are consistent with those reported for q0 cells via rhodamine- based evaluation of Dwm [21]. Collectively, these ﬁndings demonstrate that either genetic or pharmacological decreases in Dwm cause complete fragmentation of the mitochondrial network, consistent with previous ﬁndings [4, 16]. Results Cells below a threshold of 34% TMRE ﬂuorescence cannot maintain mitochondrial interconnection Cells with low Dwm lack reticular mitochondrial organization WT 0 WT+CCCP A detail WT+CCCP WT 0 A TMRE fluorescence (a.u.) 4000 3000 2000 1000 WT 0 WT+CCCP 0 E E B C WT 0 WT+CCCP MTCO2 -tubulin WT 0 WT+CCCP mtDNA nt7130-8113 E B WT 0 WT+CCCP D D provides loading control. d Representative histograms of WT, q0, and CCCP-treated WT cells incubated with 100 nM TMRE and assayed via ﬂow cytometry. 50,000 cells assayed for TMRE ﬂuorescence (X- axis) in each experiment. Y-axis indicates number of cells at ﬂuorescence values expressed in arbitrary units (a.u.). e Average TMRE ﬂuorescence of WT, q0, and CCCP-treated WT cells in arbitrary units (a.u.). Values represent average TMRE ﬂuorescence of indicated cell lines in n [ 3 experiments ± standard error (SE). Statistical signiﬁcance, p \ 0.01, Tukey’s post hoc test following one-way ANOVA Fig. 1 Mitochondrial morphology and bioenergetics in human 143B osteosarcoma cells. a Cultured 143B cells visualized by confocal ﬂuorescence microscopy. WT, mtDNA-depleted q0, and CCCP- treated (10 lM, 1 h) WT cells were immunolabeled for mitochondrial TOM20 (green). Nuclei stained with DAPI (blue). Outlined box in Merge (white) is enlarged in Detail panel. Size bar 2 lm. n = 3 experiments. b Polymerase chain reaction of mtDNA. MtDNA- speciﬁc primers were used to amplify nt7130-8113 of human mtDNA from 100 ng of total cellular DNA isolated from WT, q0, and CCCP- treated WT cells. n = 3 experiments. c Anti-MTCO2 Western blotting of lysates from cultured 143B cell lines. Anti-a-tubulin morphologies (51 ± 15%), with a small (4 ± 3%) pro- portion of cells with predominantly fragmented mitochondria. On the other hand, cells treated with 10 lM CCCP showed 100% fragmented morphology (Fig. 2c; Table 1), demonstrating the spectrum of mitochondrial morphology and Dwm in 143B cells. Strikingly, cells with intermediate Dwm levels showed a clear threshold effect: at 0, 2, and 4 lM CCCP, [90% of cells assayed had either reticular or intermediate morphologies, with less than 15% of cells having a fragmented morphology at these mitochondrial morphology, cells treated with the indicated concentrations of CCCP were immunolabeled for TOM20. mitochondrial morphology, cells treated with the indicated concentrations of CCCP were immunolabeled for TOM20. For quantitation, individual cells were scored as having predominantly reticular, fragmented, or intermediate mitochondrial morphologies, as elsewhere [34, 38–40]. Cells with low Dwm lack reticular mitochondrial organization 2a), indicating that mitochondrial ﬁssion/fusion balance is lost between 1 and 10 lM CCCP. To further probe this hypothesis, cells were treated with CCCP from 2 to 10 lM, and TMRE ﬂow cytometry was used to assay Dwm as in Fig. 1e. When normalized to untreated WT cells, CCCP at increasing concentrations produced stepwise decreases in TMRE ﬂuorescence, deﬁning a range from untreated (100%) to 10 lM CCCP-treated (9%). Critically, cells incubated with 4.75 lM CCCP showed a normalized TMRE signal of 34 ± 5% relative to untreated cells, while cells incubated with 5 lM CCCP had TMRE signal of 25 ± 5% and q0 cells 29 ± 4% (Fig. 2b). To explore the impact of these stepwise decreases in Dwm on 12 1350 E. Jones et al. WT 0 WT+CCCP WT 0 WT+CCCP TMRE fluorescence (a.u.) 4000 3000 2000 1000 WT 0 WT+CCCP 0 A B E C D WT 0 WT+CCCP MTCO2 -tubulin WT 0 WT+CCCP mtDNA nt7130-8113 detail Mitochondrial morphology and bioenergetics in human 143B arcoma cells. a Cultured 143B cells visualized by confocal cence microscopy. WT, mtDNA-depleted q0, and CCCP- (10 lM, 1 h) WT cells were immunolabeled for mitochondrial 0 (green). Nuclei stained with DAPI (blue). Outlined box in (white) is enlarged in Detail panel. Size bar 2 lm. n = 3 ments. b Polymerase chain reaction of mtDNA. MtDNA- c primers were used to amplify nt7130-8113 of human mtDNA 00 ng of total cellular DNA isolated from WT, q0, and CCCP- WT cells. n = 3 experiments. c Anti-MTCO2 Western g of lysates from cultured 143B cell lines. Anti-a-tubulin provides loading control. d Representative histograms of WT, q0, and CCCP-treated WT cells incubated with 100 nM TMRE and assayed via ﬂow cytometry. 50,000 cells assayed for TMRE ﬂuorescence (X- axis) in each experiment. Y-axis indicates number of cells at ﬂuorescence values expressed in arbitrary units (a.u.). e Average TMRE ﬂuorescence of WT, q0, and CCCP-treated WT cells in arbitrary units (a.u.). Values represent average TMRE ﬂuorescence of indicated cell lines in n [ 3 experiments ± standard error (SE). Statistical signiﬁcance, p \ 0.01, Tukey’s post hoc test following one-way ANOVA E. Jones et al. Cells with low Dwm lack reticular mitochondrial organization When scoring cell proﬁles, individual cells with more than two regions of either reticular or fragmented mitochondrial organization were scored as intermediate. As expected, untreated cells showed a high proportion of cells with predominantly reticular (44 ± 17%) and intermediate For quantitation, individual cells were scored as having predominantly reticular, fragmented, or intermediate mitochondrial morphologies, as elsewhere [34, 38–40]. When scoring cell proﬁles, individual cells with more than two regions of either reticular or fragmented mitochondrial organization were scored as intermediate. As expected, untreated cells showed a high proportion of cells with predominantly reticular (44 ± 17%) and intermediate 123 A threshold of transmembrane potential is required for mitochondrial dynamic balance… 1351 A B D C E WT CCCP: 0 0.1 M 1 M 10 M WT CCCP: 4 M 5 M detail detail TMRE (%) 100 80 60 40 CCCP: 0 2 4 4.25 4.5 4.75 5 6 8 10 M 0 20 100 80 60 40 20 % of cells retic. frag. intermed. CCCP: 0 2 4 4.25 4.5 4.75 5 6 8 10 M TMRE fluorescence (a.u.) 4000 3000 2000 1000 WT +5 M +10 M CCCP CCCP 0 1 hr. incub. 4 hrs. incub. NS NS NS Fig. 2 Mitochondrial morphology and Dwm monitored across CCCP titration. a WT cells incubated with 0, 0.1, 1, or 10 lM CCCP for 1 h were then ﬁxed and immunolabeled for mitochondrial TOM20 (green), followed by DAPI staining of nuclei (blue). Outlined boxes (white) enlarged in Detail image. Size bar 2 lm. n = 3 experiments. b WT cells were incubated with 0, 2, 4, 4.25, 4.5, 4.75, 5, 6, 8, or 10 lM CCCP for 1 h, as well as q0 cells, and incubated with 100 nM TMRE for 20 min. Average TMRE ﬂuorescence given for each condition, expressed as % of untreated (0 lM CCCP) ± SE. n C 3 experiments for each condition, 50,000 cells assayed per experiment. All values analyzed by one-way ANOVA, p \ 0.01. c Quantitation of mitochondrial morphology across CCCP titration. WT cells were incubated with CCCP as in b for 1 h and processed for TOM20 immunolabeling, as in a. In three independent experiments, cell imaged and scored as having predominantly reticular ( predominantly fragmented (white), or intermediate (gray) mit drial morphology. Average % of cells with indicated morp shown across CCCP concentrations indicated. For analy statistical signiﬁcance, see Table 1. Cells with low Dwm lack reticular mitochondrial organization d WT cells incubated presence of 4 or 5 lM CCCP and immunolabeled for TOM20 (g Size bar 2 lm. e Cells were incubated in the absence or presen or 10 lM CCCP for 1 or 4 h and assayed by TMRE as in b. experiments for each condition, 50,000 cells assayed per exper All values analyzed by one-way ANOVA, p \ 0.01. NS d p [ 0.05, Tukey’s post hoc test A threshold of transmembrane potential is required for mitochondrial dynamic balance… A WT CCCP: 0 0.1 M 1 M 10 M detail A 1 M 0.1 M B TMRE (%) 100 80 60 40 CCCP: 0 2 4 4.25 4.5 4.75 5 6 8 10 M 0 20 NS B D WT CCCP: 4 M 5 M D C E detail 100 80 60 40 20 % of cells retic. frag. intermed. CCCP: 0 2 4 4.25 4.5 4.75 5 6 8 10 M TMRE fluorescence (a.u.) 4000 3000 2000 1000 WT +5 M CCCP 0 1 hr. in 4 hrs. NS E detail TMRE fluorescence (a.u.) 4000 3000 2000 1000 WT +5 M +10 M CCCP CCCP 0 1 hr. incub. 4 hrs. incub. NS NS E C 100 80 60 40 20 % of cells retic. frag. intermed. CCCP: 0 2 4 4.25 4.5 4.75 5 6 8 10 M C Fig. 2 Mitochondrial morphology and Dwm monitored across CCCP titration. a WT cells incubated with 0, 0.1, 1, or 10 lM CCCP for 1 h were then ﬁxed and immunolabeled for mitochondrial TOM20 (green), followed by DAPI staining of nuclei (blue). Outlined boxes (white) enlarged in Detail image. Size bar 2 lm. n = 3 experiments. b WT cells were incubated with 0, 2, 4, 4.25, 4.5, 4.75, 5, 6, 8, or 10 lM CCCP for 1 h, as well as q0 cells, and incubated with 100 nM TMRE for 20 min. Average TMRE ﬂuorescence given for each condition, expressed as % of untreated (0 lM CCCP) ± SE. n C 3 experiments for each condition, 50,000 cells assayed per experiment. All values analyzed by one-way ANOVA, p \ 0.01. c Quantitation of mitochondrial morphology across CCCP titration. WT cells were incubated with CCCP as in b for 1 h and processed for TOM20 immunolabeling, as in a. Cells with low Dwm lack reticular mitochondrial organization In three independent experiments, cells were imaged and scored as having predominantly reticular (black), predominantly fragmented (white), or intermediate (gray) mitochon- drial morphology. Average % of cells with indicated morphology shown across CCCP concentrations indicated. For analysis of statistical signiﬁcance, see Table 1. d WT cells incubated in the presence of 4 or 5 lM CCCP and immunolabeled for TOM20 (green). Size bar 2 lm. e Cells were incubated in the absence or presence of 5 or 10 lM CCCP for 1 or 4 h and assayed by TMRE as in b. n C 3 experiments for each condition, 50,000 cells assayed per experiment. All values analyzed by one-way ANOVA, p \ 0.01. NS denotes p [ 0.05, Tukey’s post hoc test observed between cells treated with 4.75 lM and 5 lM CCCP: at 4.75 lM CCCP, 70% of cells had either reticular or intermediate morphology, while cells treated with 5 lM observed between cells treated with 4.75 lM and 5 lM CCCP: at 4.75 lM CCCP, 70% of cells had either reticular or intermediate morphology, while cells treated with 5 lM concentrations. Conversely, at 5, 6, 8, and 10 lM, at least 80% of cells had predominantly fragmented mitochondrial morphology (Fig. 2c; Table 1). A sharp dropoff was concentrations. Conversely, at 5, 6, 8, and 10 lM, at least 80% of cells had predominantly fragmented mitochondrial morphology (Fig. 2c; Table 1). A sharp dropoff was 12 3 1352 E. Jones et al. Table 1 Quantitation of mitochondrial morphology in CCCP-treated cells CCCP (lM) Reticular Intermediate Fragmented 0 44.4 ± 17 51.3 ± 15 4.3 ± 3 2 39.1 ± 10 58.9 ± 11 1.9 ± 1 4 5.7 ± 3* 83.5 ± 6 14.1 ± 7 4.25 15 ± 6 69 ± 4 16 ± 5 4.5 14.7 ± 2 72.3 ± 1 13 ± 3 4.75 4.7 ± 1 65 ± 3 30.3 ± 3 5 1 ± 1 19.6 ± 4 79.6 ± 5 6 0 ± 0 20.5 ± 8 79.5 ± 2 8 0 ± 0 5.6 ± 2 94.4 ± 2 10 0 ± 0 0 ± 0 100 ± 0 Values represent the average percentage of cells with the indicated morphology ± standard error in three independent experiments,[150 cells scored. Statistical analysis: data analyzed by one-way ANOVA, p \ 0.05. Cells with low Dwm lack reticular mitochondrial organization versus 1197 ± 197, respectively, not signiﬁcant (NS) (Fig. 2b)], and both have fragmented mitochondrial morphology (Figs. 1, 2d). Additional experiments explored whether longer treatments with CCCP alter Dwm status: cells incubated with 5 lM CCCP for 1 versus 4 h showed equivalent TMRE values, as did cells incubated with 10 lM CCCP (Fig. 2e), indicating that increasing CCCP treatment beyond 1 h does not cause further decreases in Dwm. This is consistent with ﬁndings elsewhere that indicate that protonophores such as CCCP act rapidly to dissipate Dwm [41], while longer incubations do not produce additional decreases in Dwm [42]. Cells with low Dwm lack reticular mitochondrial organization * p \ 0.05, ** p \ 0.01, compared to value immediately above, Tukey’s post hoc test. These values were used to generate the bar graph of these data corresponding to Fig. 2c Table 1 Quantitation of mitochondrial morphology in CCCP-treated cells Values represent the average percentage of cells with the indicated morphology ± standard error in three independent experiments,[150 cells scored. Statistical analysis: data analyzed by one-way ANOVA, p \ 0.05. * p \ 0.05, ** p \ 0.01, compared to value immediately above, Tukey’s post hoc test. These values were used to generate the bar graph of these data corresponding to Fig. 2c and the plasma membrane [45]. Gerencser et al. [46] found that the resting Dwm of cultured mammalian cells in high- glucose medium is -139 mV, while Springett et al. have successfully employed the oxidation state of mitochondrial bc1 heme groups to quantitatively explore Dwm, but note the lack of additional direct methods to cross-correlate their measurements [47]. Given these caveats, discussion of Dwm levels are here restricted to TMRE ﬂuorescence values, rather than extrapolating these differences to mV. TMRE values are normalized to untreated WT cells (Fig. 2c) to reﬂect the relative change between samples. Assuming this value as 100%, our results show that 4.75 lM CCCP, 5 lM CCCP, and q0 cells have relative TMRE levels of 34, 25 and 29%, respectively. These results indicate that both pharma- cological and genetic loss of Dwm to levels below that of 4.75 lM-treated WT cells cause dramatic loss of reticular mitochondrial organization, representing a critical threshold required for mitochondrial ﬁssion/fusion balance. As mito- chondrial dynamics are governed by distinct ﬁssion and fusion pathways, we next explored the involvement of OPA1, OMA1, and DRP1 in maintaining Dwm-dependent mitochondrial dynamic balance. CCCP showed 80% fragmented morphology (Fig. 2c; Table 1). Representative images demonstrate this striking threshold: at 4 lM CCCP, cells showed extensive regions of mitochondrial interconnection, while at 5 lM CCCP, the mitochondrial network was largely fragmented (Fig. 2d). Similar results were obtained for human HeLa and murine 3T3 cell lines: at 4 lM CCCP, both cell lines displayed clear evidence of mitochondrial interconnection when visualized by anti-TOM20 immunolabeling, while HeLa and 3T3 cells treated with 5 lM CCCP displayed near-complete fragmentation (Suppl. Figure 1). Strikingly, TMRE ﬂuorescence of WT cells treated with 5 lM CCCP and untreated q0 cells are statistically equivalent [1023 ± 194 a.u. OMA1-mediated OPA1 cleavage is required for Dwm-sensitive mitochondrial dynamic balance 2c, d: WT cells treated with 4.25, 4.5, and 4.75 lM CCCP maintain similar L-OPA1 levels to untreated WT cells (45 ± 2, 44 ± 5, and 46 ± 4%, respectively), while WT cells treated with 5 lM CCCP have a signiﬁcantly lower proportion of L-OPA1 (33 ± 3%) (Fig. 3b). These results suggest that [40% L-OPA1 (of total OPA1) is necessary for mitochondrial fusion capability, and further suggests that the Dwm threshold observed for mitochondrial interconnection (Fig. 2) reﬂects the available pool of fusion-active L-OPA1 isoforms. 544 ± 21, respectively (Fig. 3c). Using EtBr treatment per King and Attardi [36], q0 MEF lines were generated in both OMA1?/? and OMA1-/- backgrounds. In both cases, the q0 line had a dramatically decreased Dwm relative to the control MEFs, as assayed by TMRE ﬂow cytometry: OMA1?/? q0 cells had average TMRE ﬂuorescence of 925 ± 42 versus 2298 ± 294 a.u. for control OMA1?/? cells, while OMA1-/- q0 cells had TMRE signal of 503 ± 118 a.u. versus 1227 ± 132 for control OMA1-/- cells (Fig. 3c). We next examined OPA1 status in OMA1?/ ? and OMA1-/- cells in the absence or presence of CCCP. While untreated OMA1?/? and OMA1-/- cells showed equivalent levels of L- and S-OPA1 isoforms, CCCP- treated OMA1?/? cell lysates showed loss of L-OPA1 isoforms and concomitant increase in S-OPA1 isoforms. Conversely, OMA1-/- cells treated with CCCP showed no change in OPA1 isoforms versus untreated OMA1-/- control cell lysates (Fig. 3d). When examined by anti-TOM20 immunolabeling, OMA1?/? MEFs show the expected mix of fragmented and reticular morphologies (Fig. 3d). Conversely, OMA1-/- cells show extensive interconnection (Fig. 3d), indicating that cells lacking the OMA1 metalloprotease retain full mitochondrial fusion capability, despite a low basal Dwm. When treated with CCCP, OMA1?/? cells show the expected fragmented morphology, while OMA1-/- cells retain extensive mitochondrial interconnection in the presence of CCCP (Fig. 3d), consistent with ﬁndings elsewhere [51, 52]. Similarly, OMA1?/? q0 cells show a fragmented morphology, consistent with the results in Figs. 1 and 2, and previous ﬁndings [16, 18]. Strikingly, however, OMA1-/- q0 cells show extensive mitochondrial interconnection (Fig. 3d), despite the loss of Dwm. Quan- titation of morphology conﬁrms this: while OMA1?/? cells show dramatic loss of reticular and intermediate mor- phologies when treated with CCCP, compared with untreated OMA?/? OMA1-mediated OPA1 cleavage is required for Dwm-sensitive mitochondrial dynamic balance As a Nernstian dye that is taken up by actively respiring mitochondria, TMRE has been extensively used to assay Dwm via ﬂow cytometry and quantitative microscopy [43, 44]. Estimation of Dwm from TMRE or other ﬂuores- cence methods involves a range of factors, including the concentration of dye within the mitochondria, the cytosol, and the extracellular space, as well as the overall mass of mitochondria within the cell [44]. Moreover, protonophores such as CCCP impact the potential at both the mitochondria As the mediator of mitochondrial inner membrane fusion, OPA1 has emerged as a key Dwm-sensitive factor in mitochondrial dynamics. OPA1 exists as ﬁve isoforms in human cells (a–e): the a and b long isoforms (L-OPA1) mediate inner membrane fusion, while the c, d, and e short isoforms (S-OPA1) are fusion-inactive [48]. Dissipation of Dwm causes cleavage of fusion-mediating L-OPA1 iso- forms to inactive S-OPA1 by the OMA1 protease, causing 123 123 1353 A threshold of transmembrane potential is required for mitochondrial dynamic balance… loss of mitochondrial fusion [32, 33], while expression of S-OPA1 activates mitochondrial ﬁssion [34]. We, there- fore, next explored OPA1 status in q0 and CCCP-treated 143B models, as in Figs. 1 and 2. Anti-OPA1 Western blotting revealed that WT cells have prominent bands for the L-OPA1 a and b isoforms, with the fusion-inactive S-OPA1 showing a strong band for the d short isoform and minor bands for the c and e isoforms (Fig. 3a). q0 cells show dramatically decreased L-OPA1 isoforms, with increased levels of the c and e S-OPA1 isoforms. CCCP- treated WT cells show even more pronounced loss of L-OPA1 isoforms, with a near-complete loss of the a iso- form and only a faint band for the b isoform, while the S-OPA1 isoforms are increased, with a major band for the e short isoform (Fig. 3a). Quantitation of OPA1 isoforms conﬁrms this: untreated WT cells maintain 43 ± 3% L-OPA1 of total OPA1 signal, while q0 cells (33 ± 2% L-OPA1) and 10 lM CCCP-treated WT cells (20 ± 3%), show signiﬁcant decreases in the proportion of L-OPA1 relative to untreated WTs. OPA1 blotting at intermediate CCCP concentrations mirrors the morphological threshold observed in Fig. OMA1-mediated OPA1 cleavage is required for Dwm-sensitive mitochondrial dynamic balance d Anti-OPA1 Western blotting of lysates from OMA1?/ ? and -/- MEFs. MEFs were incubated in the absence or presence of 20 lM CCCP for 1 h and lysates were prepared, followed by blotting to PVDF and blotting with anti-OPA1. n = 3 experiments. e Mito- chondrial morphology of OMA1?/? and OMA1-/- MEFs. Untreated, CCCP-treated (20 lM, 1 h), or mtDNA-depleted q0 MEFs in each background were immunolabeled for mitochondrial TOM20 (green) and counterstained with DAPI (blue). n = 3 experiments. Size bar 2 lm. f Quantitation of mitochondrial morphology OMA1?/? and OMA1-/- MEFs in the absence or presence of CCCP. As in Fig. 2c, cells were imaged and scored as having predominantly reticular (black), predominantly fragmented (white), or intermediate (gray) mitochondrial morphology. Average % of cells with indicated morphology in n = 3 experiments A OPA1 WT 0 WT+CCCP L S B A C TMRE fluorescence (a.u.) OMA1+/+ OMA1-/- * * 2000 1500 1000 500 0 2500 * * untreated 0 +CCCP C E E D OMA1+/+ OMA1-/- CCCP: - + - + OPA1 D F 100 80 60 40 20 % of cells retic. frag. intermed. OMA1+/+ OMA1-/- CCCP: - + - + F F signiﬁcance, p \ 0.01, one-way ANOVA followed by Dunnett’s post hoc test. d Anti-OPA1 Western blotting of lysates from OMA1?/ ? and -/- MEFs. MEFs were incubated in the absence or presence of 20 lM CCCP for 1 h and lysates were prepared, followed by blotting to PVDF and blotting with anti-OPA1. n = 3 experiments. e Mito- chondrial morphology of OMA1?/? and OMA1-/- MEFs. Untreated, CCCP-treated (20 lM, 1 h), or mtDNA-depleted q0 MEFs in each background were immunolabeled for mitochondrial TOM20 (green) and counterstained with DAPI (blue). n = 3 experiments. Size bar 2 lm. f Quantitation of mitochondrial morphology OMA1?/? and OMA1-/- MEFs in the absence or presence of CCCP. As in Fig. 2c, cells were imaged and scored as having predominantly reticular (black), predominantly fragmented (white), or intermediate (gray) mitochondrial morphology. Average % of cells with indicated morphology in n = 3 experiments Fig. 3 Role of OPA1 and OMA1 in Dwm-dependent mitochondrial dynamics. a Anti-OPA1 Western blotting of 143B cells. Lysates were prepared from WT, q0, and 10 lM CCCP-treated WT cells, followed by blotting with anti-OPA1 monoclonal antibody. L-OPA1 (a, b) and S-OPA1 (c–e) isoforms labeled as indicated. n = 3 experiments. OMA1-mediated OPA1 cleavage is required for Dwm-sensitive mitochondrial dynamic balance cells, OMA1-/- cells do not show a signiﬁcant change in the proportion of cells with reticular or intermediate morphologies when treated with CCCP (Fig. 3f). These results demonstrate that cells lacking OMA1 are insensitive to loss of Dwm, retaining a fused mitochondrial network when challenged with either phar- macological or genetic loss of Dwm. However, we note that CCCP-treated OMA1-/- cells show an overall decrease in the proportion of cells’ reticular mitochondria (21 ± 13 versus 40 ± 3% for untreated OMA1-/- cells) (Fig. 3f). While this decrease is not statistically signiﬁcant, it does suggest that additional factors may be involved. As the OMA1 metalloprotease has emerged as a critical regulator of stress-sensitive OPA1-mediated mitochondrial fusion, we next examined the role of OMA1 in this process. While OPA1 is cleaved by a variety of proteases including YME1 [48–50], AFG3L1, and AFG3L2 [33], only OMA1 has been shown to mediate Dwm-sensitive cleavage of OPA1 [32, 33], suggesting OMA1 as a likely regulator of the observed Dwm threshold of mitochondrial ﬁssion/fusion balance. To explore this, experiments examined pharma- cological and genetic Dwm loss, as above, in mouse embryonic ﬁbroblasts (MEFs) either carrying (OMA1?/?) or ablated for (OMA1-/-) the OMA1 gene (kind gift of Dr. Carlos Lopez-Otin, University of Oviedo). When assayed via TMRE ﬂow cytometry, OMA?/? cells show an average ﬂuorescence of 2298 ± 294 a.u. Strikingly, OMA1-/- cells show a signiﬁcantly decreased TMRE signal of 1227 ± 132 a.u., relative to OMA1?/? cells (Fig. 3c), indicating that these cells have a low basal Dwm. Both OMA1?/? and OMA1-/- cell lines showed massive decreases in TMRE ﬂuorescence when treated with 20 lM CCCP, with average ﬂuorescence values of 567 ± 50 and These ﬁndings strongly indicate that L-OPA1 is a key determinant of the Dwm-dependent mitochondrial ﬁs- sion/fusion dynamics observed in Fig. 2. To explore this further, we incubated WT cells with 5 and 10 lM CCCP, as in Fig. 2, followed by recovery in CCCP-free media, and 123 12 3 E. Jones et al. 1354 A B C E D F OPA1 WT 0 WT+CCCP * % L-OPA1 (L-OPA1/ total OPA1) 40 30 20 10 0 50 * * WT 4.25 4.5 4.75 5 M 0 10 M CCCP CCCP L-OPA1 S-OPA1 TMRE fluorescence (a.u.) OMA1+/+ OMA1-/- * * 2000 1500 1000 500 0 2500 * * 100 80 60 40 20 % of cells retic. frag. intermed. OMA1-mediated OPA1 cleavage is required for Dwm-sensitive mitochondrial dynamic balance OMA1+/+ OMA1-/- CCCP: - + - + OMA1+/+ OMA1-/- CCCP: - + - + OPA1 OMA1+/+ OMA1-/- untreated +CCCP 0 untreated +CCCP 0 untreated 0 +CCCP detail detail Fig. 3 Role of OPA1 and OMA1 in Dwm-dependent mitochondrial dynamics. a Anti-OPA1 Western blotting of 143B cells. Lysates were prepared from WT, q0, and 10 lM CCCP-treated WT cells, followed by blotting with anti-OPA1 monoclonal antibody. L-OPA1 (a, b) and S-OPA1 (c–e) isoforms labeled as indicated. n = 3 experiments. b Quantitation of Western blot densitometry using Image J, ± SE. n = 3 experiments. Statistical signiﬁcance, p \ 0.05, one-way ANOVA followed by Dunnett’s post hoc test. c TMRE analysis of OMA1?/? and OMA1-/- mouse embryonic ﬁbroblasts (MEFs). MEFs with (OMA1?/?) and lacking (OMA1-/-) the murine OMA1 gene were incubated in the absence or presence of 20 lM CCCP for 1 h, or in the absence or presence of EtBr for 10 days. Untreated, CCCP- treated, or q0 MEFs were incubated with 100 nM TMRE and TMRE ﬂuorescence quantitated via ﬂow cytometry. Average TMRE ﬂuo- rescence expressed in a.u. ± SE. n C 3 experiments for each condition, 50,000 cells assayed per experiment. Statistical signiﬁcance, p \ 0.01, one-way ANOVA followed by Dunnett’s post hoc test. d Anti-OPA1 Western blotting of lysates from OMA1?/ ? and -/- MEFs. MEFs were incubated in the absence or presence of 20 lM CCCP for 1 h and lysates were prepared, followed by blotting to PVDF and blotting with anti-OPA1. n = 3 experiments. e Mito- chondrial morphology of OMA1?/? and OMA1-/- MEFs. Untreated, CCCP-treated (20 lM, 1 h), or mtDNA-depleted q0 MEFs in each background were immunolabeled for mitochondrial TOM20 (green) and counterstained with DAPI (blue). n = 3 experiments. Size bar 2 lm. f Quantitation of mitochondrial morphology OMA1?/? and OMA1-/- MEFs in the absence or presence of CCCP. As in Fig. 2c, cells were imaged and scored as having predominantly reticular (black), predominantly fragmented (white), or intermediate (gray) mitochondrial morphology. Average % of cells with indicated morphology in n = 3 experiments 1354 E. Jones et al. E * % L-OPA1 (L-OPA1/ total OPA1) 40 30 20 10 0 50 * * WT 4.25 4.5 4.75 5 M 0 10 M CCCP CCCP OMA1+/+ OMA1-/- untreated +CCCP 0 untreated +CCCP 0 detail detail signiﬁcance, p \ 0.01, one-way ANOVA followed by Dunnett’s post hoc test. OMA1-mediated OPA1 cleavage is required for Dwm-sensitive mitochondrial dynamic balance b Quantitation of Western blot densitometry using Image J, ± SE. n = 3 experiments. Statistical signiﬁcance, p \ 0.05, one-way ANOVA followed by Dunnett’s post hoc test. c TMRE analysis of OMA1?/? and OMA1-/- mouse embryonic ﬁbroblasts (MEFs). MEFs with (OMA1?/?) and lacking (OMA1-/-) the murine OMA1 gene were incubated in the absence or presence of 20 lM CCCP for 1 h, or in the absence or presence of EtBr for 10 days. Untreated, CCCP- treated, or q0 MEFs were incubated with 100 nM TMRE and TMRE ﬂuorescence quantitated via ﬂow cytometry. Average TMRE ﬂuo- rescence expressed in a.u. ± SE. n C 3 experiments for each condition, 50,000 cells assayed per experiment. Statistical 123 12 A threshold of transmembrane potential is required for mitochondrial dynamic balance… 1355 contribution of DRP1 to Dwm-dependent mitochondrial dynamic balance. contribution of DRP1 to Dwm-dependent mitochondrial dynamic balance. examined mitochondrial morphology. Untreated WT cells maintained the expected balance of mitochondrial ﬁssion and fusion, while cells incubated with 5 and 10 lM CCCP both showed the extensive mitochondrial fragmentation noted in Fig. 2 (Fig. 4a). When CCCP-treated cells (either 5- or 10 lM-treated) were allowed to recover in fresh media lacking CCCP, extensive mitochondrial interconnection was observed in both, with highly elongated, networked mito- chondria observed (Fig. 4a). Western blotting of CCCP- treated cells showed the expected loss of L-OPA1 isoforms, while CCCP-treated cells allowed to recover in CCCP-free media failed to restore L-OPA1 (Fig. 4b), consistent with the results of Griparic et al. [48]. Thus, while the results of Fig. 3 indicate that L-OPA1 levels correlate with the observed fusion threshold, with OMA1 playing a major role in medi- ating Dwm-dependent mitochondrial dynamic balance, Fig. 4 shows that extensive mitochondrial interconnection can nevertheless be demonstrated in the absence of an abundant pool of fusion-active L-OPA1. Similarly, CCCP- treated OMA1-/- cells show a decreased (though not sta- tistically signiﬁcant) percentage of cells with reticular morphology, relative to untreated OMA1-/- cells (Fig. 3f). Taken together, these ﬁndings suggest that other factors likely play a role in Dwm-dependent mitochondrial dynamic balance. Notably, DRP1 is actively recruited to mitochondria of CCCP-treated cells [53], suggesting that CCCP treatment and recovery cause effective inhibition of DRP1-mediated ﬁssion, causing the observed extensive mitochondrial interconnection in Fig. 4. We, therefore, next explored the Fig. 4 CCCP treatment and recovery restores mitochondrial interconnection. a WT cells were incubated with either 5 or 10 lM CCCP for 1 h without or with subsequent recovery in media lacking CCCP (wash) for 3.5 h [48] followed by anti- TOM20 immunolabeling, n = 3 experiments. b Western blotting of WT cells treated as in a. Lysates were prepared from untreated WT, 10 lM CCCP- treated WT cells, and 10 lM CCCP-treated WT cells allowed to recover in CCCP-free media, followed by blotting with anti- OPA1 and anti-tubulin monoclonal antibodies. L-OPA1 (a, b) and S-OPA1 (c– e) isoforms labeled as indicated. n = 3 experiments DRP1 is required for Dwm-dependent mitochondrial dynamic balance To further explore the mechanism of Dwm-dependent mitochondrial dynamics, we next tested the role of DRP1- mediated ﬁssion in this process. Western blotting for DRP1 in WT, q0, and CCCP-treated WT cell lysates revealed no appreciable differences in DRP1 expression between the different cell lines (Fig. 5a). To explore the functional role of DRP1 in Dwm-sensitive ﬁssion/fusion balance, we examined Dwm loss under both pharmacological inhibition and genetic knockout of DRP1. Mitochondrial morphology was examined in WT and q0 cells in the presence or absence of mdivi-1, a small molecular agent that prevents the oligomerization of DRP1 at the mitochondrial outer membrane [13]. Untreated WT cells displayed the expected mixture of fusion and ﬁssion, while many mdivi-1-treated WT cells showed elaborately interconnected mitochondria, as visualized using MitoTracker (Suppl. Figure 2A). Quantitation of morphology, as in Fig. 2, reﬂected this: 36 ± 3% of untreated WT cells had a predominantly reticular morphology, but mdivi-1 treatment signiﬁcantly increased this to 56 ± 6%. Similarly, the proportion of cells with fragmented mitochondria signiﬁcantly decreased, falling from 11 ± 1% in untreated WT cells to 4 ± 2% in response to mdivi-1 (Suppl. Figure 2B). These A B 5 M CCCP 10 M CCCP wash: - + - + untreated WT detail Tubulin OPA1-S OPA1-L Untr. + CCCP + CCCP + recov. 1 3 A B 5 M CCCP 10 M CCCP wash: - + - + untreated WT detail Tubulin OPA1-S OPA1-L Untr. + CCCP + CCCP + recov. Fig. 4 CCCP treatment and recovery restores mitochondrial interconnection. a WT cells were incubated with either 5 or 10 lM CCCP for 1 h without or with subsequent recovery in media lacking CCCP (wash) for 3.5 h [48] followed by anti- TOM20 immunolabeling, n = 3 experiments. b Western blotting of WT cells treated as in a. Lysates were prepared from untreated WT, 10 lM CCCP- treated WT cells, and 10 lM CCCP-treated WT cells allowed to recover in CCCP-free media, followed by blotting with anti- OPA1 and anti-tubulin monoclonal antibodies. L-OPA1 (a, b) and S-OPA1 (c– e) isoforms labeled as indicated. n = 3 experiments 1 3 A 5 M CCCP 10 M CCCP wash: - + - + untreated WT detail Fig. 4 CCCP treatment and recovery restores mitochondrial interconnection. DRP1 is required for Dwm-dependent mitochondrial dynamic balance a WT cells were incubated with either 5 or 10 lM CCCP for 1 h without or with subsequent recovery in media lacking CCCP (wash) for 3.5 h [48] followed by anti- TOM20 immunolabeling, n = 3 experiments. b Western blotting of WT cells treated as in a. Lysates were prepared from untreated WT, 10 lM CCCP- treated WT cells, and 10 lM CCCP-treated WT cells allowed to recover in CCCP-free media, followed by blotting with anti- OPA1 and anti-tubulin monoclonal antibodies. L-OPA1 (a, b) and S-OPA1 (c– e) isoforms labeled as indicated. n = 3 experiments Fig. 4 CCCP treatment and recovery restores mitochondrial interconnection. a WT cells were incubated with either 5 or 10 lM CCCP for 1 h without or with subsequent recovery in media lacking CCCP (wash) for 3.5 h [48] followed by anti- TOM20 immunolabeling, n = 3 experiments. b Western blotting of WT cells treated as in a. Lysates were prepared from untreated WT, 10 lM CCCP- treated WT cells, and 10 lM CCCP-treated WT cells allowed to recover in CCCP-free media, followed by blotting with anti- OPA1 and anti-tubulin monoclonal antibodies. L-OPA1 (a, b) and S-OPA1 (c– e) isoforms labeled as indicated. n = 3 experiments 5 M CCCP A B B 12 123 1356 E. Jones et al. demonstrate that mdivi-1 inhibits ﬁssion in this causing increased mitochondrial fusion. Untreated ultrastructure, as in Fig. 1. Strikingly, however, mdivi-1- treated q0 cells also show a completely fragmented mor- 100 80 60 40 20 % of cells retic. frag. intermed. A D B C E WT 0 WT+CCCP DRP1 -tubulin HCT116 untreated + CCCP 0 detail DRP1 KO untreated + CCCP 0 DRP1 -tubulin control DRP1 KO CCCP: - + - + TMRE fluorescence (a.u.) 6000 4000 2000 HCT116 DRP1 KO 0 untreated +CCCP detail HCT116 DRP1 KO CCCP: - + - + Dwm-sensitive mitochondrial dynamics require DRP1. a Anti- Western blotting of lysates from cultured 143B cell lines. Anti- n provides loading control. n = 3 experiments. b Anti-DRP blotting of control and DRP1 knockout HCT116 cells or with CCCP treatment (10 lM, 1 h). Anti-a-tubulin loading control. n = 3 experiments. c TMRE ﬂow cytometry 16 and DRP1 knockout cell lines in the absence or presence P (10 lM, 1 h). Average TMRE ﬂuorescence expressed in E. n C 3 experiments for each condition, 50,000 cells per experiment. DRP1 is required for Dwm-dependent mitochondrial dynamic balance Statistical signiﬁcance from corresponding d cell lines, p \ 0.01, Tukey’s post hoc test following one- way ANOVA. d Immunoﬂuorescence of HCT116 control and DRP1 knockout cells. Untreated, CCCP-treated (10 lM, 1 h), and mtDNA- depleted q0 cells in each background were immunolabeled for TOM20 (green) and stained with DAPI (blue). Outlined boxes (white) enlarged in Detail image. Size bar 2 lm. n = 3 experiments. e Quantitation of mitochondrial morphology in HCT116 and DRP1 knockout cells in the absence or presence of CCCP. Cells were imaged and scored as having predominantly reticular (black), predominantly fragmented (white), or intermediate (gray) mitochon- drial morphology, as above (Figs. 2c, 3f). Average % of cells with indicated morphology in n = 3 experiments A D B C WT 0 WT+CCCP DRP1 -tubulin HCT116 untreated + CCCP 0 detail DRP1 KO untreated + CCCP 0 DRP1 -tubulin control DRP1 KO CCCP: - + - + TMRE fluorescence (a.u.) 6000 4000 2000 HCT116 DRP1 KO 0 untreated +CCCP detail A WT 0 WT+CCCP DRP1 -tubulin D A HCT116 B DRP1 -tubulin control DRP1 KO CCCP: - + - + B C DRP1 KO untreated + CCCP 0 DRP1 -tubulin TMRE fluorescence (a.u.) 6000 4000 2000 HCT116 DRP1 KO 0 untreated +CCCP detail C TMRE fluorescence (a.u.) 6000 4000 2000 HCT116 DRP1 KO 0 untreated +CCCP C 0 100 80 60 40 20 % of cells retic. frag. intermed. E HCT116 DRP1 KO CCCP: - + - + E E way ANOVA. d Immunoﬂuorescence of HCT116 control and DRP1 knockout cells. Untreated, CCCP-treated (10 lM, 1 h), and mtDNA- depleted q0 cells in each background were immunolabeled for TOM20 (green) and stained with DAPI (blue). Outlined boxes (white) enlarged in Detail image. Size bar 2 lm. n = 3 experiments. e Quantitation of mitochondrial morphology in HCT116 and DRP1 knockout cells in the absence or presence of CCCP. Cells were imaged and scored as having predominantly reticular (black), predominantly fragmented (white), or intermediate (gray) mitochon- drial morphology, as above (Figs. 2c, 3f). Average % of cells with indicated morphology in n = 3 experiments way ANOVA. d Immunoﬂuorescence of HCT116 control and DRP1 knockout cells. Untreated, CCCP-treated (10 lM, 1 h), and mtDNA- depleted q0 cells in each background were immunolabeled for TOM20 (green) and stained with DAPI (blue). Outlined boxes (white) enlarged in Detail image. Size bar 2 lm. DRP1 and OMA1 coordinately impact mitochondrial fusion and OPA1 Collectively, our ﬁndings indicate that both OMA1 and DRP1 are required for Dwm-dependent mitochondrial dynamic balance, as cells lacking either factor maintain mitochondrial interconnection when challenged with loss of Dwm (Figs. 3, 5). Moreover, WT cells challenged with CCCP followed by recovery in CCCP-free media show extensive reticular mitochondria, despite the lack of abundant L-OPA1 in these cells (Fig. 4). These ﬁndings suggest that OMA1 and DRP1 cooperatively act to mediate Dwm-dependent mitochondrial fusion/ﬁssion balance. However, mdivi-1 may not be sufﬁciently potent to completely inhibit DRP1 ﬁssion activity, particularly if loss of Dwm stimulates DRP1 functional activity, as found elsewhere [39] via phosphorylation at residue Ser656 [54]. To more rigorously test the requirement for DRP1 in Dwm- sensitive mitochondrial dynamics, we examined human HCT116 cells containing or knocked out for DRP1 (kind gift of Dr. Richard Youle, NIH) with pharmacological (CCCP) or genetic (mtDNA-depleted) loss of Dwm. Anti- DRP1 Western blotting of HCT116 control and DRP1 knockout cells conﬁrmed that DRP1 is present in both untreated and CCCP-treated HCT116 cells, while DRP1 knockout cells (without or with CCCP treatment) lack DRP1 (Fig. 5b). TMRE ﬂow cytometry was used as above to monitor Dwm: HCT116 cells maintained a TMRE signal of 5368 ± 551 a.u. while CCCP treatment lowered their TMRE value to 1438 ± 230 a.u. DRP1 knockout cells, however, had an average TMRE ﬂuorescence of 3379 ± 85 a.u., signiﬁcantly lower than control HCT116s, consistent with bioenergetic functional defects in cells with genetically altered ﬁssion or fusion dynamics [55]. CCCP- treated DRP1 knockout cells showed a dramatic decrease in TMRE signal to 1092 ± 72 a.u., conﬁrming that CCCP dissipates Dwm in HCT116 and DRP1KO cells (Fig. 5c). Mitochondrial morphology of HCT116 and DRP1KO cells was monitored via confocal microscopy with anti-TOM20 immunolabeling, as above. While control HCT116 cells maintained a largely intermediate morphology, with regions of both mitochondrial interconnection and frag- mentation, DRP1 knockout cells showed elaborately interconnected mitochondria, as visualized by TOM20 imaging (Fig. 5d). In response to either CCCP treatment or depletion of mtDNA (via EtBr treatment), control HCT116 cells show fragmentation of the mitochondrial network, as in Fig. 1. Conversely, DRP1 knockout cells maintain an extensively interconnected mitochondrial network when treated with CCCP or under mtDNA depletion (Fig. 5d). Quantitation of morphology, as in Figs. DRP1 is required for Dwm-dependent mitochondrial dynamic balance n = 3 experiments. e Quantitation of mitochondrial morphology in HCT116 and DRP1 knockout cells in the absence or presence of CCCP. Cells were imaged and scored as having predominantly reticular (black), predominantly fragmented (white), or intermediate (gray) mitochon- drial morphology, as above (Figs. 2c, 3f). Average % of cells with indicated morphology in n = 3 experiments Fig. 5 Dwm-sensitive mitochondrial dynamics require DRP1. a Anti- DRP1 Western blotting of lysates from cultured 143B cell lines. Anti- a-tubulin provides loading control. n = 3 experiments. b Anti-DRP Western blotting of control and DRP1 knockout HCT116 cells without or with CCCP treatment (10 lM, 1 h). Anti-a-tubulin provides loading control. n = 3 experiments. c TMRE ﬂow cytometry of HCT116 and DRP1 knockout cell lines in the absence or presence of CCCP (10 lM, 1 h). Average TMRE ﬂuorescence expressed in a.u. ± SE. n C 3 experiments for each condition, 50,000 cells assayed per experiment. Statistical signiﬁcance from corresponding untreated cell lines, p \ 0.01, Tukey’s post hoc test following one- results demonstrate that mdivi-1 inhibits ﬁssion in this system, causing increased mitochondrial fusion. Untreated q0 cells show a completely fragmented mitochondrial ultrastructure, as in Fig. 1. Strikingly, however, mdivi-1- treated q0 cells also show a completely fragmented mor- phology, conﬁrmed by quantitation: no q0 cells were 12 123 123 A threshold of transmembrane potential is required for mitochondrial dynamic balance… 1357 observed to have a reticular morphology in either the absence or presence of mdivi-1, (Suppl. Figure 2A, B). Similar results were obtained in CCCP-treated WT cells: mdivi-1-treated WT cells challenged with 5 or 10 lM CCCP also showed total fragmentation of the mitochon- drial network, with no observable organellar interconnection (Suppl. Figure 2C), suggesting that Dwm- dependent fragmentation of the mitochondrial network might be independent of DRP1-mediated ﬁssion. that DRP1 knockout cells are insensitive to loss of Dwm, and indicate that DRP1 plays a major role in maintaining Dwm-dependent mitochondrial ﬁssion/fusion balance. DRP1 and OMA1 coordinately impact mitochondrial fusion and OPA1 50 40 30 20 10 % of reticular cells OMA1-/- 0 untreated +CCCP +recov * A B C D DRP1 >34% m OMA1 DRP1 < 34% m OMA1 OMA1-/- untreated +CCCP +recov detail HCT116 DRP1 KO CCCP: - + - + OPA1 Tubulin 50 40 30 20 10 % of reticular cells OMA1 0 untreated + * A B OMA1-/- untreated +CCCP +recov detail A OMA1-/- untreated +CCCP +recov detail 50 40 30 20 10 % of reticular cells OMA1-/- 0 untreated +CCCP +recov * B B A C HCT116 DRP1 KO CCCP: - + - + OPA1 Tubulin C D DRP1 >34% m OMA1 D DRP1 >34% m OMA1 DRP1 < 34% m OMA1 < 34% m D Fig. 6 Cooperative roles for OMA1 and DRP1 in Dwm-dependent mitochondrial dynamics. a Untreated and CCCP-treated ? recovery OMA1-/- cells were incubated with MitoTracker and visualized by confocal microscopy, n = 3 experiments. b Quantitation of mito- chondrial morphology of cells in a. Percentage of cells with predominantly reticular morphology in untreated versus CCCP- treated ? recovery OMA1-/- cells, n = 3 experiments. Statistical signiﬁcance, p \ 0.01, Student’s t test. c Anti-OPA1 blotting of control and DRP1 knockout HCT116 cells. Cells were incubated in the absence or presence of 10 lM CCCP for 1 h and lysates were Fig. 6 Cooperative roles for OMA1 and DRP1 in Dwm-dependent mitochondrial dynamics. a Untreated and CCCP-treated ? recovery OMA1-/- cells were incubated with MitoTracker and visualized by confocal microscopy, n = 3 experiments. b Quantitation of mito- chondrial morphology of cells in a. Percentage of cells with predominantly reticular morphology in untreated versus CCCP- treated ? recovery OMA1-/- cells, n = 3 experiments. Statistical signiﬁcance, p \ 0.01, Student’s t test. c Anti-OPA1 blotting of control and DRP1 knockout HCT116 cells. Cells were incubated in the absence or presence of 10 lM CCCP for 1 h and lysates were prepared, followed by blotting with anti-OPA1. n = 3 experiments. d Model of Dwm-dependent ﬁssion/fusion balance. An intact Dwm (at or above the 34% TMRE threshold) acts to inhibit both L-OPA1 cleavage by OMA1 and recruitment of DRP1 to mitochondria, resulting in intact mitochondrial fusion. When Dwm falls below the observed threshold, ﬁssion is activated via increased DRP recruitment to the mitochondria, while OMA1 proteolytic activity is activated, rapidly depleting L-OPA1, resulting in a completely fragmented population of individual organelles (Fig. 5d, e). DRP1 and OMA1 coordinately impact mitochondrial fusion and OPA1 2c and 3f, supports this: CCCP-treated HCT116 cells show a dramatic increase in cells with fragmented mitochondria (98 ± 1%) com- pared with untreated HCT116s (24 ± 6%). Conversely, DRP1 knockout cells show overwhelmingly reticular morphology (96 ± 2%) that does not change in response to CCCP treatment (97 ± 2%). These results demonstrate To explore this possibility, we hypothesized that CCCP challenge and recovery would cause an additive increase in mitochondrial interconnection even in the presence of a stable pool of L-OPA1. To test this, we challenged OMA1-/- cells with CCCP treatment and recovery (in CCCP-free media) as in Fig. 4. Untreated OMA1-/- cells showed the expected mixture of reticular and intermediate morphologies expected, as per Fig. 3. Strikingly, however, OMA1-/- cells challenged with CCCP and recovery showed a robust increase in reticular mitochondrial mor- phology, with elaborately interconnected mitochondria apparent (Fig. 6a). Quantitation conﬁrmed this: while 28 ± 2% of untreated OMA1-/- cells had a reticular mitochondrial morphology, OMA1-/- cells given CCCP challenge and recovery showed a signiﬁcant increase, with 45 ± 5% of cells showing a reticular mitochondrial mor- phology. Thus, despite the presence of a stable pool of available Dwm-independent L-OPA1 (Fig. 3d), OMA1-/- cells show an additive increase in mitochondrial intercon- nection when challenged with CCCP and recovery (Fig. 6a, b), strongly indicating that DRP1 and OMA1 together modulate mitochondrial dynamic balance. While OMA1 has been extensively characterized as a key mediator of Dwm-dependent OPA1 cleavage [32–34], we examined OPA1 status in DRP1 knockout and control cells to see whether DRP1 impacts OPA1. To explore this, we examined HCT116 control and DRP1 knockout cells via OPA1 Western blotting. Control HCT116 cells show the expected distribution of OPA1 isoforms, with prominent bands for the b and d isoforms. Upon incubation with CCCP, HCT116 cells show cleavage of L-OPA1 isoforms, resulting in accumulation of S-OPA1 isoforms (Fig. 6c), consistent with the mitochondrial fragmentation found in CCCP-treated HCT116s in Fig. 5d. Untreated DRP1 knockout cells show the ﬁve OPA1 iso- forms, with comparatively lower L-OPA1 levels than 12 123 3 1358 E. Jones et al. OMA1 and DRP1 coordinately mediate Dwm- dependent mitochondrial dynamics As mitochondrial ﬁssion and fusion pathways have distinct sets of mediating factors, either pathway (or both) could be mechanistically responsible for this tipping point threshold. Mitochondrial fusion has been shown to be dependent on Dwm [4], mediated by L-OPA1 isoforms [48] that are cleaved to fusion-inactive S-OPA1 by OMA1 in response to loss of Dwm [32, 33]. These ﬁndings suggested that fusion is the major mediator of Dwm-dependent mito- chondrial dynamics, somewhat de-emphasizing the role of DRP1-mediated ﬁssion. However, our results show that cells lacking either OMA1 or DRP1 are insensitive to loss of Dwm. If OMA1 alone controlled Dwm-dependent mito- chondrial dynamics, DRP1 knockout cells would fragment in q0 or CCCP-treated lines. The highly interconnected mitochondrial morphology of DRP1 knockout cells (Fig. 4) despite their lack of abundant L-OPA1 (Fig. 5) demon- strates the importance of DRP1. Similarly, cells treated with CCCP, with subsequent recovery in fresh media, do not show restoration of L-OPA1 (Fig. 4b), but nevertheless show striking mitochondrial interconnection (Fig. 4a). Taken together, these data support a strong role for DRP1 and ﬁssion per se in maintaining Dwm-dependent mito- chondrial dynamic balance. DRP1 and OMA1 coordinately impact mitochondrial fusion and OPA1 While DRP1 knockouts have decreased L-OPA1 relative to control HCT116s, they do not show any activation of OPA1 cleavage in response to CCCP treatment (Fig. 6c). These ﬁndings are consistent with loss of L-OPA1 in response to DRP1 downregulation [56], and suggest that the DRP1-mediated ﬁssion machinery interacts control HCT116 cells. Strikingly, however, DRP1 knock- out cells treated with CCCP show no appreciable change in OPA1 status: L- and S-OPA1 levels are essentially iden- tical in control versus CCCP-treated DRP1 knockout cells. This is consistent with the extensive mitochondrial inter- connection shown in CCCP-treated DRP1 knockouts 123 1359 A threshold of transmembrane potential is required for mitochondrial dynamic balance… below 34% TMRE signal have disrupted ﬁssion/fusion homeostasis. CCCP titration demonstrates that at concen- trations above 4.75 lM, cells have obligate fragmentation of the mitochondrial network (Fig. 2). This loss of mito- chondrial interconnection correlates with loss of fusion- active L-OPA1 isoforms (Fig. 3a, b), strongly indicating that OPA1-mediated inner membrane fusion per se is lost beyond this point. These ﬁndings demonstrate a key bioenergetic determinant of mitochondrial ﬁssion/fusion dynamics. Further research may employ alternate methods such as live-cell imaging [43] and heme oxidation state [47] to conﬁrm the Dwm threshold in mV. In addition, this Dwm threshold likely contributes to the canonical mito- chondrial genetic threshold effect: our model predicts that any mtDNA mutation that pushes Dwm below this thresh- old commits the organelle to obligate ﬁssion. As such, this parameter is likely to be of critical importance to mito- chondrial pathology in a range of human diseases, as well as therapeutic strategies seeking to eliminate Dwm-im- pacting mutant mtDNAs through pathways such as autophagy [58]. with OPA1 and the mitochondrial fusion factors to coop- eratively mediate Dwm-dependent mitochondrial dynamic balance. Discussion The interaction of bioenergetic function with fusion and ﬁssion pathways drives the structure/function dynamics of mitochondria as an organellar network. While it has been shown that complete dissipation of Dwm causes mito- chondrial fragmentation, we here examine (1) the level of Dwm required, and (2) the contribution of both ﬁssion and fusion pathways, in maintaining Dwm-dependent mito- chondrial dynamic balance in human cells. Our ﬁndings indicate that a threshold of 34% TMRE-assayed Dwm, strongly correlating with available fusion-active L-OPA1, is required to maintain mitochondrial ﬁssion/fusion bal- ance. Surprisingly, cells lacking either OMA1 or DRP1 have obligate mitochondrial fusion and fail to cleave long OPA1 isoforms in response to pharmacological or genetic loss of Dwm. These results suggest that OMA1 and DRP1 cooperatively act to mediate mitochondrial Dwm-dependent dynamic balance, and provide further evidence in support of a proposed mechanistic link between DRP1-mediated ﬁssion and OPA1 [34, 57]. As mitochondrial dynamic balance is crucial to cellular processes such as apoptosis, this ‘tipping point’ mechanism of mitochondrial dynamic balance will have far-reaching impacts on these key pathways. Mitochondrial dynamic balance requires a threshold of Dwm Here, we address a key question of mitochondrial struc- ture/function relationships: to what extent can the mitochondrial network lose bioenergetic function before dynamic balance is compromised? While total collapse of Dwm has been shown to cause fragmentation of the mito- chondrial network [4, 32, 33], the functional tipping point of Dwm-dependent mitochondrial dynamics has remained unknown. Mitochondrial threshold effects have been extensively documented in mtDNA-derived neuromuscular diseases: cells or tissues carrying greater than 80–90% mutant mtDNA typically show collapse of bioenergetic capacity [30]. Previously, we found that cells carrying a mutation load greater than 90% D-mtDNA lost the ability to maintain mitochondrial interconnection [17]. Here, we explore the functional and mechanistic tipping point behind this threshold using TMRE ﬂow cytometry to assay Dwm, and confocal imaging to monitor mitochondrial organiza- tion. When normalized against the TMRE value of untreated WT cells as 100%, our data indicate that cells Moreover, our results support both functional coordi- nation and mechanistic interaction of OPA1-mediated fusion and DRP1-mediated ﬁssion. While experiments in cells lacking OMA1 (Fig. 3) or DRP1 (Fig. 5) show that both are necessary for mitochondrial dynamic balance, the 12 3 1360 E. Jones et al. targeting the organelle to the autophagosome [64], while mitochondrial ﬁssion is directly activated by AMP kinase signaling [65]. These ﬁndings thus have direct relevance to a wide range of prevalent diseases that include mitochon- drial dysfunction in their pathogenesis. Mitochondrial dynamics are increasingly found to be critical for ener- getically dependent tissues such as heart [66] and skeletal muscle [67], with decreased OxPhos function and disrupted ﬁssion/fusion balance emerging in patient samples and disease models of neuromuscular diseases [30], neurode- generative aging and Parkinson’s disease [68, 69], diabetes [70–72], and heart failure [28]. As such, our ﬁndings illustrate a basic mechanism of mitochondrial struc- ture/function homeostasis with major implications for the pathogenesis and translational treatment of these disorders. decreased L-OPA1 observed in DRP1 knockout cells (Fig. 6c) indicates that DRP1 is necessary to maintain L-OPA1 stability. This enhanced OPA1 processing is consistent with Mopert et al. [57], who found similar results in response to transient knockdown of DRP1, while Huang et al. found that DRP interacts with mitofusin 2 [59], consistent with DRP1 interactions with the mito- chondrial fusion machinery. These ﬁndings suggest a role for DRP1 in stabilizing mitochondrial ﬁssion/fusion factors via direct and indirect protein–protein interactions span- ning the outer and inner mitochondrial membranes. Cell culture Human 143B osteosarcoma cell lines FLP6a39.2 (WT) and 143B206 (q0) were described previously [16, 19], as have OMA1?/? and OMA1-/- mouse embryonic ﬁbroblasts (MEFs) [52] and human colorectal carcinoma HCT116 control and DRP1 knockout cell lines [73]. Cells were grown in high-glucose Dulbecco’s Modiﬁed Eagle’s med- ium (DMEM) with 10% fetal bovine serum supplemented with 50 lg/mL uridine in 5% CO2 at 37 C. CCCP and mdivi-1 (Sigma, St. Louis, MO, USA) were dissolved as stock solutions in DMSO for dilution in complete media. All cell culture reagents were from ThermoFisher (Wal- tham, MA, USA). Mitochondrial dynamic balance requires a threshold of Dwm Thus, while mitochondrial ﬁssion and fusion have fre- quently been discussed as mechanistically distinct pathways, these results support a model in which an intact (i.e., above threshold) Dwm provides a check to both OMA1 and DRP1, allowing a balance of ﬁssion and fusion with both DRP1 and OMA1 interacting (directly or indi- rectly) with OPA1. Loss of Dwm to levels below the observed 34% TMRE threshold simultaneously activates OMA1 cleavage of L-OPA1 and mitochondrial recruitment of DRP1, collapsing mitochondrial organization to an obligately fragmented state (Fig. 6d). It is likely that the loss of Dwm below threshold causes profound changes in conformation and enzymatic activity of multiple proteins at the mitochondrial inner and outer membranes, altering the protein–protein interactions that determine mitochondrial structural dynamics. OMA1 has been shown to undergo self-cleavage and activation of L-OPA1 proteolysis during membrane depolarization [60], while Anand et al. found that expression of S-OPA1 is sufﬁcient to induce mito- chondrial ﬁssion, with S-OPA1 frequently colocalizing with DRP1 at ER-mitochondrial sites of contact [34]; the authors postulated that S-OPA1 may stimulate DRP1 ﬁs- sion activity. Dwm-dependent DRP1 ﬁssion activity is activated by phosphorylation at S637 [27], and can be activated by calcineurin in response to increased cytosolic calcium [53]. Recruitment of DRP1 to mitochondria is mediated by actin [61, 62]. 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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. Acknowledgements The authors gratefully acknowledge Carlos Lopez-Otin and Pedro Quiros (University of Oviedo) for providing OMA1?/? and OMA1-/- MEFs, Richard Youle (National Institutes Fluorescence microscopy Cultured cells were seeded to 18 mm2 glass coverslips. Coverslips were incubated with MitoTracker CMXRos (Invitrogen Molecular Probes, Carlsbad, CA, USA), fol- lowed by ﬁxation in 4% paraformaldehyde in PBS. For immunolabeling of the translocase of the outer mitochon- drial membrane-20 protein (TOM20), cells were permeabilized with 0.1% TX-100 in PBS, followed by blocking in 10% normal goat serum and incubation with anti-TOM20 monoclonal antibody FL-145 at 1:100 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Coverslips were incubated with goat anti-mouse Alexa488- conjugated secondary antibody at 1:100 dilution (Invitro- gen Molecular Probes, Carlsbad, CA, USA), followed by staining with diaminophenylindole (DAPI) and mounting in 50% glycerol in PBS. Coverslips were imaged on an Olympus Fluoview FV-10i (Olympus, Center Valley, PA, USA) with a 609 UPLSAP60xW objective with aperture Mitochondrial ﬁssion/fusion status directly impacts a broad range of cellular processes such as apoptosis. Con- sistent with this, cells below the Dwm threshold show dramatically decreased viability when challenged with galactose-containing media (Suppl. Figure 3). 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https://openalex.org/W2261270772	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0148180&type=printable	English	null	Reorganization of Respiratory Descending Pathways following Cervical Spinal Partial Section Investigated by Transcranial Magnetic Stimulation in the Rat	PloS one	2,016	cc-by	8,596	RESEARCH ARTICLE OPEN ACCESS Citation: Vinit S, Keomani E, Deramaudt TB, Bonay M, Petitjean M (2016) Reorganization of Respiratory Descending Pathways following Cervical Spinal Partial Section Investigated by Transcranial Magnetic Stimulation in the Rat. PLoS ONE 11(2): e0148180. doi:10.1371/journal.pone.0148180 Stéphane Vinit1,2, Emilie Keomani1,2, Therese B. Deramaudt1,2,3, Marcel Bonay1,2,3,4☯, Michel Petitjean1,2,4☯* 1 Université de Versailles Saint-Quentin-en-Yvelines, UFR des Sciences de la Santé – Simone Veil, Montigny-le-Bretonneux, France, 2 Inserm U1179, End:icap, Laboratoire de Physiologie TITAN, Montigny- le-Bretonneux, France, 3 LIA-BAHN (Laboratoire International Associé – Biologie Appliquée Handicap Neuromusculaire), CSM (Centre Scientifique de Monaco), 8 Quai Antoine Ier - 98000, Monaco, 4 Service de Physiologie-Explorations Fonctionnelles; Hôpital Ambroise Paré, Assistance Publique-Hôpitaux de Paris (AP-HP), Groupe Hospitalier Paris Ile-de-France Ouest, Boulogne-Billancourt, France ☯These authors contributed equally to this work. * michel.petitjean134@gmail.com ☯These authors contributed equally to this work. * michel.petitjean134@gmail.com Reorganization of Respiratory Descending Pathways following Cervical Spinal Partial Section Investigated by Transcranial Magnetic Stimulation in the Rat Stéphane Vinit1,2, Emilie Keomani1,2, Therese B. Deramaudt1,2,3, Marcel Bonay1,2,3,4☯, Michel Petitjean1,2,4☯* Stéphane Vinit1,2, Emilie Keomani1,2, Therese B. Deramaudt1,2,3, Marcel Bonay1,2,3,4☯, Michel Petitjean1,2,4☯* Editor: Antal Nógrádi, University of Szeged, HUNGARY Editor: Antal Nógrádi, University of Szeged, HUNGARY Received: September 17, 2015 Accepted: January 14, 2016 Published: February 1, 2016 Copyright: © 2016 Vinit et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Antal Nógrádi, University of Szeged, HUNGARY Received: September 17, 2015 Accepted: January 14, 2016 Published: February 1, 2016 Copyright: © 2016 Vinit 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: © 2016 Vinit et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Abstract High cervical spinal cord injuries lead to permanent respiratory deficits. One preclinical model of respiratory insufficiency in adult rats is the C2 partial injury which causes unilateral diaphragm paralysis. This model allows the investigation of a particular population of respi- ratory bulbospinal axons which cross the midline at C3-C6 spinal segment, namely the crossed phrenic pathway. Transcranial magnetic stimulation (TMS) is a non-invasive tech- nique that can be used to study supraspinal descending respiratory pathways in the rat. Interestingly, a lateral C2 injury does not affect the amplitude and latency of the largest motor-evoked potential recorded from the diaphragm (MEPdia) ipsilateral to the injury in response to a single TMS pulse, compared to a sham animal. Although the rhythmic respira- tory activity on the contralateral diaphragm is preserved at 7 days post-injury, no diaphragm activity can be recorded on the injured side. However, a profound reorganization of the MEPdia evoked by TMS can be observed. The MEPdia is reduced on the non-injured rather than the injured side. This suggests an increase in ipsilateral phrenic motoneurons excitabil- ity. Moreover, correlations between MEPdia amplitude and spontaneous contralateral dia- phragmatic activity were observed. The larger diaphragm activity correlated with a larger MEPdia on the injured side, and a smaller MEPdia on the non-injured side. This suggests, for the first time, the occurrence of a functional neuroplasticity process involving changes in motoneuron excitability balance between the injured and non-injured sides at a short post- lesional delay. Editor: Antal Nógrádi, University of Szeged, HUNGARY Introduction Yvelines. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. High cervical spinal cord injuries often lead to respiratory insufficiencies, and ventilatory assistance is necessary in order for the patients to survive [1]. However, few patients can be weaned off the ventilatory assistance, demonstrating the ability of the respiratory system to exert tremendous and spontaneous post-lesional neuroplasticity. One of the most studied preclinical models in respiratory insufficiency induced by cervical spinal injury is the C2 injured rat [2–8]. The initial spinal injury, performed at the cervical 2 segment induces a diaphragm paralysis due to a disruption of the main descending respiratory path- ways which connect to the phrenic motoneuron pool. The contralateral side remains intact and allows animal to recover. This particular model expresses some spontaneous neuroplas- ticity leading to a partial reactivation of the ipsilateral phrenic and diaphragm activities. This partial recovery is sustained by activation of the silent spared contralateral bulbospinal respiratory pathways that cross the midline at the segmental level C3-C6 after a short post-lesional delay [7, 9]. These pathways are called the crossed phrenic pathways (CPP) [2, 10]. However, this reactivation is marginal and does not directly participate in the animal ventilation [11]. To date, only invasive techniques have been used to evaluate the excitability and reorganization of such pathways in vivo at various post-lesional delays. Transcranial magnetic stimulation (TMS) is a promising technique consisting of applying a brief and high intensity magnetic field at supraspinal level with the aim to depolarize the cor- tical and subcortical areas to elicit volleys of action potentials, which propagate to the phrenic motoneuron and diaphragm. Supraspinal connections from cortical and bulbar neu- rons innervating the phrenic motoneuron pool have been demonstrated in cats [12], rats [13], and humans [14, 15]. TMS in humans has been proven to be effective in the study of diaphragmatic motor evoked potential (MEP) [16–19]. Recently, specific diaphragmatic MEP (MEPdia) has been recorded following a single pulse of TMS with a large figure-of- eight coil on a respiratory insufficiency model in adult male Sprague-Dawley rats [20]. This non-invasive technique allows the assessment of the respiratory supraspinal excitability and plasticity in the rat. Introduction In the present study, using a preclinical animal model of respiratory insufficiency induced by a cervical spinal injury, we proposed to investigate 1) the early reor- ganization of the silent crossed phrenic pathway excitability by TMS single pulse 1 hour and 7 days post-injury, and 2) the potential correlations of the MEPdia with the remaining spared pathways and/or the functional outcome from the contralateral intact side. This novel non-invasive technique could be used as a diagnosis tool to evaluate the remaining functional spinal fibers spared by the initial injury and the putative functional prognosis of respiratory recovery. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. TMS and Respiratory Crossed Pathways Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by funding from the European Union Seventh framework Programme (FP7/2007-2013) under grant agreement No. 246556 (European project RBUCE-UP), HandiMedEx allocated by the French Public Investment Board, the Chancellerie des Universités de Paris (Legs Poix), the «Centre d’Assistance Respiratoire à Domicile d’Ile de France (CARDIF)», the «Fonds de Dotation de Recherche en Santé Respiratoire», LVL Medical, and Université de Versailles Saint-Quentin-en- 1 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 Transcranial magnetic stimulation (TMS) TMS was performed using the magnetic stimulator MAGPRO X100 (Magventure, Farum, Denmark) connected to a figure-of-eight coil (CB60; dimensions: 165x85x20 mm) delivering a unique biphasic pulse (380 μs in duration) with an intensity of the stimulus expressed as a per- centage of maximum output of the stimulator (% MO). The MO was set-up at 90%, and the center of the coil placed at -6 mm from Bregma. This position is known to elicit a specific dia- phragmatic Motor Evoked Potential (MEPdia) from supraspinal stimulation transiting through the spinal cord in rats [20]. Each animal received about 100 TMS single pulses across the different experimental conditions. As described previously [20], interpulse duration was always above 10s to avoid low frequency repetitive TMS like effects. Chronic C2 hemisection Tramadol (analgesic, 15 mg/kg), carpofen (anti-inflammatory, 5 mg/kg), enrofloxacin (antibi- otics, 4 mg/kg), and medetomidine (100 μg/kg) were administered sub-cutaneously 10–20 mn before inducing isoflurane anesthesia in a closed chamber (in 100% O2). Rats were intubated and ventilated with a rodent ventilator (model 683; Harvard Apparatus, South Natick, MA, USA). Anesthesia was maintained throughout the procedure (1.5–2% Isoflurane in 100% O2). Skin and muscles were retracted, and a C2 laminectomy and durotomy were performed. The spinal cord was sectioned unilaterally just caudal to the C2 dorsal roots with micro-scissors fol- lowed with a micro-scalpel to ensure the section of all the remaining fibers, as described previ- ously [5]. We deliberately chose not to do a complete hemisection, but a graded section on the injured side to leave enough ventral white matter. The wounds and skin were sutured closed. Sham rats underwent the same procedures without hemisection. Atipamezole (400 μg/kg, intra muscular) was given to reverse medetomidine. The Isoflurane vaporizer was turned off, the endotracheal tube was removed, and the rats were monitored throughout recovery. Rats received analgesic (tramadol, 15 mg/kg), antibiotics (enrofloxacin, 4 mg/kg) and anti-inflam- matory (carpofen, 5 mg/kg) drugs for 2 days post-surgery. Materials and Methods Ethics statement All experiments reported in this manuscript conformed to policies laid out by the National Institutes of Health (USA) in the Guide for the Care and Use of Laboratory Animals. These experiments were performed on 2 months-old male Sprague—Dawley rats (Janvier, France). The animals were dual-housed in individually ventilated cages in a state-of-the-art animal care facility (2CARE animal facility, accreditation A78-322-3, France), with access to food and water ad libitum with a 12h light/dark cycle. The Ethics committee of the RBUCE-UP Chair of Excellence (University of Paris-Sud, grant agreement No. 246556) and the University of Ver- sailles Saint-Quentin-en-Yvelines approved these experiments. 2 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 TMS and Respiratory Crossed Pathways PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 Electrophysiological recordings A total of 39 animals were used in this study for terminal electrophysiological recordings. They were randomly divided into 3 different groups: Sham (n = 21), 1 hour post-injury (1h P.I. n = 10) and 7 days post-injury (7d P.I. n = 8). As described previously [20, 21], anesthesia was induced using isoflurane (3.5% in 21% O2 balanced) through a nose cone. A 25G catheter was placed in the tail vein. The rats were tracheotomized and pump ventilated (Rodent Ventilator, model 683; Harvard Apparatus, South Natick, MA, USA). The ventilation rate was adjusted below the animal central apneic threshold throughout the experiment. EtCO2 was monitored using an infrared capnograph (Viamed, VM-2500-M). Animals were placed on a heating pad to maintain a constant body temperature and their rectal temperature was monitored through- out the experiment. Arterial pressure was measured through a catheter inserted in the right femoral artery. Arterial and tracheal pressures were monitored continuously with transducers connected to a bridge amplifier (AD Instruments, Dunedin, New Zealand). Isoflurane anesthe- sia was then slowly converted to urethane anesthesia (1.8 g/kg, i.v.; Sigma-Aldrich). The depth of anesthesia was confirmed by the absence of any response to toe pinch. A laparotomy was performed, and the liver was gently moved caudally to access the diaphragm. Gauze soaked with warm phosphate-buffered saline was placed on the liver to prevent dehydration. The head of the animal was placed on a non-magnetic custom-made stereotaxic apparatus which allowed its positioning from the center of the figure-of-eight coil to -6 mm from Bregma, at an angle of 0°, as shown previously [20]. Two custom-made hooked bipolar electrodes were placed on 3 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 TMS and Respiratory Crossed Pathways each mid-costal part of the diaphragm and left there for the entire duration of the experiment. Diaphragm spontaneous poïkilocapnic normoxic or transient mild asphyxia (by pinching the animal nose for 15 s) EMGs and MEPdia induced by a single pulse of TMS were amplified (gain, 1k; A-M Systems, Everett, WA, USA) and band pass-filtered (100 Hz to 10 kHz). The signals were digitized with an 8-channel Powerlab data acquisition device (Acquisition rate: 100 k/s; AD Instruments, Dunedin, New Zealand) connected to a computer and analyzed using LabChart 7 Pro software (AD Instruments, Dunedin, New Zealand). The bilateral dia- phragmatic EMGs were integrated (50 ms decay). Electrophysiological recordings At the end of the experiment (around 3 hours under urethane anesthesia), animals were euthanized with an i.v. overdose of urethane. Histological reconstruction of the extent of C2 injury Immediately after euthanasia, the cervical spinal cord was dissected (C1 to C4) and placed overnight in cold 4% paraformaldehyde (in 0.1 M Phosphate Buffered Saline). The segment was then cryoprotected in ascending concentration of sucrose solution and frozen. Transverse sections (30 μm thickness) were collected and the injury was assessed with cresyl violet stain- ing. A light microscope was used to draw the extent of the injury and the latter was reported on a stereotaxic transverse plane as described previously [5, 6]. Each injury was digitized and ana- lyzed with Image J software (NIH). The percentage of spared ventral white matter on the injured side was calculated by reference to a complete hemisection (which is 0% of spared white matter, as previously described [22]). Data processing The amplitude and frequency of 10 integrated diaphragm EMG inspiratory bursts during nor- moxia and mild asphyxia was calculated for each animal from the injured and intact sides with LabChart 7 Pro software (AD Instruments). The average of at least 5 MEPdia was calculated with LabChart Pro software (AD Instru- ments). The baseline-to-peak amplitude of the first negative wave (N1) of each averaged MEP- dia was measured. MEPdia latency was defined as the first electrical (positive or negative) deviation following the magnetic pulse artefact (as previously described in rodent TMS studies [20, 23]). Normality of the data distribution was assessed using a Kolmogorov-Smirnov test and log transformation was performed when required. Two way ANOVA with Bonferoni correction for multiple comparisons was performed between animal groups followed by post-hoc Student t-test. A Student paired t-test was performed to demonstrate the reorganization of the MEPdia between both sides (injured vs. intact) in the same animal at various post-injury delays (1 hour and 7 days). All the data are presented as mean ± SEM. Individual values of the MEPdia ampli- tude are represented as well as the mean value to highlight the difference between the intact and injured side in the same animal. A test was considered significant if p<0.05. A linear regression has been performed to compare the MEPdia, diaphragm contralateral activity and remaining spared white matter. Regression coefficient R2 and p values are indicated in the fig- ure legend. Physiological effects of a C2 injury Physiological effects of a C2 injury The body weight of the animal groups was similar for Sham animals and 1h post-injury (sham: 380±51g, 1h P.I.: 398±13g). However, at 7d P.I., the body weight declined significantly (342 4 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 TMS and Respiratory Crossed Pathways ±8g) compared to Sham and 1h P.I. groups. The body temperature increased significantly in 1h P.I. animals compared to sham and 7d P.I. animals (37.8±0.2°C compared to 36.7±0.1°C and 36.8±0.2°C respectively). End-tidal CO2 was similar in all groups (30±0.6 mmHg for sham, 29.6±3 mmHg for 1h P.I. and 30±1 mmHg for 7 d P.I.). Mean arterial blood pressure (MAP) was maintained throughout the experiment for all groups (MAP start: 92±2 mmHg, MAP End: 99±3 mmHg for sham; MAP start: 70±7 mmHg, MAP End: 68±7 mmHg for 1h P. I.; MAP start: 97±5 mmHg, MAP End: 96±4 mmHg for 7d P.I.). However, the corresponding MAP start and MAP end decreased significantly in 1h P.I. animal group compared to sham and 7d P.I. (Table 1). Effect of a C2 injury on diaphragm activity The size of the C2 injury was similar in the lateral part in all injured animals (7d P.I.). We deliberately chose not to perform a complete hemisection and left some ventro-medial part of the spinal cord, allowing some spared ventral white matter (Fig 1A). A lateral C2 injury was sufficient to abolish the diaphragmatic activity on the injured side at 1h P.I., and it persisted at 7d P.I. (Fig 1B). The diaphragmatic electromyogram on the intact side did not show any differ- ence in the frequency and amplitude in all groups (Fig 1B). Quantitatively, sham group showed an integrated diaphragm average amplitude of 16.3±1.5 μV/s, similar to the intact side of the 7d P.I. group (12.3±1.8 μV/s) whereas at 1h P.I., a statistically significant increase in diaphragm integrated amplitude emerged compared to the other groups in the intact side (36.9±10 μV/s, Fig 1C). No diaphragm activity was observed at 1h and 7d P.I. on the injured side in normoxic poïkilocapnic breathing and during mild asphyxia (Fig 1C, data not shown for the mild asphyxia). Similar respiratory frequencies were recorded among these groups during normoxic poïkilocapnic breathing (sham: 28.1±0.8 breaths/mn; 1h P.I.: 31.4±3 breaths/mn; 7d P.I.: 24.8 ±2.7 breaths/mn). The diaphragm EMG amplitude during normoxia and mild asphyxia corre- lated strongly with the ventral spared white matter (Fig 2A). Interestingly, less ventral spared white matter led to a larger contralateral diaphragm activity during normoxia and mild asphyxia (Fig 2B), whereas no ipsilateral diaphragm activity was recorded in our animals in both conditions (not shown). PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 Correlation among the injury size, MEPdia, and contralateral diaphragm activity As expected, the injuries extents in 7d P.I. animal group varied from 0% to 38% of ventral spared white matter (Fig 4A, x axis). A correlation was observed between both intact and injured MEPdia amplitudes and the ventral spared white matter (express as a percentage of the injured side) at 7d P.I. More ventral spared white matter led to a higher recorded MEPdia induced by TMS in both injured and intact sides. For a given ventral spared white matter value, the MEPdia from the injured side was systematically higher compared to that of the intact side (Fig 4A). The intact side integrated diaphragm amplitude during poïkilocapnic normoxic breathing in 7d P.I. group correlated with the recorded MEPdia amplitude in the injured and Fig 1. Diaphragm activity following C2 spinal cord injury. A. Representative extent of injury in 8 animals at 7 days post-injury. B. Representative traces of raw diaphragm EMG for Sham, 1 hour post-injury (1 h P.I.) and 7 days post-injury (7 d P.I.). Note the absence of rhythmic activity on the injured side at 1h and 7d P.I. The deflexion of the signal recorded on the injured side is due to the parasitic recording of the electrocardiogram. C. Integrated diaphragm amplitude for Sham, 1h P.I. and 7d P.I. animals. Note the absence of diaphragm activity on the injured side for 1h and 7d P.I. groups. D. Diaphragm frequency in poïkilocapnic breathing Sham, 1h and 7d P.I. animals. * p<0.05 compared to Sham and 7d P.I. groups; # p<0.05 between sides (injured vs. spared side), two-way ANOVA with Bonferonni correction tested. doi:10.1371/journal.pone.0148180.g001 Fig 1. Diaphragm activity following C2 spinal cord injury. A. Representative extent of injury in 8 animals at 7 days post-injury. B. Representative traces of raw diaphragm EMG for Sham, 1 hour post-injury (1 h P.I.) and 7 days post-injury (7 d P.I.). Note the absence of rhythmic activity on the injured side at 1h and 7d P.I. The deflexion of the signal recorded on the injured side is due to the parasitic recording of the electrocardiogram. C. Integrated diaphragm amplitude for Sham, 1h P.I. and 7d P.I. animals. Note the absence of diaphragm activity on the injured side for 1h and 7d P.I. groups. D. Diaphragm frequency in poïkilocapnic breathing Sham, 1h and 7d P.I. animals. * p<0.05 compared to Sham and 7d P.I. groups; # p<0.05 between sides (injured vs. Effect of a TMS pulse on the MEPdia A TMS single pulse (90% MO) at -6 mm over the Bregma in a urethane anesthetized sham ani- mal induced an ample MEPdia on both sides of the diaphragm (Fig 3A). Surprisingly, at 1h and 7d P.I., a MEPdia could be recorded on both the injured and the intact side of the dia- phragm in each animal (Fig 3A, S1 Supporting Information). Comparable average MEPdia amplitude was recorded in all groups (Sham: 16.4±2 μV; 1h P.I.: Intact side = 13.9±3.6 μV; Injured side = 14.8±2.2 μV; 7d P.I.: Intact side = 8.1±1.4 μV; Injured side = 13.8±1.9 μV) with a non-significant trend indicating amplitude reduction on the intact side in 7d P.I. group. No dif- ference side by side in the amplitude of MEPdia was observed in sham animals (paired t-test, two-tailed p value = 0.191, data not shown), and we decided to average the amplitude of each side and present the data per animal. However, when 1h P.I. and 7d P.I. groups were compared individually side by side (intact side vs. injured side), a significant increase in MEPdia ampli- tude was observed at 7d P.I. for each animal at the injured side compared to the intact side (paired t-test, two-tailed p value = 0.0022) whereas no significant difference was observed at 1h P.I. (paired t-test, two-tailed p value = 0.803) (Fig 3B). MEPdia latencies were similar in all groups (Sham: 7.7±0.2 ms; 1h P.I.: Intact side = 6.9±0.2 ms; Injured side = 7.2±0.2 ms; 7d P.I.: Intact side = 7.5±0.5 ms; Injured side = 8.5±0.5 ms) (Fig 3C). 5 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 Body weight (g) Temperature (°C) EtCO2 (mmHg) MAP Start (mmHg) MAP End (mmHg) Treatment Group Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Sham 380 51 36.7 0.1 30 0.6 92 2 99 3 1h C2HS 398 13 37.8b 0.2 29.6 3 70b 7 68b 7 7d C2HS 342a 8 36.8 0.2 30 1 97 5 96 4 Table 1. Physiological parameters. TMS and Respiratory Crossed Pathways Correlation among the injury size, MEPdia, and contralateral diaphragm activity As expected, the injuries extents in 7d P.I. animal group varied from 0% to 38% of ventral spared white matter (Fig 4A, x axis). A correlation was observed between both intact and injured MEPdia amplitudes and the ventral spared white matter (express as a percentage of the injured side) at 7d P.I. More ventral spared white matter led to a higher recorded MEPdia induced by TMS in both injured and intact sides. For a given ventral spared white matter value, the MEPdia from the injured side was systematically higher compared to that of the intact side (Fig 4A). The intact side integrated diaphragm amplitude during poïkilocapnic normoxic breathing in 7d P.I. group correlated with the recorded MEPdia amplitude in the injured and Table 1. Physiological parameters. Body weight (g) Temperature (°C) EtCO2 (mmHg) MAP Start (mmHg) MAP End (mmHg) Treatment Group Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Sham 380 51 36.7 0.1 30 0.6 92 2 99 3 1h C2HS 398 13 37.8b 0.2 29.6 3 70b 7 68b 7 7d C2HS 342a 8 36.8 0.2 30 1 97 5 96 4 MAP: Mean arterial pressure, EtCO2: End-tidal CO2, a p<0.05 compared to Sham and 1h, b p<0.05 compared to Sham and 7d. doi:10.1371/journal.pone.0148180.t001 Fig 1. Diaphragm activity following C2 spinal cord injury. A. Representative extent of injury in 8 animals at 7 days post-injury. B. Representative traces of raw diaphragm EMG for Sham, 1 hour post-injury (1 h P.I.) and 7 days post-injury (7 d P.I.). Note the absence of rhythmic activity on the injured side at 1h and 7d P.I. The deflexion of the signal recorded on the injured side is due to the parasitic recording of the electrocardiogram. C. Integrated diaphragm amplitude for Sham, 1h P.I. and 7d P.I. animals. Note the absence of diaphragm activity on the injured side for 1h and 7d P.I. groups. D. Diaphragm frequency in poïkilocapnic breathing Sham, 1h and 7d P.I. animals. * p<0.05 compared to Sham and 7d P.I. groups; # p<0.05 between sides (injured vs. spared side), two-way ANOVA with Bonferonni correction tested. doi:10.1371/journal.pone.0148180.g001 Table 1. Physiological parameters. TMS and Respiratory Crossed Pathways Fig 2. Correlation between diaphragm activity and extent of C2 injury in normoxic and mild asphyxic rats. A. Representative EMG recording during normoxia and mild asphyxia with corresponding extent of injury. Note a greater EMG amplitude with less spared white matter. B. Correlation between integrated contralateral diaphragm amplitude and ventral spared white matter (during normoxia (white dots, R2:0.138, p = 0.011) and mild asphyxia (black dots, R2:0.609, p<0.001)). Note higher contralateral diaphragm amplitude with smaller spared white matter during mild asphyxia. Fig 2. Correlation between diaphragm activity and extent of C2 injury in normoxic and mild asphyxic rats. A. Representative EMG recording during normoxia and mild asphyxia with corresponding extent of injury. Note a greater EMG amplitude with less spared white matter. B. Correlation between integrated contralateral diaphragm amplitude and ventral spared white matter (during normoxia (white dots, R2:0.138, p = 0.011) and mild asphyxia (black dots, R2:0.609, p<0.001)). Note higher contralateral diaphragm amplitude with smaller spared white matter during mild asphyxia. doi:10.1371/journal.pone.0148180.g002 intact sides. Higher intact side diaphragmatic integrated amplitude corresponds to a lower MEPdia whereas a lower amplitude in integrated diaphragm recording corresponded to a higher MEPdia recorded in the intact side. Similar trends were observed when comparing the MEPdia obtained from the injured side with the intact side integrated diaphragm amplitude (Fig 4B). The MEPdia amplitude induced by a TMS pulse could predict the amplitude of dia- phragmatic activity on the intact side 7d P.I. Fig 3. Diaphragm motor evoked potentials in sham, 1h P.I. and 7d P.I. animals. A. Representative MEPdia recordings for sham, 1h P.I. spared and injured sides and 7d P.I. spared and injured sides. Note the diminution of the MEPdia amplitude in the spared side compared to the injured side at 7d P.I. B. MEPdia amplitude in Sham, 1h P.I. and 7d P.I. animals. Note the reorganization of the MEPdia at 7d P.I. (increased in the injured side compared to the spared side) when compared to 1h P.I. group. C. MEPdia latency in sham, 1h P.I and 7d P.I. group. Note the trend of higher latency in the 7d P.I. injured side group. * p<0.05 compared to spared side. d i 10 1371/j l 0148180 003 Fig 3. Diaphragm motor evoked potentials in sham, 1h P.I. and 7d P.I. animals. A. Representative MEPdia recordings for sham, 1h P.I. spared and injured sides and 7d P.I. Correlation among the injury size, MEPdia, and contralateral diaphragm activity spared side), two-way ANOVA with Bonferonni correction tested. doi:10.1371/journal.pone.0148180.g001 6 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 spared and injured sides. Note the diminution of the MEPdia amplitude in the spared side compared to the injured side at 7d P.I. B. MEPdia amplitude in Sham, 1h P.I. and 7d P.I. animals. Note the reorganization of the MEPdia at 7d P.I. (increased in the injured side compared to the spared side) when compared to 1h P.I. group. C. MEPdia latency in sham, 1h P.I and 7d P.I. group. Note the trend of higher latency in the 7d P.I. injured side group. * p<0.05 compared to spared side. doi:10.1371/journal.pone.0148180.g003 doi:10.1371/journal.pone.0148180.g003 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 7 / 13 TMS and Respiratory Crossed Pathways Fig 4. Correlations among MEPdia amplitude, extent of injury, and contralateral diaphragm activity at 7d P.I. A. Correlation of the extent of injury (represented as the ventral spared white matter) with MEPdia amplitude on the injured (black dots, R2:0.16, p = 0.034) and spared (white dots, R2:0.364, p = 0.149) side. Note the higher MEPdia recording with bigger spared white matter. B. Correlation between contralateral diaphragm amplitude and MEPdia amplitude on injured (black dots, R2:0.559, p = 0.005)/spared (white dots, R2:0.495, p = 0.014)sides. Note higher diaphragm EMG with lower MEPdia amplitude. doi:10 1371/journal pone 0148180 g004 Fig 4. Correlations among MEPdia amplitude, extent of injury, and contralateral diaphragm activity at 7d P.I. A. Correlation of the extent of injury (represented as the ventral spared white matter) with MEPdia amplitude on the injured (black dots, R2:0.16, p = 0.034) and spared (white dots, R2:0.364, p = 0.149) side. Note the higher MEPdia recording with bigger spared white matter. B. Correlation between contralateral diaphragm amplitude and MEPdia amplitude on injured (black dots, R2:0.559, p = 0.005)/spared (white dots, R2:0.495, p = 0.014)sides. Note higher diaphragm EMG with lower MEPdia amplitude. doi:10.1371/journal.pone.0148180.g004 doi:10.1371/journal.pone.0148180.g004 doi:10.1371/journal.pone.0148180.g004 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 Indeed, a profound morphological reorganization of these crossed phrenic pathways, especially an increase in dendrodendritic appositions and synaptically active zones in the ipsilateral phrenic motor nucleus could be observed as early as 2h post-C2 injury [26] (see Fig 5B and 5C). These anatomical events could be a part of the neuronal substrate to sustain the MEPdia evoked by TMS at 1h post-injury on the injured side. Moreover, intercon- nections between the ipsilateral and contralateral phrenic motoneurons as well as projections from the rostral ventral respiratory group with contralateral crossed connections to the injured part at the segmental level could also be involved in the activation of the crossed phrenic path- ways and potentially activated after acute and chronic injured animals [27]. A profound and spontaneous reorganization of descending respiratory bulbospinal pathways was observed fol- lowing C2 injury, contributing to the functional spontaneous restoration of the diaphragmatic activity at longer post-lesional delays [28]. In this present study, we chose to study the early reorganization and changes in spinal excitability following C2 injury. The lack of difference in MEPdia amplitude in Sham and 1h P.I. group observed in our study could be explained by the fact that the C2 initial injury induces a spinal shock at the injured side and an immediate massive release of glutamate from the axonal stubs projecting to the deafferented phrenic motoneurons. The massive amount of glutamate is excitotoxic and modifies the intraspinal excitability on and around the phrenic motoneurons, probably due to the removal of inhibitory spontaneously active immediately after the acute C2 injury, a MEPdia could be recorded on the side of injury, suggesting the presence of pre-existing silent synaptic crossed connections, which could be revealed by TMS. An evoked response induced by a contralateral C1 intraspinal stimulation could also be recorded on the ipsilateral side following a chronic C2 injury at a lon- ger time post-injury [24, 25]. However, no differences in the evoked crossed phrenic pathways between 2 weeks and 4 weeks post-injury have been demonstrated [25], suggesting that spinal plasticity occurs earlier. Indeed, a profound morphological reorganization of these crossed phrenic pathways, especially an increase in dendrodendritic appositions and synaptically active zones in the ipsilateral phrenic motor nucleus could be observed as early as 2h post-C2 injury [26] (see Fig 5B and 5C). TMS and Respiratory Crossed Pathways Fig 5. Proposed model of the correlation of early respiratory bulbospinal descending pathways rewiring with extent of injury. A. Representation of the bulbospinal descending tract 1h P.I. The contralateral pathways remain intact and allow the transit of the MEPdia induced by a single TMS pulse. Crossed phrenic pathways can also be activated with TMS. B. Early reorganization of bulbospinal respiratory pathway following a large C2 injury. The evoked potential by TMS transit through less spared fibers, allowing a smaller recorded MEPdia on the ipsilateral and contralateral sides (less connections from respiratory related fibers to the contralateral side). C. Reorganization of respiratory bulbospinal descending tracts with a partial lateral C2 section. More respiratory related tracts are spared and activated with TMS, inducing a bigger MEPdia. However, these pathways remain silent when the animal breathes by itself, suggesting a potential neuroanatomical substrate for inducing functional respiratory recovery. d i 10 1371/j l 0148180 005 Fig 5. Proposed model of the correlation of early respiratory bulbospinal descending pathways rewiring with extent of injury. A. Representation of the bulbospinal descending tract 1h P.I. The contralateral pathways remain intact and allow the transit of the MEPdia induced by a single TMS pulse. Crossed phrenic pathways can also be activated with TMS. B. Early reorganization of bulbospinal respiratory pathway following a large C2 injury. The evoked potential by TMS transit through less spared fibers, allowing a smaller recorded MEPdia on the ipsilateral and contralateral sides (less connections from respiratory related fibers to the contralateral side). C. Reorganization of respiratory bulbospinal descending tracts with a partial lateral C2 section. More respiratory related tracts are spared and activated with TMS, inducing a bigger MEPdia. However, these pathways remain silent when the animal breathes by itself, suggesting a potential neuroanatomical substrate for inducing functional respiratory recovery. doi:10.1371/journal.pone.0148180.g005 spontaneously active immediately after the acute C2 injury, a MEPdia could be recorded on the side of injury, suggesting the presence of pre-existing silent synaptic crossed connections, which could be revealed by TMS. An evoked response induced by a contralateral C1 intraspinal stimulation could also be recorded on the ipsilateral side following a chronic C2 injury at a lon- ger time post-injury [24, 25]. However, no differences in the evoked crossed phrenic pathways between 2 weeks and 4 weeks post-injury have been demonstrated [25], suggesting that spinal plasticity occurs earlier. Discussion This study demonstrated for the first time the use of a non-invasive transcranial magnetic stim- ulation technique, in the assessment of the phrenic motoneuron excitability in a preclinical model of respiratory insufficiency. These results support the feasibility of using TMS to study respiratory dysfunction associated with neurological injury and disease. Interestingly, following a cervical spinal partial cord injury at C2, a MEPdia can be recorded after a TMS single pulse stimulation on both sides at 1h and 7d P.I. despite the absence of dia- phragmatic spontaneous activity during poïkilocapnic and transient asphyxia breathing. TMS transits induced the volleys of descending action potentials supraspinally through the contra- lateral bulbospinal main respiratory pathways, and these volleys crossed the midline at the seg- mental level C3-C6 (Fig 5A). Despite the fact that these crossed phrenic synapses were not 8 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 They could modulate the crossed phrenic pathways and the respiratory descending activities [29], which may explain longer latency observed at 7d P.I. in our study. At longer P.I. time (weeks to months), several events take place in the injured spinal cord. Increased occupational area in activated microglia around the phrenic motoneurons at 7d P.I. and an increase in respiratory plasticity associated molecules, BDNF and its high-affinity receptor TrkB (along with its phosphorylated form pTrkb), in and around the phrenic motoneurons were observed following C2 lateral injury [30, 31]. The BDNF/TrkB cascade allows microglia to control the excitability of neuronal network and it can unmask hidden neuronal circuitry that is normally silent [32]. This can contribute to the changes in phrenic motoneuron excitability at 7d P.I. in response to TMS. Nevertheless, no difference in breathing frequency on the intact side has been observed between 1h and 7d P.I., suggesting that the plasticity occurs preferentially at the spinal segmental level instead of the brainstem respiratory structures above the injury side. We cannot exclude the fact that follow- ing a chronic C2 hemisection, profound morphological changes and reorganization occur at the diaphragm neuromuscular junction overtime [33], and they may play a role in the excitabil- ity reorganization of MEPdia following TMS single pulse at 7d P.I. Following a cervical hemisection, the blood respiratory gazes and pH oxygenation were not affected despite the fact that one hemidiaphragm was paralyzed [34, 35]. The implications of other respiratory related muscles (i.e. intercostals and abdominals, Fig 5B and 5C) for main- taining a certain respiratory gazes exchange homeostasis and for compensating for the mainte- nance of respiratory function following a chronic C2 hemisection have already been demonstrated [36, 37]. Interestingly, the injury size contributes to the spontaneous functional recovery as well as to the respiratory deficit. In fact, a larger injury induces more respiratory deficits measured by plethysmograph, suggesting an effect on other respiratory related path- ways in rats [22] and mice [38]. In our study, we observed a perfect inverse correlation between the extent of injury and contralateral diaphragm activity during poïkilocapnic and mild asphyxia breathing at 7d P.I. A small injury will lead to a smaller contralateral diaphragm activ- ity, suggesting that the respiratory descending pathways to the abdominal and intercostal muscles are less affected by the initial injury and that they further contribute to the whole respi- ration (Fig 5C). These anatomical events could be a part of the neuronal substrate to sustain the MEPdia evoked by TMS at 1h post-injury on the injured side. Moreover, intercon- nections between the ipsilateral and contralateral phrenic motoneurons as well as projections from the rostral ventral respiratory group with contralateral crossed connections to the injured part at the segmental level could also be involved in the activation of the crossed phrenic path- ways and potentially activated after acute and chronic injured animals [27]. A profound and spontaneous reorganization of descending respiratory bulbospinal pathways was observed fol- lowing C2 injury, contributing to the functional spontaneous restoration of the diaphragmatic activity at longer post-lesional delays [28]. In this present study, we chose to study the early reorganization and changes in spinal excitability following C2 injury. The lack of difference in MEPdia amplitude in Sham and 1h P.I. group observed in our study could be explained by the fact that the C2 initial injury induces a spinal shock at the injured side and an immediate massive release of glutamate from the axonal stubs projecting to the deafferented phrenic motoneurons. The massive amount of glutamate is excitotoxic and modifies the intraspinal excitability on and around the phrenic motoneurons, probably due to the removal of inhibitory pontaneously active immediately after the acute C2 injury, a MEPdia could be recorded on the de of injury, suggesting the presence of pre-existing silent synaptic crossed connections, 9 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 TMS and Respiratory Crossed Pathways connections and/or hyperexcitability of the spinal stub [20]. This could contribute to the hyperexcitability of the crossed phrenic pathways observed 1h after the injury. An interesting observation is that the latency of MEPdia evoked by TMS single pulse is slightly diminished 1h P.I. compared to Sham and 7d P.I., suggesting a change in spinal excitability in favor of the sig- nal transit. Similar MEPdia diminished latencies have been recorded in response to a TMS sin- gle pulse applied at the spinal stub following a complete C2 transection [20]. The absence of significant differences in MEPdia latencies across groups and sides suggests that the number of synapses, which the signal transits through from stimulus location (-6 mm from Bregma), and the response recording in the diaphragm are similar. However, following a C2 hemisection, spi- nal interneurons participate in spinal plasticity following the lesion. PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 Author Contributions Conceived and designed the experiments: SV MP. Performed the experiments: SV EK MP. Analyzed the data: SV EK TBD MP. Wrote the paper: SV TBD MB MP. Conceived and designed the experiments: SV MP. Performed the experiments: SV EK MP. Analyzed the data: SV EK TBD MP. Wrote the paper: SV TBD MB MP. Supporting Information S1 Supporting Information. Individual traces (diaphragm activities, MEPdia) for each ani- mal included in this study. (PDF) S1 Supporting Information. Individual traces (diaphragm activities, MEPdia) for each ani- mal included in this study. Acknowledgments This work was supported by funding from the European Union Seventh framework Pro- gramme (FP7/2007-2013) under grant agreement No. 246556 (European project RBUCE-UP), HandiMedEx allocated by the French Public Investment Board, the Chancellerie des Univer- sités de Paris (Legs Poix), the « Centre d’Assistance Respiratoire à Domicile d’Ile de France (CARDIF) », the « Fonds de Dotation de Recherche en Santé Respiratoire », LVL Medical and Université de Versailles Saint-Quentin-en-Yvelines. The supporters had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. TMS and Respiratory Crossed Pathways in synaptic connectivity could contribute to the early changes in phrenic motoneuron excitabil- ity and reorganization demonstrated at 7d P.I. by TMS. Another interesting point to discuss is the discrepancy observed between larger contralateral amplitude of MEPdia and smaller contralateral spontaneous diaphragm activity at 7d P.I. The excitability induced by TMS at the spinal level does not reflect the physiological supraspinal drive (i.e., spontaneous descending respiratory drive from the brainstem areas). This suggests that TMS recruits more supraspinal existing silent pathways connected to the contralateral phrenic motoneurons with inhibiting projections to the brainstem. These inhibiting projec- tions from cortical areas have been observed in humans and rats [40, 41]. These pre-existing anatomical pathways could be the targets for innovative therapeutics to enhance the respiratory function following a C2 injury. To conclude, TMS could be used as a functional diagnostic tool to investigate the remaining silent and functional spared pathways and it could be used to assess the reorganization of syn- aptic excitability following a cervical spinal cord injury as well as new therapeutics. PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 By contrast, if the injury affects more respiratory related pathways (less spared white matter), the contralateral diaphragm will adapt its activity (bigger amplitude) to main- tain the respiratory homeostasis. Another interesting point is that the CPP plays a marginal role in the tidal volume following a chronic C2 injury, suggesting that others respiratory related pathways are implicated in the generation of inspiration [11]. A lateral C2 injury is sufficient to permanently abolish the ipsilateral diaphragm activity, allowing more bulbospinal descending respiratory related spared pathways located medially in the spinal cord to re-connect the deaf- ferented phrenic motoneurons [9]. In addition, a hyperactivity of phrenic motoneuron is observed also in supraspinal areas following a chronic C2 hemisection, suggesting the estab- lishment of supraspinal compensatory routes which were initially inactive [39]. These increases 10 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0148180 February 1, 2016 References Intermittent hypoxia induces functional recovery following cervical spinal injury. Respiratory physiology & neurobiology. 2009; 169(2):210–7. Epub 2009/08/05. doi: 10. 1016/j.resp.2009.07.023 PMID: 19651247; PubMed Central PMCID: PMC2783733. 9. Vinit S, Kastner A. Descending bulbospinal pathways and recovery of respiratory motor function follow- ing spinal cord injury. 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https://openalex.org/W3010533361	https://link.springer.com/content/pdf/10.1007/s00500-020-04784-0.pdf	English	null	One more look on visualization of operation of a root-finding algorithm	Soft computing	2,020	cc-by	14,145	Soft Computing (2020) 24:14135–14155 https://doi.org/10.1007/s00500-020-04784-0 Soft Computing (2020) 24:14135–14155 https://doi.org/10.1007/s00500-020-04784-0 METHODOLOGIES AND APPLICATION Abstract Many algorithms that iteratively ﬁnd solution of an equation require tuning. Due to the complex dependence of many algorithm’s elements, it is difﬁcult to know their impact on the work of the algorithm. The article presents a simple root- ﬁnding algorithm with self-adaptation that requires tuning, similarly to evolutionary algorithms. Moreover, the use of various iteration processes instead of the standard Picard iteration is presented. In the algorithm’s analysis, visualizations of the dynamics were used. The conducted experiments and the discussion regarding their results allow to understand the inﬂuence of tuning on the proposed algorithm. The understanding of the tuning mechanisms can be helpful in using other evolutionary algorithms. Moreover, the presented visualizations show intriguing patterns of potential artistic applications. Keywords Self-adaptation · Root ﬁnding · Dynamics · Iterations · Visualization One more look on visualization of operation of a root-ﬁnding algorithm Ireneusz Go´sciniak1 · Krzysztof Gdawiec1 Published online: 4 March 2020 © The Author(s) 2020 Published online: 4 March 2020 © The Author(s) 2020 1 Institute of Computer Science, University of Silesia, B¸edzi´nska 39, 41-200 Sosnowiec, Poland 1 Introduction 2006 and distance to global best position Suresh et al. 2008). The value of inertia weight mostly presented in the literature is within the range of [0.4, 0.9] (see Jordehi and Jasni 2013). Both the inertia weight and the acceleration constants allow to control the particle behaviour within a wider range. The test function (problem being solved) and the selected method of iteration also inﬂuence particle’s behaviour. The particle dynamics is determined by the adaptation mechanics resulting mostly from particles’ cooperation. The method of algorithm param- eters selecting is not deterministic—these values are selected by a tuning process depending on a solved problem and the iteration method that is used (similarly to evolutionary algo- rithms Bansal et al. 2011; Sengupta and Mishra 2014). g The analysis of dynamics of dynamical systems is a very important problem (Sayama 2015), because it gives us an understanding of the changes that occur in the system. To analyse the dynamics, we can use many different methods (Broer and Takens 2011). One of such methods is a graphical presentation of the dynamics. The graphical representation provides a lot of intuitive, geometrical insight into the sys- tem’s dynamics, which would be hard to infer if we had just looked at algebraic equations (Sayama 2015). In the analysis of the complex polynomial root-ﬁnding process, the graph- ical presentation of dynamics is a very popular method and it even got its own name, namely polynomiography (Kalan- tari 2009). In polynomiography we visualize the root-ﬁnding process by using the number of iterations needed to obtain the root of a given polynomial and some colour map. The obtained images show the dynamics of the process. For the visualization and the analysis of algorithm’s behaviour, we can use methods similar to these used in polynomiography (Kalantari 2004). The polynomiography visualizes the com- plex nature of the relationships between parameters. The images generated using polynomiography very often reveal aesthetic patterns with a very complex structure (Gdawiec 2017). Because these complex patterns are gen- erated with a simple method polynomiography, besides its analytic purposes, it also found application in computer- aided design and in the arts (Kalantari 2004). 1 Introduction Gdawiec 14136 formly in the [0, 1] interval, x pb i—the best position of the ith particle and xgb—the global best position of the particles. roots, cube roots, etc.)? The answer to this question is that for polynomials of degree greater than four we cannot ﬁnd such formulas (Grant and Kleiner 2015). The formulas for quadratic polynomial are known since the Babylonians. For the cubic polynomial the formulas were found by G. Car- dano, whereas for the quartics by L. Ferrari both in the XVI century. In 1779 P. Rufﬁni and in 1826 N.-H. Abel proved the unsolvability by radicals of the polynomial equa- tion of degree greater than four—the Abel–Rufﬁni theorem (Grant and Kleiner 2015). Thus, to solve a general poly- nomial equation we need to use some numerical methods, which are iterative methods and deﬁne discrete dynamical systems. In the literature, we can ﬁnd many such methods, e.g. Newton’s method (Kalantari 2009) and Halley’s method (Kalantari 2009), or even the gradient descent method can be used for root ﬁnding (Kotarski and Lisowska 2018). g The inertia weight (ω) and acceleration constants (η1, η2) play a very important role in the algorithm—they are respon- sible for the particle dynamics. The acceleration constants direct the particle towards its own best position or the global best position (Sengupta and Mishra 2014). The particle can be trapped in a local optima for too high values of the acceler- ation constants or cannot reach the solution for too low values of these constants. Balance of the exploration and exploita- tion is also controlled by the inertia weight (Bansal et al. 2011). The control rules of inertia weight can be classiﬁed as follows: constant (Shi and Eberhart 1998); random (Eberhart and Shi 2001); time varying (linear decreasing Shi and Eber- hart 1999, sigmoid increasing/decreasing Malik et al. 2007, simulated annealing Al-Hassan et al. 2006, Sugeno func- tion Lei et al. 2006, exponential decreasing law Chen et al. 2006; Li and Gao 2009, logarithmic decreasing law Gao et al. 2008); adaptive control using feedbacks of the optimization process (best ﬁtness Saber et al. 2006; Shi and Eberhart 2001, ﬁtness of the current and previous iterations Yang et al. 2007, global best and average local best ﬁtness Arumugam and Rao 2008, particle rank Panigrahi et al. 2008, distance to parti- cle and global best positions Qin et al. 1 Introduction are well known (Klein et al. 2009; Koneˇcný and Richtárik 2017; Senov and Granichin 2017). Methods for determining function’s local minimum are based on the value of the function or its gradient (mostly numer- ically determined). The gradient method determines the direction of particle movement based on the knowledge of the gradient of the objective function (at the point reached in the previous step), and on this basis, it calculates the next posi- tion of the particle (Polak 1997)—a typical example of the gradient method is described by the expression (the gradient descent): A group of algorithms solving such problems includes evolutionary algorithms. The analysis of evolutionary algo- rithm is a very complex task (Gosciniak 2008, 2017). Some optimization algorithms can have similar behavioural characteristics as evolutionary algorithms (Weise 2009). A particular attention should be paid to the particle swarm opti- mization (PSO) algorithms (Zhang et al. 2014). The complex nature of particles movement in these algorithms does not allow a precise deﬁnition of their effect on the algorithm. Particle’s movement in the PSO algorithm is described by the following equation (Yang and Li 2010): x′ i = xi −γ ∇f (xi), (1) (1) x′ i = xi −γ ∇f (xi), where −∇f (xi) is the negative gradient of f , γ —step size, x′i—the current position of the ith particle in a D- dimensional environment and xi—the previous position of the ith particle. Many modiﬁcations of this method are described in the literature, and their operation mechanisms x′ i = xi + v′ i, (2) (2) where x′ i is the current position of the ith particle in a D- dimensional environment, xi—the previous position of the ith particle and v′ i—the current velocity of the ith particle in a D-dimensional environment that is given by the following formula: Communicated by V. Loia. B Ireneusz Go´sciniak ireneusz.gosciniak@us.edu.pl Krzysztof Gdawiec kgdawiec@ux2.math.us.edu.pl 1 Institute of Computer Science, University of Silesia, B¸edzi´nska 39, 41-200 Sosnowiec, Poland v′ i = ωvi + η1r1(x pb i −xi) + η2r2(xgb −xi), (3) (3) where vi is the previous velocity of the ith particle, ω—the inertia weight (ω ∈[0, 1]), η1, η2—acceleration constants (η1, η2 ∈(0, 1]), r1, r2—random numbers generated uni- 1 Institute of Computer Science, University of Silesia, B¸edzi´nska 39, 41-200 Sosnowiec, Poland 12 I. Go´sciniak, K. 123 1 Introduction where x0 ∈RD is a starting position, v0 = [0, 0, . . . , 0] is a starting velocity, vn+1 is the current velocity of the parti- cle (vn+1 = [v1 n+1, v2 n+1, . . . , vD n+1]) and xn is the previous position of the particle (xn = [x1 n, x2 n, . . . , x D n ]). The algo- rithm uses a similar methodology as the PSO algorithm, i.e. it sums the position of the particle xn with its current velocity vn+1. The aim of the article is to show the dynamics of parti- cle motion and the inﬂuence of the parameters on particle’s behaviour. For this purpose, we use a visualization method and analysis similar to the polynomiography. The used visu- alization method—presented in the article—can also have an artistic meaning. According to the authors, the understanding of the dynamics of particle’s motion is a very important for its use in the group of evolutionary algorithms. The proper selection of particle dynamics may not cause the inﬂuence of the environment on the algorithm—as it is realized in the algorithm presented in Gosciniak (2017). The current velocity of the particle is determined by the component of velocity of the previous iteration (inertia) and the component resulting from its current position (accelera- tion): vk n+1 = ωvk n + η f (xn) f (xn) −f (xn + εk) D j=1 \| f (xn) −f (xn + ε j)\| , (6) (6) Let’s try to summarize. What cannot be found in this arti- cle? The article is not a recipe how to effectively adjust the parameters of the algorithm. (Tuning an algorithm is a complex problem to be solved by other optimization algorithms—for instance by a genetic algorithm.) So what is this article about? Its main task is to visualize and analyse the complex nature of particle dynamics resulting from the selec- tion of algorithm’s coefﬁcients and its interaction with the environment. However, if we analyse the behaviour of parti- cles in evolutionary algorithms working in multidimensional spaces, a fractal analysis method can be proposed (Gosciniak 2017). What are the beneﬁts of the article? Besides the men- tioned visualization and the analysis of the complex nature of particle dynamics, the results of the paper can ﬁnd appli- cation in the generative art. 3 Iteration processes Let T : RD →RD be given by the following formula: T (xn) = xn + vn+1. (7) (7) T (xn) = xn + vn+1. Thus, algorithm (5) can be written in the following form 1 Introduction (Another example of this kind of algorithm can be the Newton’s method, which is at the forefront in this ﬁeld.) where k ∈{1, 2, . . . , D}, vk n+1—the current velocity of the particle in the direction k, vk n—the previous velocity of the particle in the direction k, ω ∈[0, 1)—inertia weight, η ∈(0, 1]—acceleration constant, εk—the step in the direc- tion k (ε1 = [τ, 0, . . . , 0], ε2 = [0, τ, 0, . . . , 0], . . ., εD = [0, . . . , 0, τ], where τ > 0). where k ∈{1, 2, . . . , D}, vk n+1—the current velocity of the particle in the direction k, vk n—the previous velocity of the particle in the direction k, ω ∈[0, 1)—inertia weight, η ∈(0, 1]—acceleration constant, εk—the step in the direc- tion k (ε1 = [τ, 0, . . . , 0], ε2 = [0, τ, 0, . . . , 0], . . ., εD = [0, . . . , 0, τ], where τ > 0). Determining the particle motion towards the root is sim- ilar to the gradient method—the marked part of Eq. (6). The acceleration is inﬂuenced by the adaptation mecha- nism resulting from the value of the function in the previous position of the particle— f (xn). (It works effectively at deter- mining a root of the function.) For this reason (when ω = 0) for xn fulﬁlling the condition f (xn) = 0, the next particle position is xn+1 = xn. The algorithm’s tuning involves the selecting inertia weight (ω) and acceleration constant (η)— similarly as in the PSO algorithm. The effect of changes in these parameters, i.e. particle dynamics, will be visualized using the method described in Sect. 4. The rest of the paper is organized as follows. Section 2 introduces a root-ﬁnding algorithm that is based on the gra- dient method. This method will be used to illustrate the inﬂuence of the parameters on its behaviour. Next, Sect. 3 introduces the iteration processes known in ﬁxed-point the- ory for ﬁnding the ﬁxed points of different types of functions. Section 4 presents a method of visualization of algorithm’s operation. And next, in Sect. 5 based on the obtained poly- nomiographs we discuss the research results. Finally, in Sect. 6 we give some concluding remarks. To solve (4), we use the following algorithm: 1 Introduction Polynomiogra- phy assists the designer in the creation of aesthetic patterns, because the designer must select only some parameters used in the method and the computer generates the pattern, whereas without polynomiography to create the same pattern the designer must spend a lot of time and put a lot of work in the creation process. Due to the particle motion mechanism and particle’s behaviour control parameters, we can use the wording—a particle dynamics. The tuning—selection of the proper val- ues of the parameters—is a very important problem for the algorithm. In most cases, the algorithm tuning process is intu- itive and it is based on user experience. Finding of roots of a given function f , i.e. solving the equation f (x) = 0, is a very important problem in prac- tical applications, e.g. physics (Franklin 2013), electronics (Chun and Kwasinski 2011) or computer graphics (Chen and Ma 2015). In many of these applications, the function is a polynomial one. So, the following question arises: can we solve the polynomial equation by its radicals (i.e. giving the formula for the roots in terms of the polynomial’s coef- ﬁcients, the four algebraic operations: addition, subtraction, multiplication, division and the extraction of roots, i.e. square The PSO-based gradient-like method is presented in the paper. In this simple root-ﬁnding algorithm, tuning parame- ters such as inertia weight and acceleration constant, and an adaptive mechanism depending on the location of the particle are proposed. This algorithm is used to show the inﬂuence 123 123 One more look on visualization of operation of a root-ﬁnding algorithm 14137 xn+1 = xn + vn+1, (5) (5) of the tuning parameters on particle’s behaviour. Moreover, we propose the use of different iteration processes instead of the standard Picard iteration that is used in the root-ﬁnding methods and optimization algorithms. xn+1 = xn + vn+1, where x0 ∈RD is a starting position, v0 = [0, 0, . . . , 0] is a starting velocity, vn+1 is the current velocity of the parti- cle (vn+1 = [v1 n+1, v2 n+1, . . . , vD n+1]) and xn is the previous position of the particle (xn = [x1 n, x2 n, . . . , x D n ]). The algo- rithm uses a similar methodology as the PSO algorithm, i.e. it sums the position of the particle xn with its current velocity vn+1. 1. The Das–Debata iteration (Das and Debata 1986): xn+1 = (1 −αn)xn + αnT2(yn), yn = (1 −βn)xn + βnT1(xn), n = 0, 1, 2, . . . , (12) The pseudocode of the visualization algorithm is pre- sented in Algorithm 1. In the algorithm, the selected iteration method is denoted as Iq, where q is a vector of parameters of the iteration. (12) where αn ∈(0, 1] and βn ∈[0, 1] for all n ∈N. 2. The Khan–Cho–Abbas iteration (Khan et al. 2011): where αn ∈(0, 1] and βn ∈[0, 1] for all n ∈N. 2. The Khan–Cho–Abbas iteration (Khan et al. 2011): 2. The Khan–Cho–Abbas iteration (Khan et al. 2011): The solution space A is contained in a D-dimensional space, so Algorithm 1 will return visualization in this space. If D = 2, then we obtain a single image that can be easily presented on a screen. When D > 2, then it is hard to visu- alize the result on a 2D surface of the screen. In this case, we make cross section of A with a two-dimensional plane and visualize this cross section. xn+1 = (1 −αn)T1(xn) + αnT2(yn), yn = (1 −βn)xn + βnT1(xn), n = 0, 1, 2, . . . , (13) (13) where αn ∈(0, 1] and βn ∈[0, 1] for all n ∈N. 3. The generalized Agarwal iteration (Khan et al. 2011): xn+1 = (1 −αn)T3(xn) + αnT2(yn), yn = (1 −βn)xn + βnT1(xn), n = 0, 1, 2, . . . , (14) where αn ∈(0, 1] and βn ∈[0, 1] for all n ∈N. xn+1 = (1 −αn)T3(xn) + αnT2(yn), yn = (1 −βn)xn + βnT1(xn), n = 0, 1, 2, . . . , (14) 2 The root-ﬁnding algorithm xn+1 = T (xn). (8) (8) Let f : RD →R be some function. We want to ﬁnd zeroes of f , i.e. to solve the following equation In the literature, this type of iteration is called the Picard iter- ation and it is widely used in many optimization algorithms and in ﬁnding ﬁxed points of a given function. f (x) = 0. (4) (4) f (x) = 0. In ﬁxed-point theory, some other iteration processes were introduced to ﬁnd the ﬁxed points of different types of func- tions: To solve (4), we use the following algorithm: 12 3 14138 I. Go´sciniak, K. Gdawiec 1. The Mann iteration (Mann 1953): the generalized Agarwal iteration reduces to the Khan–Cho– Abbas iteration when T1 = T3 and to the Agarwal iteration when T1 = T2 = T3. xn+1 = (1 −αn)xn + αnT (xn), n = 0, 1, 2, . . . , (9) (9) Having such a variety of different iteration processes, we can use them in our algorithm. In the iterations with a single mapping we use (7) as the mapping, and in the case of itera- tion with several mappings we use also (7), but with different values of ω and η parameters for each of the mappings. where αn ∈(0, 1] for all n ∈N. 2. The Ishikawa iteration (Ishikawa 1974): xn+1 = (1 −αn)xn + αnT (yn), yn = (1 −βn)xn + βnT (xn), n = 0, 1, 2, . . . , (10) 4 Visualization of the dynamics where αn ∈(0, 1] and βn ∈[0, 1] for all n ∈N. 3. The Agarwal iteration (Agarwal et al. 2007) (S-iteration): 3. The Agarwal iteration (Agarwal et al. 2007) (S-iteration): To visualize the dynamics of the proposed algorithm, we will use method that is very similar to the method used in polynomiography (Gdawiec et al. 2015). Because of this sim- ilarity, we will call the obtained images polynomiographs, although our algorithm ﬁnds roots of arbitrary functions and not only of polynomials as in polynomiography. xn+1 = (1 −αn)T (xn) + αnT (yn), yn = (1 −βn)xn + βnT (xn), n = 0, 1, 2, . . . , (11) where αn ∈[0, 1] and βn ∈[0, 1] for all n ∈N. In the algorithm, we choose iteration method, e.g. one of the methods presented in Sect. 3, parameters ω, η for a single mapping T or ω1, ω2, ω3 and η1, η2, η3 for T1, T2, T3 (dependingonthechoseniteration). Moreover, wechoosethe maximum number of iterations m which the algorithm should realize, the accuracy of the computations r and a colouring function C : N →{0, 1, . . . , 255}3. Then, for each x0 in the solution space A we use our algorithm. The iterations of the algorithm proceed till the convergence criterion: Let us notice that the Mann iteration for αn = 1 reduces to the Picard iteration, the Ishikawa iteration reduces to the Mann iteration when βn = 0 and to the Picard iteration when αn = 1, βn = 0, and the S-iteration reduces to the Picard iteration when αn = 0, or αn = 1 and βn = 0. A review of various iteration processes and their dependencies can be found in Gdawiec and Kotarski (2017). All the presented iterations used only one mapping, but in the ﬁxed-point theory exist iterations that use several mappings and are used to ﬁnd common ﬁxed points of the mappings. Let us recall some of them. (15) \|xn+1 −xn\| < r (15) is satisﬁed or the maximum number of iterations is reached. When the algorithm ends, we assign a colour to x0 using colouring function C and the number of performed iterations. 1. The Das–Debata iteration (Das and Debata 1986): Algorithm 1: Visualization of the dynamics Input: f – function, A ⊂RD – solution space, m – the maximum number of iterations, Iq – iteration method, q ∈[0, 1]N – parameters of the iteration Iq, ω, ω1, ω2, ω3, η, η1, η1, η2, η3 – parameters deﬁning functions T , T1, T2, T3, C – colouring function, r – accuracy Fig. 1 Colour map used in the experiments The functions (20)–(23) have the same roots as the fol- lowing functions: g y Output: visualization of the dynamics 1 foreach x0 ∈A do 2 i = 0 3 v0 = [0, 0, . . . , 0] 4 while i ≤m do 5 xn+1 = Iq(xn) 6 if \|xn+1 −xn\| < r then 7 break 8 i = i + 1 9 colour x0 with C(i) g1(z) = z3 −1, (24) g2(z) = z4 −10z2 + 9, (25) g3(z) = z5 −z, (26) g4(z) = z6 + 10z3 −8. (27) (24) (27) Similar approach, to the one presented in this paper, for function z3−1 for which visualization of the dynamics using Mann and Ishikawa iteration in the Newton’s method can be found in Kotarski et al. (2012). f1(x, y) = (−x3 + 3xy2 + 1)2 + (y3 −3x2y)2, (16) (16) In all experiments, the same colour map was used to colour the obtained images. The colour map is presented in Fig. 1. The colour map has 256 colours (ordered from left to right). The colour number represents the number of iterations needed to reach the solution. Moreover, in every experiment the following parameters were used: τ = 1.0e−3, m = 256, r = 1.0e−2, image resolution 800 × 800 pixels. The solu- tion spaces depend on the function and are the following: A1 = A3 = [−2.0, 2.0]2, A2 = [−4.0, 4.0] × [−2.0, 2.0], A4 = [−2.3, 1.7] × [−2.0, 2.0]. the function f1 has three roots, namely [1, 0], [−0.5, −0.866025], [−0.5, 0.866025]; f2(x, y) = (x4 −6x2y2 + y4 −10x2 + 10y2 + 9)2+ + (4x3y −4xy3 −20xy)2 , (17) the function f2 has four roots, namely [−3, 0], [−1, 0], [1, 0], [3, 0]; f3(x, y)= (x5−10x3y2+5xy4−x)2+(5x4y−10x2y3+y5−y)2, (18) the function f2 has four roots, namely [−3, 0], [−1, 0], [1, 0], [3, 0]; The software used in the research was implemented in the C++ programming language. Algorithm 1: Visualization of the dynamics The experiments were real- ized on a computer with the Intel Core i5-2520M processor, 4 GB RAM, and Linux 3.16.7-42-desktop openSUSE 13.2 (Harlequin 64-bits, KDE Platform version 4.14.9). f3(x, y)= (x5−10x3y2+5xy4−x)2+(5x4y−10x2y3+y5−y)2, (18) In all experiments, the polynomiographs are generated using Algorithm 1 in 2D space. Thus, looking at the gener- ated images we can read two important characteristics. The ﬁrst one is the speed of convergence of the algorithm, i.e. the colour of each point gives us information on how many iterations were performed by the algorithm to reach the root. The second characteristic is the dynamics of the algorithm. Low dynamics is in areas where the variation of colours is small, whereas in areas with a large variation of colours the dynamics is high. the function f3 has ﬁve roots, namely [−1, 0], [0, −1], [0, 0], [0, 1], [1, 0]; the function f3 has ﬁve roots, namely [−1, 0], [0, −1], [0, 0], [0, 1], [1, 0]; f4(x, y) = (x6−15x4y2+15x2y4−y6+10x3−30xy2−8)2+ + (6x5y−20x3y3+6xy5+30x2y−10y3)2 , (19) (19) the function f4 has six roots, namely [−2.207, 0], [−0.453, −0.785], [−0.453, 0.785], [0.906, 0], [1.103, −1.911], [1.103, 1.911]. the function f4 has six roots, namely [−2.207, 0], [−0.453, −0.785], [−0.453, 0.785], [0.906, 0], [1.103, −1.911], [1.103, 1.911]. 5 Discussion on the research results (14) where αn ∈(0, 1] and βn ∈[0, 1] for all n ∈N. In this section, we present and discuss the results of visualiz- ing the dynamics of the method introduced in Sect. 2 together with the various iteration methods presented in Sect. 3. In our study, we used functions fi : R2 →R for i ∈{1, 2, 3, 4} given by the following formulas: Let us notice that the Das–Debata iteration for T1 = T2 reduces to the Ishikawa iteration, the Khan–Cho–Abbas iter- ation reduces to the Agarwal iteration when T1 = T2, and 123 One more look on visualization of operation of a root-ﬁnding algorithm 14139 Algorithm 1: Visualization of the dynamics Input: f – function, A ⊂RD – solution space, m – the maximum number of iterations, Iq – iteration method, q ∈[0, 1]N – parameters of the iteration Iq, ω, ω1, ω2, ω3, η, η1, η1, η2, η3 – parameters deﬁning functions T , T1, T2, T3, C – colouring function, r – accuracy Output: visualization of the dynamics 1 foreach x0 ∈A do 2 i = 0 3 v0 = [0, 0, . . . , 0] 4 while i ≤m do 5 xn+1 = Iq(xn) 6 if \|xn+1 −xn\| < r then 7 break 8 i = i + 1 9 colour x0 with C(i) 5.1 The Picard iteration 3 Magniﬁcation of the marked areas in Fig. 2f try similar areas, create the corresponding dynamics of the particle. As was already mentioned, the parameters of the examples have been chosen so that in addition to the visu- alization of particle’s dynamics, the polynomiograph also represents (according to the authors) an aesthetic image. In Fig. 2 visualizations of the dynamics for the f1 and the Picard iteration using ω = 0 and varying η are presented. The acceleration constant is the only factor inﬂuencing a par- ticle motion—it affects its speed. (The larger the value of η, the greater the speed vn+1.) Moreover, the generation time of each image is shown in Fig. 2. Looking at the results, we see that the increase in particle’s velocity can shorten the creation time of the image. The black colour shows places which have not reached a solution in the given number of 256 iterations—Fig. 2a. The areas of the same colour indi- cate the same number of iterations required to determine the f (xn) = 0—they look similar to the contour lines on the map (Fig. 2f). The magniﬁcations of the marked areas in Fig. 2f are presented in Fig. 3. Fig. 3 Magniﬁcation of the marked areas in Fig. 2f Figure 5 presents examples of visualizations obtained for f1 and the Picard iteration using η = 0.025 and varying ω, and also their generation times. From the results, it is possible to observe that the higher the value of ω, the lower the generation time—the minimal value of time is obtained by polynomiograph in Fig. 5c. In this case, both too low and too high inertia may cause the extension of the time. Inertia helps the particle to escape from a not promis- ing area of solution space. Figure 4 presents examples of visualizations obtained for f1 and the Picard iteration using rather high inertia, i.e. ω = 0.7 and varying η, and also their generation times. The shortest time of the creation of poly- nomiographs in Fig. 4 is obtained by the polynomiograph from Fig. 4c. Both too low and too high velocities may cause the increase in the time of polynomiograph creation. Figures 6 and 7 show examples of visualizations obtained for η = 0.1 and η = 0.3 (respectively) and various values of ω for function f1. 5.1 The Picard iteration Because R2 is isomorphic with C and for each [x, y] ∈ R2 we have x + iy = z ∈C, then the functions fi for i ∈{1, 2, 3, 4} can be written in the following form: The behaviour of particles is affected by the speed and inertia. They are controlled by two parameters: the acceleration con- stant (η) and the inertia weight (ω) for the described above benchmark functions. The visualization of method’s dynam- ics allows to analyse their impact on the algorithm’s work. f1(z) = \|z3 −1\|, (20) f2(z) = \|z4 −10z2 + 9\|, (21) f3(z) = \|z5 −z\|, (22) f4(z) = \|z6 + 10z3 −8\|. (23) f1(z) = \|z3 −1\|, (20) f2(z) = \|z4 −10z2 + 9\|, (21) f3(z) = \|z5 −z\|, (22) f4(z) = \|z6 + 10z3 −8\|. (23) We start the experiments with the f1 function given by (16). This function has three roots that, thanks to the symme- 12 3 14140 I. Go´sciniak, K. Gdawiec 14140 I. Gosciniak, K. Gdawiec Fig. 2 Polynomiographs of f1 and the Picard iteration for ω = 0 and varying η try similar areas, create the corresponding dynamics of the particle. As was already mentioned, the parameters of the examples have been chosen so that in addition to the visu- alization of particle’s dynamics, the polynomiograph also represents (according to the authors) an aesthetic image. In Fig. 2 visualizations of the dynamics for the f1 and the Picard iteration using ω = 0 and varying η are presented. The acceleration constant is the only factor inﬂuencing a par- ticle motion—it affects its speed. (The larger the value of η, the greater the speed vn+1.) Moreover, the generation time of each image is shown in Fig. 2. Looking at the results, we see that the increase in particle’s velocity can shorten the creation time of the image. The black colour shows places which have not reached a solution in the given number of 256 iterations Fig 2a The areas of the same colo r indi Fig. 3 Magniﬁcation of the marked areas in Fig. 2f Figure 5 presents examples of visualizations obtained for f d h Pi d i i i 0 025 d i Fig. 2 Polynomiographs of f1 and the Picard iteration for ω = 0 and varying η Fig. 2 Polynomiographs of f1 and the Picard iteration for ω = 0 and varying η Fig. 5.1 The Picard iteration Looking at the ﬁgures, we see that for the higher velocity of particle (acceleration constant increases in the following ﬁgures) the high value of inertia weight causes the increase in the creation time of the image. From all the images obtained for f1 and the Picard iter- ation, we see that the dynamics of the method changes in 123 One more look on visualization of operation of a root-ﬁnding algorithm 14141 Fig. 4 Polynomiographs of f1 and the Picard iteration for ω = 0.7 and varying η Fig. 5 Polynomiographs of f1 and the Picard iteration for η = 0.025 and varying ω Fig. 6 Polynomiographs of f1 and the Picard iteration for η = 0.1 and varying ω Fig. 4 Polynomiographs of f1 and the Picard iteration for ω = 0.7 and varying η Fig. 5 Polynomiographs of f1 and the Picard iteration for η = 0.025 and varying ω Fig. 5 Polynomiographs of f1 and the Picard iteration for η = 0.025 and varying ω Fig. 6 Polynomiographs of f1 and the Picard iteration for η = 0.1 and varying ω Polynomiographs of the Picard iteration for the f2 func- tion are shown in Fig. 8. Two groups of areas forming a different dynamics are clearly visible in Fig. 8a. The proper value of ω is responsible for creating a particle dynamics balance—it is evident in Fig. 8c. In this case, we observe a Polynomiographs of the Picard iteration for the f2 func- tion are shown in Fig. 8. Two groups of areas forming a different dynamics are clearly visible in Fig. 8a. The proper value of ω is responsible for creating a particle dynamics balance—it is evident in Fig. 8c. In this case, we observe a a signiﬁcant way when we change two parameters of the method (ω, η). A smooth image represents low dynamics of the particles. Moreover, we see that using the proposed method we obtain interesting patterns and more diverse pat- terns are observed for particles with greater dynamics. a signiﬁcant way when we change two parameters of the method (ω, η). A smooth image represents low dynamics of the particles. Moreover, we see that using the proposed method we obtain interesting patterns and more diverse pat- terns are observed for particles with greater dynamics. 123 12 3 14142 I. Go´sciniak, K. Gdawiec Fig. 5.1 The Picard iteration 7 Polynomiographs of f1 and the Picard iteration for η = 0.3 and varying ω Fig. 8 Polynomiographs of f2 and the Picard iteration for η = 0.01 and varying ω Fig. 9 Polynomiographs of f3 and the Picard iteration for ω = 0.5 and varying η Fig. 7 Polynomiographs of f1 and the Picard iteration for η = 0.3 and varying ω ig. 7 Polynomiographs of f1 and the Picard iteration for η = 0.3 and varying ω Polynomiographs of f1 and the Picard iteration for η = 0.3 and varying ω Fig. 8 Polynomiographs of f2 and the Picard iteration for η = 0.01 and varying ω ig. 8 Polynomiographs of f2 and the Picard iteration for η = 0.01 and varying ω Fig. 9 Polynomiographs of f3 and the Picard iteration for ω = 0.5 and varying η particle—it shortens the time of creation of the polynomio- graph (Fig. 10a, b). The excessive value of inertia weight can be compensated by a decrease in the value of acceler- ation constant—consequently, similar particle dynamics is obtained in the whole area (Fig. 10c, d). reduction in the time of the polynomiograph creation. How- ever, an excessive increase in the inertia coefﬁcient extends this time. The next ﬁgure—Fig. 9—shows polynomiographs of the Picard iteration for the f3 function. The function f3, thanks to the symmetry of roots placement, has four areas that cre- ate similar particle dynamics. The increase in the value of the acceleration constant (the particle moves faster—it increases in its dynamics) can shorten the time of creation of the poly- nomiograph. The dynamics of the particle is limited in the whole area of the polynomiograph—it is shown in Fig. 8b–d. However, areas from which the particle does not reach the solution (black colour) arise. 5.2 The Mann iteration The Mann iteration is enhanced by the α parameter in relation to the Picard iteration. It gives the possibility to adjust the dynamic of the particle behaviour independently of the ω and η. Figure 11 presents the dynamics of the method for ω = 0, η = 0.025 using the Mann iteration and f1. For α = 1, we obtain the Picard iteration (Fig. 11a). From the images, we see that the decrease in the value of the parameter α, which is responsible for a linear combination in the Mann itera- Two groups of areas with different dynamics are vis- ible in Fig. 10. The images in this ﬁgure were created using the f4 function and the Picard iteration. Increasing the value of the inertia weight increases the dynamics of the 123 123 One more look on visualization of operation of a root-ﬁnding algorithm 14143 Fig. 10 Polynomiographs of f4 and the Picard iteration for varying ω and η Fig. 11 Polynomiographs of f1 and the Mann iteration for ω = 0, η = 0.025 and varying α tion, reduces the dynamics of particles. We can also observe th t d i th d i f ti l th l k f dynamic of the particle. The high particle dynamics allows t bt i i t ti tt Fig. 10 Polynomiographs of f4 and the Picard iteration for varying ω and η Fig. 10 Polynomiographs of f4 and the Picard iteration for varying ω and η Fig. 11 Polynomiographs of f1 and the Mann iteration for ω = 0, η = 0.025 and varying α dynamic of the particle. The high particle dynamics allows to obtain interesting patterns. dynamic of the particle. The high particle dynamics allows to obtain interesting patterns. tion, reduces the dynamics of particles. We can also observe that reducing the dynamics of particles can cause the lack of ﬁnding a solution (black colour), even for large areas of the solution space. Moreover, we see that the lower the value of α, the greater the generation time. tion, reduces the dynamics of particles. We can also observe that reducing the dynamics of particles can cause the lack of ﬁnding a solution (black colour), even for large areas of the solution space. Moreover, we see that the lower the value of α, the greater the generation time. Signiﬁcantly lower particles dynamics is presented in Fig. 15. 5.2 The Mann iteration The polynomiographs in this ﬁgure were obtained for f1, ω = 0.3, η = 0.3 and varying α in the Mann itera- tion. These images are similar to the images obtained for the Picard iteration. The decrease in particle’s dynamics smooths out the images—it is also visible in the remaining images. Figure 15a, b and Fig. 15c, d—although different in particle’s dynamics—are similar in the colouring. The acceleration constant (η) affects particle dynamics. Using a wide range of changes in α = 0.2, 0.5, 0.7, 0.9 (which limits the particle dynamics) in Fig. 12, there are no areas where the algorithm does not reach the solution. It is a result of increased particle dynamics by increasing the acceleration constant (η = 0.35). Tuning the algorithm is a multidimensional problem. It means that an increase in the number of parameters compli- cates this task. The polynomiographs from Fig. 16 (generated for the f2 function) show the particle dynamics constituting the intersection of the algorithm tuning space for the α. The increase in the α parameter value increases the dynamics of the particles. Properly selected value of this parameter Relatively large dynamics of particles for f1 is presented in Figs. 13 and 14 obtained for different values of parameters ω and η deﬁning T and the α parameter in the Mann iteration. From the images, we can observe that the decrease in the value of the parameter α, which reduces the proportion of the part speciﬁed by T in the Mann iteration, reduces the 12 3 14144 I. Go´sciniak, K. Gdawiec 14144 I. Go´sciniak, K. Gda Fig. 12 Polynomiographs of f1 and the Mann iteration for ω = 0, η = 0.35 and varying α Fig. 13 Polynomiographs of f1 and the Mann iteration for ω = 0.8, η = 0.1 and varying α Fig. 14 Polynomiographs of f1 and the Mann iteration for ω = 0.6, η = 0.2 and varying α Fig. 15 Polynomiographs of f1 and the Mann iteration for ω = 0.3, η = 0.3 and varying α (Fig. 16c) minimizes the time of the polynomiograph cre- ation. As in the previous example, in Fig. 17 polynomiographs generated using a varying α parameter are presented, but this time the f3 function was used A limitation of the dynamic range observed in Fig. 17c occurs for the shortest tim a polynomiograph creation. 5.2 The Mann iteration Moreover, we can observe the change in the α parameter by 0.1 affects the dynam in different way. In Fig. 17a, b the dynamics is almos Fig. 12 Polynomiographs of f1 and the Mann iteration for ω = 0, η = 0.35 and varying α Fig. 12 Polynomiographs of f1 and the Mann iteration for ω = 0, η = 0.35 and varying α Fig. 13 Polynomiographs of f1 and the Mann iteration for ω = 0.8, η = 0.1 and varying α Fig. 12 Polynomiographs of f1 and the Mann iteration for ω = 0, η = 0.35 and varying α Fig. 13 Polynomiographs of f1 and the Mann iteration for ω = 0.8, η = 0.1 and varying α Fig. 14 Polynomiographs of f1 and the Mann iteration for ω = 0.6, η = 0.2 and varying α Fig. 14 Polynomiographs of f1 and the Mann iteration for ω = 0.6, η = 0.2 and varying α 0.2 and varying α Fig. 15 Polynomiographs of f1 and the Mann iteration for ω = 0.3, η = 0.3 and varying α range observed in Fig. 17c occurs for the shortest time of a polynomiograph creation. Moreover, we can observe that the change in the α parameter by 0.1 affects the dynamics in different way. In Fig. 17a, b the dynamics is almost the (Fig. 16c) minimizes the time of the polynomiograph cre- ation. As in the previous example, in Fig. 17 polynomiographs generated using a varying α parameter are presented, but this time the f3 function was used. A limitation of the dynamic 123 14145 One more look on visualization of operation of a root-ﬁnding algorithm Fig. 16 Polynomiographs of f2 and the Mann iteration for ω = 0.8, η = 0.01 and varying α Fig. 17 Polynomiographs of f3 and the Mann iteration for ω = 0.8, η = 0.025 and varying α Fig. 18 Polynomiographs of f4 and the Mann iteration for α = 0.1 and varying η and ω Fig. 16 Polynomiographs of f2 and the Mann iteration for ω = 0.8, η = 0.01 and varying α Fig. 17 Polynomiographs of f3 and the Mann iteration for ω = 0.8, η = 0.025 and varying α Fig. 18 Polynomiographs of f4 and the Mann iteration for α = 0.1 and varying η and ω Fig. same, whereas in Fig. 17b, c and in Fig. 17c, d the change is signiﬁcant. 5.2 The Mann iteration 19 Polynomiographs of f1 and the Ishikawa iteration for β = 0.4, ω = 0.5, η = 0.3 and varying α Fig. 20 Polynomiographs of f1 and the Ishikawa iteration for β = 0.8 ω = 0.5, η = 0.3 and varying α Fig. 16 Polynomiographs of f2 and the Mann iteration for ω = 0.8, η = 0.01 and varying α Fig. 17 Polynomiographs of f3 and the Mann iteration for ω = 0.8, η = 0.025 and varying α Fig. 18 Polynomiographs of f4 and the Mann iteration for α = 0.1 and varying η and ω Fig. 18 Polynomiographs of f4 and the Mann iteration for α = 0.1 and varying η and ω Fig. 19 Polynomiographs of f1 and the Ishikawa iteration for β = 0.4, ω = 0.5, η = 0.3 and varying α Fig. 20 Polynomiographs of f1 and the Ishikawa iteration for β = 0.8 ω = 0.5, η = 0.3 and varying α Fig. 20 Polynomiographs of f1 and the Ishikawa iteration for β = 0.8, ω = 0.5, η = 0.3 and varying α Fig. 19 Polynomiographs of f1 and the Ishikawa iteration for β = 0.4, ω = 0.5, η = 0.3 and varying α Examples of polynomiographs for the Mann iteration and function f4 arepresentedinFig.18.Thevalueoftheα param- same, whereas in Fig. 17b, c and in Fig. 17c, d the change is signiﬁcant. 12 I. Go´sciniak, K. Gdawiec 14146 Fig. 21 Polynomiographs of f1 and the Ishikawa iteration for α = 0.7, ω = 0.2, η = 0.6 and varying β Fig. 22 Polynomiographs of f1 and the Ishikawa iteration for α = 0.3, ω = 0.2, η = 0.4 and varying β Fig. 21 Polynomiographs of f1 and the Ishikawa iteration for α = 0.7, ω = 0.2, η = 0.6 and varying β eter is small, which signiﬁcantly limits the dynamics of the particle. The increase in the value of the acceleration constant slightly reduces the time of the polynomiograph creation— Fig. 18b. A signiﬁcant reduction in the inertia weight causes a reduction in particle dynamics and reduces the time needed to the polynomiograph creation—it is visible in Fig. 18d. 5.3 The Ishikawa and the Das–Debata iterations 29 Polynomiographs of f1 and the Das–Debata iteration for α = 0.4, β = 0.3, ω1 = 0.2, ω2 = 0.4, η2 = 0.4 and varying η1 Fig. 26 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.7, η1 = 0.4, ω2 = 0.4, η2 = 0.4 and varying ω1 Fig. 28 Polynomiographs of f1 and the Das–Debata iteration for α = 0.4, β = 0.1, ω1 = 0.3, ω2 = 0.3, η2 = 0.4 and varying η1 Fig. 28 Polynomiographs of f1 and the Das–Debata iteration for α = 0.4, β = 0.1, ω1 = 0.3, ω2 = 0.3, η2 = 0.4 and varying η1 Fig. 26 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.7, η1 = 0.4, ω2 = 0.4, η2 = 0.4 and varying ω1 Fig. 27 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.5, η1 = 0.5, ω2 = 0.3, η2 = 0.4 and varying ω1 Fig. 29 Polynomiographs of f1 and the Das–Debata iteration for α = 0.4, β = 0.3, ω1 = 0.2, ω2 = 0.4, η2 = 0.4 and varying η1 Fig. 29 Polynomiographs of f1 and the Das–Debata iteration for α = 0.4, β = 0.3, ω1 = 0.2, ω2 = 0.4, η2 = 0.4 and varying η1 Fig. 27 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.5, η1 = 0.5, ω2 = 0.3, η2 = 0.4 and varying ω1 Figures 19 and 20 show the reduction of the dynamics of theparticlefor f1 bytheα parameterintheIshikawaiteration. A small value of β parameter causes that the reference point is created close to the active point. The same value of the α parameter is the cause of creation of similar patterns. Polynomiographs presented in Fig. 23 for the f2 function and the Ishikawa iteration show particle dynamics for chang- ing ω and α parameters. Comparing Fig. 23a, b, an increase in dynamics due to an increase in the ω value is observed, which shortens the time of the polynomiograph creation. Then a fur- ther increase in dynamics due to an increase in the ω value results in a longer time of a polynomiograph creation—Fig. 23c.Reducingthedynamicsoftheparticlethroughtheappro- priate selection of the α parameter reduces the time of the polynomiograph creation. 5.3 The Ishikawa and the Das–Debata iterations In the Ishikawa iteration, an additional parameter β is intro- duced in relation to the Mann iteration. This parameter, like the parameter α, has an impact on the dynamics of particles. It is also involved in the creation of an additional reference point (the sample in the solution space). In this way, particle motion is a linear combination of its position and a move- ment of reference point. This gives the possibility to deﬁne two different operators (T1 and T2) for transforming the cur- rent position of the particle and the position of the reference point—the Das–Debata iteration. For the differentiation of these operators, the parameters ω1, ω2 and η1 and η2 are responsible. Fig. 21 Polynomiographs of f1 and the Ishikawa iteration for α = 0.7, ω = 0.2, η = 0.6 and varying β Fig. 21 Polynomiographs of f1 and the Ishikawa iteration for α = 0.7, ω = 0.2, η = 0.6 and varying β Fig. 22 Polynomiographs of f1 and the Ishikawa iteration for α = 0.3, ω = 0.2, η = 0.4 and varying β Two types of iterations—Ishikawa and Das–Debata—give a wide range of possibilities for the regulation of the algo- rithm’s work. The use of these iterations causes twisting contour lines. The α parameter, as in the case of the Mann iteration, limits the dynamics of a particle. A similar function as the parameter α is realized by the parameter β in relation to the reference point. Fig. 22 Polynomiographs of f1 and the Ishikawa iteration for α = 0.3, ω = 0.2, η = 0.4 and varying β Fig. 23 Polynomiographs of f2 and the Ishikawa iteration for β = 0.9, η = 0.01 and varying α, ω Fig. 24 Polynomiographs of f3 and the Ishikawa iteration for ω = 0.7, η = 0.02 and varying α, β Fig. 23 Polynomiographs of f2 and the Ishikawa iteration for β = 0.9, η = 0.01 and varying α, ω Fig. 23 Polynomiographs of f2 and the Ishikawa iteration for β = 0.9, η = 0.01 and varying α, ω Fig. 24 Polynomiographs of f3 and the Ishikawa iteration for ω = 0.7, η = 0.02 and varying α, β Fig. 5.3 The Ishikawa and the Das–Debata iterations 24 Polynomiographs of f3 and the Ishikawa iteration for ω = 0.7, η = 0.02 and varying α, β 12 3 One more look on visualization of operation of a root-ﬁnding algorithm 14147 One more look on visualization of operation of a root-ﬁnding algorithm 14147 Fig. 25 Polynomiographs of f4 and the Ishikawa iteration for ω = 0.9, η = 0.0007 and varying α, β Fig. 26 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.7, η1 = 0.4, ω2 = 0.4, η2 = 0.4 and varying ω1 Fig. 27 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.5, η1 = 0.5, ω2 = 0.3, η2 = 0.4 and varying ω1 Fig. 28 Polynomiographs of f1 and the Das–Debata iteration for α = 0.4, β = 0.1, ω1 = 0.3, ω2 = 0.3, η2 = 0.4 and varying η1 Fig. 29 Polynomiographs of f1 and the Das–Debata iteration for α = 0.4, β = 0.3, ω1 = 0.2, ω2 = 0.4, η2 = 0.4 and varying η1 = 0.0007 and varying α, β Fig. 28 Polynomiographs of f1 and the Das–Debata iteration for α = 0.4, β = 0.1, ω1 = 0.3, ω2 = 0.3, η2 = 0.4 and varying η1 Fig. 29 Polynomiographs of f1 and the Das–Debata iteration for α = 0.4, β = 0.3, ω1 = 0.2, ω2 = 0.4, η2 = 0.4 and varying η1 Fig. 25 Polynomiographs of f4 and the Ishikawa iteration for ω = 0.9, η = 0.0007 and varying α, β Fig. 25 Polynomiographs of f4 and the Ishikawa iteration for ω = 0.9, η = 0.0007 and varying α, β Fig. 25 Polynomiographs of f4 and the Ishikawa iteration for ω = 0.9, η = 0.0007 and varying α, β Fig. 26 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.7, η1 = 0.4, ω2 = 0.4, η2 = 0.4 and varying ω1 Fig. 27 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.5, η1 = 0.5, ω2 = 0.3, η2 = 0.4 and varying ω1 Fig. 28 Polynomiographs of f1 and the Das–Debata iteration for α = 0.4, β = 0.1, ω1 = 0.3, ω2 = 0.3, η2 = 0.4 and varying η1 Fig. 5.3 The Ishikawa and the Das–Debata iterations 34 Polynomiographs of f1 and the Das–Debata iteration for ω1 = 0.4, η1 = 0.2, ω2 = 0.9 Fig. 35 Polynomiographs of f1 and the Das–Debata iteration for α = 0.5, β = 0.4, η1 = 0.2, ω2 = 0.9, η2 = 0.9 and varying ω1 Fig. 31 Polynomiographs of f1 and the Das–Debata iteration for α = 0.7, β = 0.5, ω1 = 0.4, η1 = 0.2, η2 = 0.7 and varying ω2 Fig. 34 Polynomiographs of f1 and the Das–Debata iteration for ω1 = 0.4, η1 = 0.2, ω2 = 0.9 Fig. 31 Polynomiographs of f1 and the Das–Debata iteration for α = 0.7, β = 0.5, ω1 = 0.4, η1 = 0.2, η2 = 0.7 and varying ω2 Fig. 31 Polynomiographs of f1 and the Das–Debata iteration for α = 0.7, β = 0.5, ω1 = 0.4, η1 = 0.2, η2 = 0.7 and varying ω2 Fig. 34 Polynomiographs of f1 and the Das–Debata iteration for ω1 = 0.4, η1 = 0.2, ω2 = 0.9 Fig. 32 Polynomiographs of f1 and the Das–Debata iteration for α = 0.8, β = 0.4, ω1 = 0.1, η1 = 0.6, ω2 = 0.3 and varying η2 Fig. 35 Polynomiographs of f1 and the Das–Debata iteration for α = 0.5, β = 0.4, η1 = 0.2, ω2 = 0.9, η2 = 0.9 and varying ω1 Fig. 32 Polynomiographs of f1 and the Das–Debata iteration for α = 0.8, β = 0.4, ω1 = 0.1, η1 = 0.6, ω2 = 0.3 and varying η2 Fig. 35 Polynomiographs of f1 and the Das–Debata iteration for α = 0.5, β = 0.4, η1 = 0.2, ω2 = 0.9, η2 = 0.9 and varying ω1 Figure 24 shows the changes in particle dynamics for the function f3 under the inﬂuence of changes in the α and β parameters for the Ishikawa iteration. Comparing the times of polynomiographs creation in Fig. 24b, c with Fig. 24a or d, the effect of change in the α parameter on particle’s dynamics is noticeably greater than that of β. greater effect on the behaviour of the particle than the change in the β parameter. Figures 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 present polynomiographs created using the Das–Debata iteration for the f1 function. In this iteration, the ω1 inertia weight is responsible for the dynamics of the reference point creation. 5.3 The Ishikawa and the Das–Debata iterations This method of particle dynam- ics reduction is ineffective and may cause the particle not to reach a solution. The reduction of the dynamics of the reference point cre- ation for f1 by the α parameter in the Ishikawa iteration is shown in Figs. 21 and 22. A signiﬁcant decrease in the dynamics of the reference point creation weakens the effect of twisting lines—it is also visible in the direction to the cen- tre of the pattern. For large values of the β parameter, major changes occur on the periphery of the image—Fig. 22. 12 3 3 3 14148 I. Go´sciniak, K. Gdawiec Fig. 30 Polynomiographs of f1 and the Das–Debata iteration for α = 0.9, β = 0.7, ω1 = 0.4, η1 = 0.2, η2 = 0.5 and varying ω2 Fig. 31 Polynomiographs of f1 and the Das–Debata iteration for α = 0.7, β = 0.5, ω1 = 0.4, η1 = 0.2, η2 = 0.7 and varying ω2 Fig. 32 Polynomiographs of f1 and the Das–Debata iteration for α = 0.8, β = 0.4, ω1 = 0.1, η1 = 0.6, ω2 = 0.3 and varying η2 Fig. 33 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.7, ω1 = 0.1, η1 = 0.3, ω2 = 0.2 and varying η2 Fig. 34 Polynomiographs of f1 and the Das–Debata iteration for ω1 = 0.4, η1 = 0.2, ω2 = 0.9 Fig. 35 Polynomiographs of f1 and the Das–Debata iteration for α = 0.5, β = 0.4, η1 = 0.2, ω2 = 0.9, η2 = 0.9 and varying ω1 I. Go´sciniak, K. Gdawiec 14148 Fig. 33 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.7, ω1 = 0.1, η1 = 0.3, ω2 = 0.2 and varying η2 Fig. 30 Polynomiographs of f1 and the Das–Debata iteration for α = 0.9, β = 0.7, ω1 = 0.4, η1 = 0.2, η2 = 0.5 and varying ω2 Fig. 33 Polynomiographs of f1 and the Das–Debata iteration for α = 0.6, β = 0.7, ω1 = 0.1, η1 = 0.3, ω2 = 0.2 and varying η2 Fig. 30 Polynomiographs of f1 and the Das–Debata iteration for α = 0.9, β = 0.7, ω1 = 0.4, η1 = 0.2, η2 = 0.5 and varying ω2 Fig. 34 Polynomiographs of f1 and the Das–Debata iteration for ω1 = 0.4, η1 = 0.2, ω2 = 0.9 Fig. 5.3 The Ishikawa and the Das–Debata iterations Small differences of the dynamics of the reference point cre- ation can cause the creation of similar patterns—Figs. 26 and 27. A similar effect is given by the change in the refer- ence point dynamics by determining the η1 parameter—see Polynomiographs obtained for the f4 function using the Ishikawa iteration are presented in Fig. 25. The obtained results conﬁrm the observations from the previous exam- ple (Fig. 24), i.e. the change in the α parameter has a much 123 One more look on visualization of operation of a root-ﬁnding algorithm 14149 Fig. 36 Polynomiographs of f2 and the Das–Debata iteration for varying α, β, ω1, η1, ω2, η2 Fig. 37 Polynomiographs of f3 and the Das–Debata iteration for α = 0.5, β = 0.5, η1 = 0.05, ω2 = 0.9, η2 = 0.05 and varying ω1 Fig. 36 Polynomiographs of f2 and the Das–Debata iteration for varying α, β, ω1, η1, ω2, η2 Fig. 37 Polynomiographs of f3 and the Das–Debata iteration for α = 0.5, β = 0.5, η1 = 0.05, ω2 = 0.9, η2 = 0.05 and varying ω1 Fig. 38 Polynomiographs of f4 and the Das–Debata iteration for α = 0.5, β = 0.5, ω1 = 0.8, η1 = 0.05, ω2 = 0.9 and varying η2 Fig. 36 Polynomiographs of f2 and the Das–Debata iteration for varying α, β, ω1, η1, ω2, η2 Fig. 37 Polynomiographs of f3 and the Das–Debata iteration for α = 0.5, β = 0.5, η1 = 0.05, ω2 = 0.9, η2 = 0.05 and varying ω1 Fig. 38 Polynomiographs of f4 and the Das–Debata iteration for α = 0.5, β = 0.5, ω1 = 0.8, η1 = 0.05, ω2 = 0.9 and varying η2 miographs of f4 and the Das–Debata iteration for α = 0.5, β = 0.5, ω1 = 0.8, η1 = 0.05, ω2 = 0.9 and varying η2 Fig. 38 Polynomiographs of f4 and the Das–Debata iteration for α = 0.5, β = 0.5, ω1 = 0.8, η1 = 0.05, ω2 = 0 Figs. 28 and 29. Small differences of the η1 parameter are the cause of creation of similar patterns. parameters, the detailed analysis of their dependence is dif- ﬁcult. The general rule that proper selection of the dynamics of particles reduces the operating time of the algorithm is conﬁrmed. Moreover, from the images we see that the use of Das–Debata iteration allows to create interesting patterns. 5.4 The Agarwal and the Khan–Cho–Abbas iterations Fig. 39 Polynomiographs of f1 and the Agarwal iteration for α = 0.5, ω1 = ω2 = ω3 = 0.4, η1 = η2 = η3 = 0.3 and varying β The generalized Agarwal iteration is more complex than the previously presented iterations. It introduces an additional transformation T3 (with parameters ω3, η3) of particle in rela- tion to the Das–Debata iteration. Fig. 40 Polynomiographs of f1 and the Agarwal iteration for β = 0.5, ω1 = ω2 = ω3 = 0.4, η1 = η2 = η3 = 0.3 and varying α Figures 39 and 40 present images obtained with the Agar- wal iteration (T1 = T2 = T3) for f1 and varying α and β parameters. When we look at these images, we see that they have features of a twisting line, similarly to the Ishikawa iteration. Depending on the dynamics of the particles, it is possible to obtain images similar to the ones obtained with the Ishikawa iteration. Operator T3 causes the introduction of an additional dynamics of particles, the contribution of which produces the unique visual effects—the additional twisting lines are visible. The increase in dynamics of particles allows creat- ing images of an artistic meaning. Moreover, we observe that the time of generation of the images (the working time of the algorithm) is longer that in the case of the previous iterations. This is due to the complexity of the iteration and the particle’s dynamics. Fig. 40 Polynomiographs of f1 and the Agarwal iteration for β = 0.5, ω1 = ω2 = ω3 = 0.4, η1 = η2 = η3 = 0.3 and varying α Figure 37 presents polynomiographs obtained for the f3 function using the Das–Debata iteration and varying ω1 parameter. In subsequent images, it is possible to observe the effect of increasing in the value of ω1, which is responsi- ble for the dynamics of creating the reference point. Changes in the values of inertia weight of the reference point transfor- mation have a small impact on the dynamics of the particle, as well as changes in the β parameter. Appropriate selection of the ω1 parameter can shorten the time of the polynomio- graph’s generation—it is presented in Fig. 37c. Polynomiographs of the f2 function and the Agarwal iter- ation with varying α and β parameters are presented in Fig. 41. 5.3 The Ishikawa and the Das–Debata iterations Dynamics of particle’s movement has a signiﬁcant impact on the appearance of the polynomiograph. The parameters ω2 and η2 are responsible for this dynamics. As it was already mentioned, both the dynamics of the reference point and the dynamics of the particle are responsible for the creation of intriguing images. Figures 30 and 31 show the effect of the change in inertia weight ω2 on the particle dynamics. High particle dynamics allows to obtain interesting images. Figures 32 and 33 show the effect of the change in the accel- eration constant η2 on the creation of a polynomiograph. In these images, a smaller dynamics is observed. Polynomiographs of the f2 function and the Das–Debata iteration for varying all parameters are presented in Fig. 36. The obtained polynomiographs present a high dynamics of the particle. The shortest time of polynomiograph’s creation is obtained for Fig. 36c. In this case, the narrowed range of the particle’s dynamic for the entire polynomiograph area is observed. As it was already mentioned, tuning the algorithm cannot be covered by the deterministic rule due to the very complex nature of the relationship between the algorithm’s coefﬁcients and the environmental impact. The images in Figs. 34 and 35 visualize a high dynamics of particle’s movement. Due to varying of a large number of 12 3 3 I. Go´sciniak, K. Gdawiec 14150 Fig. 39 Polynomiographs of f1 and the Agarwal iteration for α = 0.5, ω1 = ω2 = ω3 = 0.4, η1 = η2 = η3 = 0.3 and varying β values of the acceleration constant parameter, but with high dynamics of the reference point creation and a high inertia weight of the particle, it is possible to obtain a sufﬁcient particle dynamics for an effective movement in the solution space. The obtained polynomiographs present very similar particle dynamics. The shortest time of polynomiograph’s creation is obtained for Fig. 38b. 5.4 The Agarwal and the Khan–Cho–Abbas iterations 45 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.5, ω1 = 0.1, ω2 = ω3 = 0.4, η2 = η3 = 0.3 (T2 = T3) and varying η1 Fig. 42 Polynomiographs of f3 and the Agarwal iteration for α = 0.9, β = 0.9, η1 = η2 = η3 = 0.01 and varying ω = ω1 = ω2 = ω3 Fig. 45 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.5, ω1 = 0.1, ω2 = ω3 = 0.4, η2 = η3 = 0.3 (T2 = T3) and varying η1 Fig. 42 Polynomiographs of f3 and the Agarwal iteration for α = 0.9, β = 0.9, η1 = η2 = η3 = 0.01 and varying ω = ω1 = ω2 = ω3 Fig. 42 Polynomiographs of f3 and the Agarwal iteration for α = 0.9, β = 0.9, η1 = η2 = η3 = 0.01 and varying ω = ω1 = ω2 = ω3 Fig. 43 Polynomiographs of f4 and the Agarwal iteration for α = 0.5, β = 0.5, ω1 = ω2 = ω3 = 0.8 and varying η = η1 = η2 = η3 Fig. 46 Polynomiographs of f1 and the Khan–Cho–Abbas iteration for α = 0.5, β = 0.5, ω1 = 0.5, η1 = 0.2, η2 = 0.5, ω3 = 0.4, η3 = 0.3 and varying ω2 Fig. 43 Polynomiographs of f4 and the Agarwal iteration for α = 0.5, β = 0.5, ω1 = ω2 = ω3 = 0.8 and varying η = η1 = η2 = η3 Fig. 43 Polynomiographs of f4 and the Agarwal iteration for α = 0.5, β = 0.5, ω1 = ω2 = ω3 = 0.8 and varying η = η1 = η2 = η3 Fig. 46 Polynomiographs of f1 and the Khan–Cho–Abbas iteration for α = 0.5, β = 0.5, ω1 = 0.5, η1 = 0.2, η2 = 0.5, ω3 = 0.4, η3 = 0.3 and varying ω2 Fig. 44 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.5, η1 = 0.4, ω2 = ω3 = 0.4, η2 = η3 = 0.3 (T2 = T3) and varying ω1 Fig. 5.4 The Agarwal and the Khan–Cho–Abbas iterations In the presented images, the shortest time of polynomio- graph’s creation is obtained for the parameters responsible for the highest dynamics of the particle (Fig. 41a). The intro- duction of the additional transformations causes that even extreme changes in the α and β parameters do not introduce drastic changes in particle’s dynamics (Fig. 41b, c). Signif- The results of using the Das–Debata iteration for f4 and a varying η2 parameter are presented in Fig. 38. Even for low Fig. 41 Polynomiographs of f2 and the Agarwal iteration for ω1 = ω2 = ω3 = 0.9, η1 = η2 = η3 = 0.01 and varying α and β Polynomiographs of f2 and the Agarwal iteration for ω1 = ω2 = ω3 = 0.9, η1 = η2 = η3 = 0.01 and varying α and 12 3 One more look on visualization of operation of a root-ﬁnding algorithm 14151 Fig. 42 Polynomiographs of f3 and the Agarwal iteration for α = 0.9, β = 0.9, η1 = η2 = η3 = 0.01 and varying ω = ω1 = ω2 = ω3 Fig. 43 Polynomiographs of f4 and the Agarwal iteration for α = 0.5, β = 0.5, ω1 = ω2 = ω3 = 0.8 and varying η = η1 = η2 = η3 Fig. 44 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.5, η1 = 0.4, ω2 = ω3 = 0.4, η2 = η3 = 0.3 (T2 = T3) and varying ω1 Fig. 45 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.5, ω1 = 0.1, ω2 = ω3 = 0.4, η2 = η3 = 0.3 (T2 = T3) and varying η1 Fig. 46 Polynomiographs of f1 and the Khan–Cho–Abbas iteration for α = 0.5, β = 0.5, ω1 = 0.5, η1 = 0.2, η2 = 0.5, ω3 = 0.4, η3 = 0.3 and varying ω2 Fig. 47 Polynomiographs of f1 and the Khan–Cho–Abbas iteration for One more look on visualization of operation of a root-ﬁnding algorithm 14151 Fig. 45 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.5, ω1 = 0.1, ω2 = ω3 = 0.4, η2 = η3 = 0.3 (T2 = T3) and varying η1 Fig. 5.4 The Agarwal and the Khan–Cho–Abbas iterations 47 Polynomiographs of f1 and the Khan–Cho–Abbas iteration for α = 0.5, β = 0.5, ω1 = 0.5, η1 = 0.2, ω2 = 0.6, ω3 = 0.4, η3 = 0.3 and varying η2 Fig. 44 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.5, η1 = 0.4, ω2 = ω3 = 0.4, η2 = η3 = 0.3 (T2 = T3) and varying ω1 Fig. 47 Polynomiographs of f1 and the Khan–Cho–Abbas iteration for α = 0.5, β = 0.5, ω1 = 0.5, η1 = 0.2, ω2 = 0.6, ω3 = 0.4, η3 = 0.3 and varying η2 icant changes in dynamics are noticeable for simultaneous change in both parameters (Fig. 41d). Polynomiographs of the Agarwal iteration for the f3 function are shown in Fig. 42. They present the limitation of particle dynamics by reducing the values of the inertia weights ω1, ω2 and ω3—limiting the dynamics results in longer polynomiograph creation time. in the acceleration constant. In this case, this results in a shortening of the polynomiograph’s creation time. The generalized Agarwal iteration fulﬁls the condition T1 ̸= T3. Operators T2 and T3 are the same for Figs. 44 and 45, which were obtained for the f1 function. The dynamics of the particle is inﬂuenced by three transformations. Even large change in the transformation T1 (by using ω1 or η1), which The last example for the Agarwal iteration—Fig. 43— presents images obtained for the f4 function. The obtained images show the particle dynamics increase by the increasing 12 14152 I. Go´sciniak, K. Gdawiec Fig. 50 Polynomiographs of f4 and the Khan–Cho–Abbas iteration for α = 0.5 and varying β, ω1, η1, ω2, η2, ω3, η3 Fig. 48 Polynomiographs of f2 and the Khan–Cho–Abbas iteration for α = 0.5, β = 0.8, and varying ω1, η1, ω2, η2, ω3, η3 Fig. 48 Polynomiographs of f2 and the Khan–Cho–Abbas iteration for α = 0.5, β = 0.8, and varying ω1, η1, ω2, η2, ω3, η3 Fig. 50 Polynomiographs of f4 and the Khan–Cho–Abbas iteration for α = 0.5 and varying β, ω1, η1, ω2, η2, ω3, η3 Fig. 49 Polynomiographs of f3 and the Khan–Cho–Abbas iteration for α = 0.5, β = 0.8 and varying ω1, η1, ω2, η2, ω3, η3 Fig. 5.4 The Agarwal and the Khan–Cho–Abbas iterations 51 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.3, ω1 = 0.3, η1 = 0.4, ω2 = 0.6, η2 = 0.7, η3 = 0.5 and varying ω3 Fig. 51 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.3, ω1 = 0.3, η1 = 0.4, ω2 = 0.6, η2 = 0.7, η3 = 0.5 and varying ω3 Fig. 49 Polynomiographs of f3 and the Khan–Cho–Abbas iteration for α = 0.5, β = 0.8 and varying ω1, η1, ω2, η2, ω3, η3 Fig. 52 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.8, β = 0.3, ω1 = 0.2, η1 = 0.5, ω2 = 0.6, η2 = 0.7, ω3 = 0.5 and varying η3 is responsible for the creation of a reference point, may have smaller effect on the appearance of the polynomiographs. Figures 46 and 47 present polynomiographs for the Khan– Cho–Abbas iteration and the f1 function. The excessive growth of the dynamics of the particles determined by the change in the parameters of the transformation T2 is clearly visible in these images. It conﬁrms only the observation that the excessive growth of dynamics of particles extends the time of image creation. Polynomiographs of the Khan–Cho–Abbas iteration for ﬁxed α, β and varying ω1, η1, ω2, η2, ω3, η3 for the f2 func- tion are presented in Fig. 48. The ω and η parameters change in the opposite directions, i.e. when ω increases/decreases, then η decreases/increases. It results in a characteristic change in dynamics presented in the polynomiographs by changing the colours. The shortest time of polynomiograph’s creation is obtained for Fig. 48b. Fig. 52 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.8, β = 0.3, ω1 = 0.2, η1 = 0.5, ω2 = 0.6, η2 = 0.7, ω3 = 0.5 and varying η3 for the f3 function, whereas the ones in Fig. 50 for the f4 function. Shortening the time of the polynomiograph, creation for Figs. 49b and 50b is obtained—these images visualize the reduction of the particle dynamic range. In all A similar change in the parameters is introduced in the examples of using the Khan–Cho–Abbas iteration that are shown in Figs. 49 and 50. The images in Fig. 49 are obtained 123 123 One more look on visualization of operation of a root-ﬁnding algorithm 14153 Fig. Fig. 55 Polynomiographs of the generalized Agarwal iteration for varying α, β, ω1, η1, ω2, η2, ω3, η3 5.4 The Agarwal and the Khan–Cho–Abbas iterations 56 Polynomiographs of f3 and the generalized Agarwal iteration for ω1 = 0.9 and varying α, β, η1, ω2, η2, ω3, η3 Fig. 53 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.5, η1 = 0.4, ω2 = 0.6, ω3 = 0.9 Fig. 53 Polynomiographs of f1 and the generalized Agarwal iteration for α = 0.5, β = 0.5, η1 = 0.4, ω2 = 0.6, ω3 = 0.9 Fig. 56 Polynomiographs of f3 and the generalized Agarwal iteration for ω1 = 0.9 and varying α, β, η1, ω2, η2, ω3, η3 Fig. 54 Polynomiographs of f1 and the Agarwal iteration for ω1 = 0.3, η1 = 0.4, ω2 = 0.6, η2 = 0.7, ω3 = 0.9, η3 = 0.5 inertia of the particle has a signiﬁcantly greater effect on the visual effects. This can be also observed in other presented images. Moreover, the images in Figs. 53 and 54 were obtained for the f1 function using the generalized Agarwal iteration and varying different parameters. The shortest times are obtained for images in Figs. 53b and 54a. These images illustrate the high particle dynamics, and they look like brush painting images. Fig. 54 Polynomiographs of f1 and the Agarwal iteration for ω1 = 0.3, η1 = 0.4, ω2 = 0.6, η2 = 0.7, ω3 = 0.9, η3 = 0.5 Polynomiographs of the generalized Agarwal iteration for the f2 function and varying all parameters are presented in Fig. 55. Shortening the time of polynomiographs creation occurs in subsequent images. (The shortest time is obtained for Fig. 55d.) The generalized Agarwal iteration gives the greatest possibility of the particle dynamics control. cases, the inﬂuence of the reference point on the movement of the particle is limited. The generalized Agarwal iteration introduces T3 transfor- mation with ω3 and η3 parameters. Figures 51 and 52 show the effect of changing in these parameters on the creation of polynomiographs for the f1 function. Due to the adaptation mechanism on which the particle acceleration depends, the The last two ﬁgures present polynomiographs generated by the generalized Agarwal iteration for the f3 (Fig. 56) and f4 (Fig. 57) functions. The generation algorithm is tuned just like in the previous example. We observe a limitation of the Fig. 55 Polynomiographs of the generalized Agarwal iteration for varying α, β, ω1, η1, ω2, η2, ω3, η3 12 123 14154 I. Go´sciniak, K. Gdawiec Fig. References Agarwal R, O’Regan D, Sahu D (2007) Iterative construction of ﬁxed points of nearly asymptotically nonexpansive mappings. J Nonlin- ear Convex Anal 8(1):61–79 dynamic range for the images having the shortest time of polynomiograph creation (Figs. 56b, 57b). dynamic range for the images having the shortest time of polynomiograph creation (Figs. 56b, 57b). Al-Hassan W, Fayek M, Shaheen S (2006) Psosa: an optimized particle swarm technique for solving the urban planning problem. In: 2006 international conference on computer engineering and systems, pp 401–405. https://doi.org/10.1109/ICCES.2006.320481 –405. https://doi.org/10.1109/ICCES.2006.320481 Arumugam M, Rao M (2008) On the improved performances of the particle swarm optimization algorithms with adaptive parameters, cross-over operators and root mean square (RMS) variants for computing optimal control of a class of hybrid systems. Appl Soft Comput 8(1):324–336. https://doi.org/10.1016/j.asoc.2007. 01.010 6 Conclusions The inertia weight and the acceleration constants are pre- sented in many optimization algorithms which require parameters tuning—it is exempliﬁed by the PSO algorithm. The discussion presented in the article allows to show their impact on the work of the proposed root-ﬁnding algorithm. Both too large and too small dynamics of the particles will have an adverse effect on the work of the algorithm. More- over, the presented experiments show that the dependence of particle’sdynamicsontheparametersusedintheroot-ﬁnding method (inertia weight and acceleration constant) and in the various iteration methods is a non-trivial function. Bansal J, Singh P, Saraswat M, Verma A, Jadon S, Abraham A (2011) Inertia weight strategies in particle swarm optimization. In: 2011 third world congress on nature and biologically inspired comput- ing, pp 633–640. https://doi.org/10.1109/NaBIC.2011.6089659 Broer H, Takens F (2011) Dynamical systems and chaos. Springer, New York ChenXD,Ma W(2015)Aplanarquadratic clippingmethodforcomput- ing a root of a polynomial in an interval. Comput Graph 46:89–98. https://doi.org/10.1016/j.cag.2014.09.014 Chen G, Huang X, Jia J, Min Z (2006) Natural exponential inertia weight strategy in particle swarm optimization. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indi- cate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitteduse,youwillneedtoobtainpermissiondirectlyfromthecopy- right holder. To view a copy of this licence, visit http://creativecomm ons.org/licenses/by/4.0/. 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https://openalex.org/W2943534188	https://bmchealthservres.biomedcentral.com/track/pdf/10.1186/s12913-019-3990-8	English	null	Delivery of integrated infectious disease control services under the new antenatal care guidelines: a service availability and readiness assessment of health facilities in Tanzania	BMC health services research	2,019	cc-by	6,427	Odjidja et al. BMC Health Services Research (2019) 19:153 https://doi.org/10.1186/s12913-019-3990-8 Odjidja et al. BMC Health Services Research (2019) 19:153 https://doi.org/10.1186/s12913-019-3990-8 Open Access Delivery of integrated infectious disease control services under the new antenatal care guidelines: a service availability and readiness assessment of health facilities in Tanzania Emmanuel Nene Odjidja1* , Ghislaine Gatasi1 and Predrag Duric2 © The Author(s). 2019, corrected publication 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. Abstract Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: emmaodjidja@gmail.com 1Village Health Works, Bururi, Burundi Full list of author information is available at the end of the article * Correspondence: emmaodjidja@gmail.com 1Village Health Works, Bururi, Burundi Full list of author information is available at the end of the article * Correspondence: emmaodjidja@gmail.com 1Village Health Works, Bururi, Burundi Full list of author information is available at the end of the article * Correspondence: emmaodjidja@gmail.co 1Village Health Works, Bururi, Burundi F ll li f h i f i i il bl Correspondence: emmaodjidja@gmail.com 1Village Health Works, Bururi, Burundi Full list of author information is available at Abstract Background: Tanzania remains among the countries with the highest burden of infectious diseases (notably HIV, Malaria and Tuberculosis) during pregnancy. In response, the country adopted World Health Organization’s (WHO) latest antenatal care (ANC) guidelines which recommend comprehensive services including diagnostic screening and treatment for pregnant women during antenatal. However, as Tanzania makes efforts to scale up these services under the existing health system resources, it is crucial to understand its capacity to deliver these services in an integrated fashion. Using the WHO’s service availability and readiness assessment(SARA) framework, this study assesses the capacity of the Tanzanian Health System to provide integrated Malaria, Tuberculosis and HIV services. Methods: Composite indicators of the five components of integration were constructed from primary datasets of the Tanzanian Service Provision Assessments (SPA) under the Demographic and Health Survey (DHS) programs. Chi-squared analysis was conducted to determine the associations of each of the defined components and background characteristics of facilities/health workers. A logistic regression model was further used to explore strength of relationships between availability of service readiness components and a pregnant women’s receipt of HIV, Malaria and TB services by reporting adjusted odds ratios. Results: Generally, capacity to integrate malaria services was significantly higher (72.3 95% CI 70.3–74.4 p = 0.02) compared to Tuberculosis (48.9 95% CI 48.4–50.7) and HIV (54.8 95% CI 53.1–56.9) services. Diagnostic capacity was generally higher than treatment commodities. Regarding the components of SARA integration, logistic regression found that the adjusted odds ratio of having all five components of integration and receiving integrated care was 1.9 (95% CI 0.8–2.7). Among these components, the strongest determinant (predictor) to pregnant women’s receipt of integrated care was having trained staff on site (AOR 2.6 95% CI 0.6–4.5). Conclusion: Toward a successful integration of these services under the new WHO guidelines in Tanzania, efforts should be channelled into strengthening infectious disease care especially HIV and TB. Channelling investments into training of health workers (the strongest determinant to integrated care) is likely to result in positive outcomes for the pregnant woman and unborn child. Keywords: Infectious diseases, Antenatal care, Service integration, Tanzania, Readiness assessment © The Author(s). 2019, corrected publication 2021. Background only 42.8% had attained the optimal number of visits (four or more visits). Additionally, median of first antenatal care visit was at 19.7 weeks. Tanzania is among countries with leading maternal and child mortality rates in the world. With an estimated under five (U5) mortality rate of 54 deaths per 1000, Tanzania ranks 51st in global U5 mortality rank [1]. Not- withstanding, it has made significant progress in combat- ing neonatal mortality. Between 2000 and 2012, Tanzania experienced a consistent annual decrease rate of 4.3% in neonatal mortality [1]. Afnan-Holmes et al. [2] forecast that if this trend should continue, Tanzania is likely to achieve child health target by 2030. In spite of the lingering health systems challenges which has resulted in lower FANC attendance [11], the Tanzanian Ministry of Health recently agreed to the im- plementation of the new model of antenatal care. This model requires pregnant women to receive a minimum of eight focused antenatal care visits in lieu of four inte- grated visits [12]. Ahead of national scale implementation of this new FANC model, we aim to understand the capacity of the health system to deliver an integrated infectious disease (HIV, Malaria and TB) control services in response to the increasing impact of infectious diseases among pregnant women. This builds on a previous study which study which employed the World Health Organisation’s service availability and readiness assessment tool and defined in- tegration based on five indicators in the context of Kenya, Malawi and Tanzania [13]. A value addition of this study is an assessment whether availability of integrated care re- sults in receipt of integrated service from health facilities under the latest ANC recommendations in Tanzania. Regarding maternal health, the United Nations Devel- opment Program’s (UNDP) report on the millennium development goals [3] indicates that Tanzania failed to achieve Millennium Development Goals (MDG) target 5.2 as maternal mortality rate stood at 133 deaths per 100,000 live births at 2015. Progress has fallen below tar- gets as annual reduction rate was only 3.4% between 1990 and 2013 [2]. While still births and pregnancy haemorrhage contribute majorly to neonatal and maternal deaths respectively, the impact of infectious diseases especially Human Immuno- deficiency Virus (HIV), Malaria and Tuberculosis (TB) during and post pregnancy has been noted by several au- thors [4–6]. Dreyfuss et al. © The Author(s). 2019, corrected publication 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. Odjidja et al. BMC Health Services Research (2019) 19:153 Page 2 of 7 Study design The original study was a cross-sectional design conducted among 1200 sample of health facilities, 6866 health workers and 4007 pregnant women across Tanzania. However, for the purposes of this study, variables were constructed from the primary data based on the WHO’s service availability and service readiness assessment (SARA) tool [14] and using indicators of integration as offered in a previous study of Mallick et al. [13]. Background [6] in a cohort study of 822 HIV positive women found that disease progression to Ac- quired Immunodeficiency Syndrome (AIDS) and low birth weight were significant outcomes experienced in Tanzania. Similar to this finding, a systematic review by Mullick et al. [7] found that HIV infection during pregnancy in South Eastern Tanzania was significantly associated to chorioam- nionitis and low birth weight. As confirmed by previous studies, the prevalence of HIV/TB comorbidity during pregnancy in Tanzania is overwhelming. Mtei et al. [8] in a trial among 94 HIV – positive women found that 71% had been comorbid with TB. Malaria during pregnancy has significant maternal and neonatal outcomes within the Tanzanian context. A study in Northern Tanzania [4] found that not only does malaria increases the risk of an- aemia and low birth weight but also, significantly increased the risk of premature delivery (OR 3.2 95% CI, 1.5–7.0). Data sources The original service provision assessment was approved by the National institute for medical research, Tanzania and the macro institutional board of Inner City Fund (ICF) International. According to the primary report of the survey [16], all participants signed a consent form. All participants were fully aware of the implications of engaging in the study including a possibility to have a secondary analysis like this study. The datasets are confi- dential and do not in any way expose study participants in a manner that affects them. For this particular study, ethical approval was granted by the Institute of Global Health and Development at the Queen Margaret Univer- sity. For the purposes of protecting identities of the study participants and/or specific facility, all analysis were conducted from district to national level only. The main source of data for this study was the service provision assessment (SPA) conducted at facilities in both the mainland Tanzania and Zanzibar (the island) in 2015/ 2016 [16]. The data were collected under the auspices of Na- tional Bureau of Statistics (NBS) and the office of the Chief government statistician in Zanzibar and funded by USAID under the demographic and health survey (DHS) program. The Tanzanian SPA was a survey of availability of ser- vices in facilities which spanned from those owned by government, private, parastatal to faith-based organisa- tion (also termed as mission facilities). Also, the survey originally collected information on facilities at all levels of care (i.e. from primary, secondary to tertiary facilities at district, regional and national level). The areas cov- ered during the survey included maternal, neonatal and child health, family planning, sexually transmitted dis- ease services, antenatal care, non-communicable diseases (i.e. cardiovascular diseases, diabetes 1 and chronic re- spiratory diseases). For each of these disease service areas, the SPA measured availability and functionality of Data analysis SPSS (version 20) and Microsoft Excel (2013) were used for all analysis in this study. Five main steps were in- volved in the analysis. Each of these are explained below. First, a background characteristics of all ANC facilities and availability of infectious diseases were computed. These were disaggregated by facility type and location. After constructing these individual integration components, a final composite indicator score of all these components was constructed to measure the readiness of facilities to integrate these disease services. This indicator was then disaggregated by disease type. Second, using the ‘countif’ excel formula, all composite indicators for each ID service area was calculated. The data was then processed in SPSS. Third, associations and emerging relationships between in- tegration and facility characteristics were explored using an appropriate test. A chi-squared test was used to determine the association between each of the five components of dis- ease integration and background characteristics of facilities. Outcome variable The main outcome variable for the study was receipt of integrated care of infectious disease services (HIV, TB and Malaria). This was based on the hypothesis that if facilities had all five components of integration present, then pregnant women should receive an integrated care during antenatal sessions. The construction of integrated care (a composite indicator) was based on three parame- ters; first pregnant women’s receipt of insecticide treated nets (ITNs) during antenatal care, second, observation of pregnant women’s consumption of antimalarial (IPTp-SP) during all antenatal sessions after 16 weeks, third was pregnant women’s receipt of HIV counselling and testing during first antenatal care. As guided by the ANC policy guidelines [15], no outcome for tuberculosis was included in this composite outcome variable. Fourth, all the five indicators of integration were put to- gether as a single indicator. For pragmatic reasons, this in- dicator was known as overall integration score for each infectious disease area. This indicator was computed for each of the three disease areas (i.e. malaria, TB and HIV). Finally, for the association between the components of integration and the outcome variable, a logistic regres- sion model was created to report the odds of association. The model was adjusted for all sociodemographic char- acteristics and facility/provider characteristics that could have a potential cofounding effect on the results. g Significance level for all tests were pegged at 95% con- fidence level (i.e. p < 0.05). Procedures for variable construction Secondary variables were constructed as a composite of the primary variables via adopting and adapting indica- tors of integration as defined by Mallick et al. [13]. This included the outcome variable under measure: pregnant woman’s receipt of integrated care; p y As a response to the devastating impact of Malaria, TB and HIV infection during pregnancy in this context, the Tanzanian Ministry of Health first adapted and imple- mented the focused antenatal care model in 2002 [9]. Under this policy, first antenatal care should commence before 16 weeks of pregnancy and should be integrated among other interventions including screening for infec- tious diseases [9]. Although focused antenatal care (FANC) is intended to mitigate utilisation barriers and improve health of women during pregnancy, challenges around optimal FANC utilisation (4+ visits) largely remain in Tanzania. According to the demographic and Health Sur- vey [10], of the 97.7% women that had first antenatal care, ‘Trained staff’ was constructed as composite of ANC health staff that had recently received training in an infectious disease area. For example, to be classified as technically trained in HIV, the health worker should be the first one that offers ANC service and secondly should be trained in HIV counselling and testing services. Another indicator, ‘availability of disease service’ was constructed as a composite indicator of having both the ID service and ANC at least three working days within the week [13]. ID service on the same Page 3 of 7 Page 3 of 7 Odjidja et al. BMC Health Services Research (2019) 19:153 the services which included a quality assessment of equip- ment, diagnostic tools and essential medicines. site with ANC was constructed having the disease service at the same place as the antenatal care. site with ANC was constructed having the disease service at the same place as the antenatal care. ‘Availability of diagnostics’ was classified as a composite indicator of all essential diagnostics of ID service available at the ANC facility [13]. Another indi- cator, ‘availability of medicines and commodities’ were also constructed as having all essential medicines for each ID service during all times of the ANC facility. Results Of all clinical officers and general medical doctors, 62.1 and 57.3% were found to have provided integrated care during antenatal sessions. Unsurprisingly, registered nurses, en- rolled nurses and nursing attendants had the least propor- tion of staff who provided integrated care. Chi-squared analysis showed that there were significant differences among cadres of staff. This result was expected as there were a 67.4% difference between the highest and lowest proportions. Results as shown in Table 3 found that malaria control obtained the highest score (72.3 95%CI 70.3–74.4) followed by HIV/AIDS and then Tuberculosis control. This insinuates that the current readiness is stronger in malaria followed with HIV/AIDS then to Tuberculosis control. It is unsurprising as components of malaria in- tegration were widely present in most ANC facilities. Similar to provision of integrated services by pro- viders, facilities that reported of having integrated ser- vices in HIV, Malaria and TB were generally low across all areas. Table 2 shows that on overall, 48.2% reported of having all services with facilities in urban areas reporting 64.0% in contrast to 41.4% in rural areas. As nurses and midwives (who reported the least of provid- ing integrated care) predominantly work in rural areas, it is unsurprising to find that rural facilities were signifi- cantly lower (p < 0.001) in having HIV, Malaria and TB services than urban facilities. Similarly, as most nurse as- sistants (who reported only 6.6% having offered inte- grated care) are found in dispensaries, it is least striking that dispensaries reported of just 8.8% having integrated services. Results Proportion of health workers and facilities that provide antenatal care services and HIV, malaria and TB services On average, as shown in Table 1, only 21.2% (895/4221) of the health staff provided care in both ANC and disease Proportion of health workers and facilities that provide antenatal care services and HIV, malaria and TB services On average, as shown in Table 1, only 21.2% (895/4221) of the health staff provided care in both ANC and disease Odjidja et al. BMC Health Services Research (2019) 19:153 Page 4 of 7 Table 1 Proportion of ANC staff that offer integrated service in HIV, Malaria and TB Proportion of health workers who provide ANC service together with both HIV, Malaria and TB services N % Sig level Value df Disaggregated by staff cadre < 0.024 5236.3 52 General Medical Doctor 51 57.3 Specialist Medical Doctor 10 26.3 Assistant Medical Officer 145 74.0 Clinical Officer 239 62.1 Assistant Clinical Officer 40 43.0 Registered Nurses 148 13.1 Enrolled Nurse 203 13.5 Nurse Assistant/Attendant 51 6.6 Other cadres 8 27.6 Total 895 21.1 (895/4221) Table 2 Proportion of ANC health facilities that offered integrated services in Malaria, HIV and Tuberculosis Proportion of ANC facilities that have HIV, Malaria and TB services N % Sig level Value df Disaggregated by managing Authority 0.011 471.1 15 Government/Public 351 46.6 Private-for-profit 20 25.0 Mission/Faith-based facilities 115 62.2 Parastatal facilities 11 91.7 Disaggregated by Facility location < 0.001 153.1 5 Rural 298 41.4 Urban 199 64.0 Disaggregated by facility type 0.031 794.5 35 National Referral Hospital 5 71.4 Regional Hospital 16 94.1 District Hospital 73 98.6 District designated hospitals 20 100 Other Hospital (Private) 96 83.5 Health Centre 250 69.6 Dispensary 37 8.8 Total 497 48.2 (497/1031) areas. Assistant medical officers topped the proportion of health staff who provided integrated care with 74.0%. Of all clinical officers and general medical doctors, 62.1 and 57.3% were found to have provided integrated care during antenatal sessions. Unsurprisingly, registered nurses, en- rolled nurses and nursing attendants had the least propor- tion of staff who provided integrated care. Chi-squared analysis showed that there were significant differences among cadres of staff. This result was expected as there were a 67.4% difference between the highest and lowest proportions. areas. Assistant medical officers topped the proportion of health staff who provided integrated care with 74.0%. Association between integration capacity and receipt of integrated care during antenatal care The main outcome variable of study was receipt of inte- grated service (defined as receipt of comprehensive HIV, TB and Malaria services during antenatal care) and the odds with which a pregnant woman would receive inte- grated services should all the components of these dis- eases be present in a health facility. Results from a logistic regression model as displayed in Table 4 found that if all components for service Table 3 Overall integration capacity of health facilities in HIV, Malaria and TB Overall disease area integration capacity Standardised score (computed on a scale of 100) 95% CI Tuberculosis control integration score 48.9 48.4–50.7 HIV/AIDS control integration score 54.8 53.1–56.9 Malaria control integration score 72.3 70.3–74.4 Table 3 Overall integration capacity of health facilities in HIV, Malaria and TB Overall integration capacity for each disease area Overall integration capacity for each disease area To measure integration of all disease areas based on the SARA tool, a standardised score of all compo- nents on a scale of 100 was computed to determine the overall readiness integration capacity of facilities in the three disease areas. Odjidja et al. BMC Health Services Research (2019) 19:153 Page 5 of 7 Table 4 Logistic regression of integration components and receipt of integrated services Component of integrated service AOR 95% CI Sig level Availability of all integration components (for HIV, TB and Malaria) 1.9 0.8–2.7 0.011 Availability of diagnostics for all disease areas 1.4 1.1–3.2 0.147 Availability of medicines and treatment commodities 1.1 0.9–2.3 < 0.001 Availability of trained staff in all disease areas 2.6 0.6–4.5 0.002 Disease service and antenatal care situated at the same site 1.0 – – Disease service available at least three times a week during antenatal care 1.3 0.9–2.4 0.033 a health facility significantly predicted receipt of care during pregnancy. The conceptualisation of receipt of integrated care during antenatal care in this study was based on five set parameters. The adjusted odds of having these parame- ters and receipt of integrated care were determined as having a positive relationship. Based on these findings, this study supports Foreit et al. [17]‘s assertion that the prime determinant of integrated care during antenatal care is having trained staff on site. It was found that the adjusted odds of having a trained staff on site and re- ceipt of integrated care by pregnant women were 2.6 times and up to 4.5 times during antenatal care. While it is inadequate to claim complete integration based on just availability of trained staff, it is an important and key step in ensuring that pregnant women receive all the requisite HIV, Malaria and TB services. Towards the im- plementation of the new WHO ANC guidelines, it is relevant for Tanzanian public health planners to con- sider training all health workers in all disease areas. integration were present for all diseases, a pregnant woman was 1.9 times (95% CI 0.8–2.7) likely to receive a comprehensive and integrated care. This therefore con- firms that there is a positive relationship between inte- gration capacity and receipt of integrated services. Overall integration capacity for each disease area Furthermore, when the model was disaggregated into the five components of integration, it found availability of a trained staff during antenatal care as the strongest predictor to receipt of integrated services (AOR 2.6 95% CI 0.6–4.5). Facilities with all recommended diagnostics were 1.4 times (AOR 1.4 95% CI 1.1–3.2) more likely to administer integrated care during ANC sessions. The ad- justed odds of administering integrated care in facilities that had essential medicines and treatment commodities was 1.1 (95% CI 0.9–2.3). Facilities with disease service available at least three times a week were 1.3 times likely (95% CI 0.9–2.4) to administer integrated care for preg- nant women during ANC sessions. The significance of training health staff in disease areas have been noted by several authors. Ouma et al. [18] noted that all components of malaria service delivery were higher among facilities that had health staff engaged in a donor funded training in malaria. Furthermore, the authors contend that training a health worker in malaria does not merely improve service delivery, but it also improves preg- nant women’s experience during antenatal care which even- tually improves coverage of future ANC services [18]. Welty et al. [19] also observed that HIV voluntary screening and counselling had increased significantly from 50.7 to 96.2% (p > 0.01) after training health workers on HIV care. Finally, the model found that having disease service at the same site was not enough to receive integrated ser- vices as the adjusted odds was one (no effect). Although organising on-the-job training programs has been recognised as a key determinant to successful ‘training’ and knowledge transfer, its sustainability would be assured when backed with quality improvement pro- cedures [20]. Batalden and Davidoff [20] identifies three quality improvement areas which could complement routine trainings of health workers. These are; 1. Gener- ating contextual evidence to guide training of health workers, 2. Routine capacity building and 3. Consistent measurement of performance improvement. Acknowledgements g The authors are thankful to Inner City Fund international and United States Agency for International Development (USAID) for granting access to the primary dataset of the Tanzania Service Provision Assessment. Funding l Not applicable. Study limitations The study encountered some limitations which were noted during the data analysis. Firstly, the nature of original ser- vice provision assessment survey being cross-sectional limits this study from claiming causality of receipt inte- grated care during antenatal session. This is due to the fact that construction of the components of integration were based on a combination of individually collected variables. In this case, when a pregnant woman receives several ID services from the same tertiary facility in different sections for example, the nature of analysis will classify it as vertical services. However, although this is likely to occur only among the five referral hospitals, a confidence interval (95%) range was calculated for all results. This was intended to reduce the impact of this limitation by com- puting the minimal and maximal boundaries upon which results could be estimated. Availability of data and materials The primary datasets of the Tanzania Service Provision Assessment is available on the Demographic Health and Survey website https:// dhsprogram.com/what-we-do/survey/survey-display-401.cfm. The data has been downloaded and available upon request from the first author on emmaodjidja@gmail.com after approval from Inner City Fund International. Competing interests Competing interests The authors declare that they have no competing interests. Discussion The aim of the study was to assess service readiness of Tanzanian health facilities to integrate HIV, Malaria and Tuberculosis under the new recommendations of the World Health Organisation. This also included the ex- tent to which the components of service integration re- sulted in actual service delivery. Based on the SARA framework, components of service integration was con- structed under five main domains. Findings from this study has demonstrated that having all components of service integration were significantly associated with re- ceipt of integrated service during antenatal care. Among the five components of integration, the study found that training an ANC health worker in all disease areas were the strongest determinant to receipt of an integrated ser- vice during ANC sessions. Consistent with this finding, Mallick et al. [13] found that having ANC providers, trained in an infectious disease area providing for care in Generating contextual evidence during trainings of health staff ensures that context-specific issues are incorporated into training programs. Context-tailored trainings responds to local needs and demands of health workers. In addition, Batalden and Davidoff [20] argues that incorporating rou- tine on-the-job capacity building strengthens quality im- provement and further ensures that policy and practice are consistent during all times of service delivery. Finally, creat- ing mechanisms to measure performance of health workers before and after training programs would ensure that indi- vidual health worker capacity is taken into consideration during planning and targeting of training programs. Page 6 of 7 Odjidja et al. BMC Health Services Research (2019) 19:153 Effective performance measurement also ensures that sub- sequent capacity building programs are tailored to respond to identified areas where majority of health workers may deem as a ‘weakness’. Allen and Brownstein [21] on an assessment of community health workers’ training programs in the United States reinforced the signifi- cance of incorporating effective performance measure- ment before and after training programs. They further recommend that adding qualitative exit interviews strengthens quality improvement even more. Finally, while complete integration of ID services at ANC may seem distant in Tanzania, health authorities should take the initial short term step of effectively co-locating these services according to the identified de- terminants. As lessons have shown, the nuanced effect- iveness of this strategy will gradually encourage other key health actors to pursue overall integration and devo- lution of health services. Abbreviations ANC: Antenatal Care; ANOVA: Analysis of Variance; AOR: Adjusted Odds Ratio; DHS: Demographic and Health Survey; FANC: Focused Antenatal Care; HIV: Human Immunodeficiency Virus; ICF: Inner City Fund; ID: Infectious Disease; MDGs: Millennium Development Goals; OR: Odds Ratio; SARA: Service Availability and Readiness Assessment; SPA: Service Provision Assessment; SPSS: Statistical Package for the Social Sciences; TB: Tuberculosis; U5: Under 5; UNDP: United Nations Development Program; USAID: United States Agency for International Development; WHO: World Health Organisation Consent for publication Not applicable. Authors’ contributions ENO l d d ENO conceptualised and designed the study, analysed the data and wrote the manuscript, GG reviewed the manuscript and made changes as applicable and PD supervised the entire study, reviewed the manuscript and incorporated changes as applicable. All authors read and approved the final version of this manuscript to submission to BMC health services research. This study has argued that integration of health ser- vices is an effective mechanism to increase coverage and to reduce the impact of infectious diseases during pregnancy. Using the SARA framework, it was found that the strongest determinant of receiving integrated care during antenatal session was having a trained staff in all disease areas on site. Unexpectedly, the model found that having antenatal care services and ID service on the same site had no association to re- ceiving integrated care. Ethics approval and consent to participate The original service provision assessment was approved by the National institute for medical research, Tanzania and the macro institutional board of Inner City Fund International. According to the primary report of the survey (2015), all participants signed a consent form. All participants were fully aware of the implications of engaging in the study including a possibility to have a secondary analysis like this study. The datasets are confidential and do not in any way expose study participants in a manner that affects them. For this particular study, ethical approval was granted by the Institute of Global Health and Development at the Queen Margaret University. For the purposes of protecting identities of the study participants and/or specific facility, all analysis were conducted from district to national level only. Ethics reference number granted to the study is Queen Margaret University/ Institute of Global Health and Development/15000647. Towards readiness to integrate infectious disease con- trol with antenatal care services under the new recom- mendations, strengthening capacity of health workers to administer this care will be crucial in higher coverage of services. Supportive quality improvement is crucial in this process and as such incorporating different tech- niques for continuous support to health workers ensures latest policy and practice are consistent. As illustrated by Batalden and Davidoff, driving this process with evi- dence even yields maximum benefits on the health sys- tem as a whole. Health workers are central to the process of supportive quality improvement and it is rele- vant to create a down-to-top heath needs assessment that continuously informs capacity building sessions. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Page 7 of 7 Page 7 of 7 Odjidja et al. BMC Health Services Research (2019) 19:153 Odjidja et al. BMC Health Services Research (2019) 19:153 18. Ouma PO, van Eijk AM, Hamel MJ, Sikuku ES, Odhiambo FO, Munguti KM, Ayisi JG, Crawford SB, Kager PA, Slutsker L. 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Tanzania human development indices and indicators: 2018 statistical update [viewed September 24]. Available from: http://hdr.undp. org/sites/all/themes/hdr_theme/country-notes/TZA.pdf. 4. Mahande AM, Mahande MJ. Prevalence of parasitic infections and associations with pregnancy complications and outcomes in northern Tanzania: a registry-based cross-sectional study. BMC Infect Dis. 2016; 16(1):78. 5. Antelman G, Msamanga GI, Spiegelman D, Urassa EJ, Narh R, Hunter DJ, Fawzi WW. Nutritional factors and infectious disease contribute to anemia among pregnant women with human immunodeficiency virus in Tanzania. J Nutr. 2000;130(8):1950–7. 6. Dreyfuss ML, Msamanga GI, Spiegelman D, Hunter DJ, Urassa EJ, Hertzmark E, Fawzi WW. Determinants of low birth weight among HIV-infected pregnant women in Tanzania. Am J Clin Nutr. 2001;74(6):814–26. 6. Dreyfuss ML, Msamanga GI, Spiegelman D, Hunter DJ, Urassa EJ, Hertzmark E, Fawzi WW. 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Dar es Salaam, Tanzania: TACAIDS, ZAC, NBS, OCGS, and ICF International; 2013. 11. Author details 1 1Village Health Works, Bururi, Burundi. 2grid.104846.fInstitute of Global Health and Development, Queen Margaret University, Edinburgh, UK. 1Village Health Works, Bururi, Burundi. 2grid.104846.fInstitute of Global Health and Development, Queen Margaret University, Edinburgh, UK. Published: 11 Mar 2019 Published: 11 Mar 2019 Published: 11 Mar 2019 19. Welty TK, Bulterys M, Welty ER, Tih PM, Ndikintum G, Nkuoh G, Nkfusai J, Kayita J, Nkengasong JN, Wilfert CM. Integrating prevention of mother-to-child HIV transmission into routine antenatal care: the key to program expansion in Cameroon. J Acquir Immune Defic Syndr. 2005; 40(4):486–93. 18. Ouma PO, van Eijk AM, Hamel MJ, Sikuku ES, Odhiambo FO, Munguti KM, Ayisi JG, Crawford SB, Kager PA, Slutsker L. Antenatal and delivery care in rural western Kenya: the effect of training health care workers to provide" focused antenatal care". Reprod Health. 2010;7(1):1. 21. White J, O'Hanlon B, Chee G, Malangalila E, Kimambo A, Coarasa J, Callahan S, Ron Levey I, McKeon K. Private health sector assessment in Tanzania: The World Bank; 2013. 20. Batalden PB, Davidoff F. What is “quality improvement” and how can it transform healthcare? Qual Saf Health Care. 2007;16(1):2. 18. Ouma PO, van Eijk AM, Hamel MJ, Sikuku ES, Odhiambo FO, Munguti KM, Ayisi JG, Crawford SB, Kager PA, Slutsker L. Antenatal and delivery care in rural western Kenya: the effect of training health care workers to provide" focused antenatal care". Reprod Health. 2010;7(1):1. 19. Welty TK, Bulterys M, Welty ER, Tih PM, Ndikintum G, Nkuoh G, Nkfusai J, Kayita J, Nkengasong JN, Wilfert CM. Integrating prevention of mother-to-child HIV transmission into routine antenatal care: the key to program expansion in Cameroon. J Acquir Immune Defic Syndr. 2005; 40(4):486–93. 20. Batalden PB, Davidoff F. What is “quality improvement” and how can it transform healthcare? Qual Saf Health Care. 2007;16(1):2. 21. White J, O'Hanlon B, Chee G, Malangalila E, Kimambo A, Coarasa J, Callahan S, Ron Levey I, McKeon K. Private health sector assessment in Tanzania: The World Bank; 2013. 20. Batalden PB, Davidoff F. What is “quality improvement” and how can it t f h lth ? Q l S f H lth C 2007 16(1) 2 References Gupta S, Yamada G, Mpembeni R, Frumence G, Callaghan-Koru J, Stevenson R, Brandes N, Baqui A. Factors associated with four or more antenatal care visits and its decline among pregnant women in Tanzania between 1999 and 2010. PLoS One. 2014;9(7):e101893. 11. Gupta S, Yamada G, Mpembeni R, Frumence G, Callaghan-Koru J, Stevenson R, Brandes N, Baqui A. Factors associated with four or more antenatal care visits and its decline among pregnant women in Tanzania between 1999 and 2010. PLoS One. 2014;9(7):e101893. 12. World Health Organisation (2017). New guidelines on antenatal care for a positive pregnancy experience. [online] World Health Organization. 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Ministry of Health and Social Welfare (MoHSW) [Tanzania Mainland], Ministry of Health (MoH) [Zanzibar], National Bureau of Statistics (NBS), Office of the Chief Government Statistician (OCGS), and ICF International 2015. Tanzania Service Provision Assessment Survey (TSPA) 2014–15. Dar es Salaam, Tanzania, and Rockville, Maryland, USA: MoHSW, MoH, NBS, OCGS, and ICF International. 17. Foreit KGF, Hardee K, Agarwal K. When does it make sense to consider integrating STI and HIV services with family planning services? Int Fam Plan Perspect. 2002:105–7. 17. Foreit KGF, Hardee K, Agarwal K. When does it make sense to consider integrating STI and HIV services with family planning services? Int Fam Plan Perspect. 2002:105–7.
https://openalex.org/W3209328251	https://www.nature.com/articles/s41467-022-32056-y.pdf	English	null	Small-world complex network generation on a digital quantum processor	Nature communications	2,022	cc-by	6,145	1National Renewable Energy Laboratory, Golden, CO 80401, USA. 2ColdQuanta Inc., Boulder, CO 80301, USA. 3Department of Physics, University of Texas, Austin, TX 78712, USA. 4Department of Physics, Colorado School of Mines, Golden, CO 80401, USA. 5NVIDIA Corporation, Boulder, CO 80302, USA. 6Google Quantum AI, Santa Barbara, CA 93117, USA. 7Quantum Engineering Program, Colorado School of Mines, Golden, CO 80401, USA. e-mail: eric.jones@coldquanta.com; ekapit@mines.edu; lcarr@mines.edu Article https://doi.org/10.1038/s41467-022-32056-y nature communications Small-world complex network generation on a digital quantum processor Eric B. Jones 1,2 , Logan E. Hillberry 3, Matthew T. Jones4,5, Mina Fasihi4, Pedram Roushan6, Zhang Jiang6, Alan Ho6, Charles Neill 6, Eric Ostby6, Peter Graf1, Eliot Kapit4,7 & Lincoln D. Carr 4,7 Eric B. Jones 1,2 , Logan E. Hillberry 3, Matthew T. Jones4,5, Mina Fasihi4, Pedram Roushan6, Zhang Jiang6, Alan Ho6, Charles Neill 6, Eric Ostby6, Peter Graf1, Eliot Kapit4,7 & Lincoln D. Carr 4,7 Received: 30 November 2021 Accepted: 13 July 2022 Check for updates Quantum cellular automata (QCA) evolve qubits in a quantum circuit depending only on the states of their neighborhoods and model how rich physical complexity can emerge from a simple set of underlying dynamical rules. The inability of classical computers to simulate large quantum systems hinders the elucidation of quantum cellular automata, but quantum compu- ters offer an ideal simulation platform. Here, we experimentally realize QCA on a digital quantum processor, simulating a one-dimensional Goldilocks rule on chains of up to 23 superconducting qubits. We calculate calibrated and error- mitigated population dynamics and complex network measures, which indi- cate the formation of small-world mutual information networks. These net- works decohere at ﬁxed circuit depth independent of system size, the largest of which corresponding to 1,056 two-qubit gates. Such computations may enable the employment of QCA in applications like the simulation of strongly- correlated matter or beyond-classical computational demonstrations. One of the most profound observations regarding the natural world is that, despite the simple set of physical laws that underpin it, the uni- verse displays a plethora of complex, emergent phenomena, encoun- tered in ﬁelds as diverse as biology, sociology, and physics1–3. Examples of classical systems where complexity arises as a result of many inter- acting degrees of freedom are ecosystems, the human brain, and power grids4. Certain classical cellular automata (CA) show how com- plexity can arise from simple rules without the controlling hand of a designer5. CA possess the ability to generate oscillatory, self-replicating structures and in some instances are themselves Turing complete6–10. It is known, however, that the laws constituting our best model of the universe are quantum mechanical rather than classical11. Therefore, in order to simulate the emergence of complexity more fundamen- tally, one ought to investigate computational models that are pre- dicated upon quantum mechanics. Article Article updated according to repeated blocks of neighborhood-local uni- tary operations along a discrete time axis. When every qubit’s state has been updated, a QCA cycle, t, is completed. After selecting 1D chains of high-quality qubits from the available hardware graph (Fig. 1a; see also Supplementary Note 2), the structure (Fig. 1b) of a 1D QCA experiment is comprised of an initialization step, followed by the application of some number of QCA cycle unitaries out to cycle t 2 f0,1, . . . , tmaxg, and the measurement of appropriate observables. Herein, we simulate a particular one-dimensional QCA on a Sycamore-class superconducting processor depicted schematically in Fig. 1a–d. Through the calculation of population dynamics and a complex network characterization of the two-body mutual informa- tion matrix, we establish that such QCA form small-world mutual information networks and thereby exhibit emergent physical com- plexity. Our results contribute to enabling the widespread use of near- term quantum processors as QCA simulators and offer a template for how to experimentally investigate QCA generally. We note that com- plex network analysis has already made a largely, though not wholly, theoretical impact on quantum information. One example arises in one-way quantum computing26 in which complex, network-structured graph states27 are irreversibly transformed using projective measure- ments. Another occurs in the context of the quantum internet where communication channels between geographically-distant quantum devices are either imposed28 by ﬁber optic networks or implied29 by satellite downlink capabilities. Notably, satellite-based quantum com- munication channels have recently been shown to support small-world connectivity30. Our work differs signiﬁcantly from these examples. Where one-way quantum computing has experimentally realized complex graph states by design via projective measurement, our work shows that QCA dynamically generate them in an emergent fashion. Where the quantum internet considers geographic networks, our networks of correlations emerge from unitary dynamics without any notion of physical distance except the locality of interactions. Finally, the complex networks occurring herein emerge on a generally-pro- grammable, gate-model quantum processor, that is, an experimental platform with real-world constraints, such as processor noise, not necessarily present in prior theoretical work and different in nature than in prior experimental work. The particular QCA that we simulate is the totalistic, three-site Goldilocks rule T6 with a uniform Hadamard activation unitary applied to each qubit and boundary conditions equivalent to ﬁxed ∣0is (see Supplementary Note 1)12. Article We note that the QCA notation T6 should not be confused with decoherence times, which we will denote ~T1 and ~T2 where applicable. Figure 1c shows how rule T6 is compiled down to quantum gates. A single, central bit ﬂip initialization is followed by one QCA cycle, and ﬁnally, measurement in the computational z-basis. The local update, represented by two non-Clifford CH gates (green box), does nothing if there are zero or two adjacent ∣1is and applies the Hadamard activator to the central qubit if there is exactly one adjacent ∣1i: this is the trade-off rule that gives rise to the Goldilocks nomenclature. Population dynamics and error mitigation The quantum processor on which we run our QCA simulations is a 53- qubit superconducting processor, Weber, which follows the design of the Sycamore architecture outlined in ref. 20 (see also Supple- mentary Note 2). Typical performance characteristics for Weber are: single-qubit gate error e1 ≈0.1%, two-qubit gate error e2 ≈1.4%, ∣0i-state readout error er0 ≈2%, ∣1i-state readout error er1 ≈7%, and population relaxation time ~T1 ≈15 μs31. Fig. 1d shows the decom- position of a single QCA cycle (red box) to the native ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ iSWAP p y two- qubit and PhXZ(a, x, z) ≡ZzZaXxZ−a family of single-qubit gates. Strictly speaking, the native two-qubit gate is better modeled by ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ iSWAP p y × CPHASE ðφÞ, where the parasitic cphase is φ ≈π/2321. We Quantum cellular automata A one-dimensional (1D) quantum elementary cellular automaton may be deﬁned as a chain of L quantum bits (qubits) whose states are Fig. 1 \| One-dimensional quantum cellular automata circuits. a Schematic for embedding one-dimensional chains into a subset of a two-dimensional Sycamore- class quantum processor. Gray crosses represent transmon qubits and blue rec- tangles represent couplers. Purple, green, yellow, and red paths are hypothetical example embeddings. b Generic structure of a one-dimensional quantum cellular automata (QCA) circuit where time ﬂows to the right. An initialization step is applied to a chain of L qubits, typically to place them into a classical product state with some number of bit ﬂips (∣1is). A number of unitary QCA update cycles, t, are applied to all L qubits before a measurement is performed. c The speciﬁc structure of a Goldilocks QCA for one QCA cycle (red box), wherein the initial state is ∣0 . . . 010 . . . 0i, the local update unitary is a controlled Hadamard gate, and mea- surement is performed in the computational basis. d After moment alignment, spin-echo insertion, and compilation down to hardware-native gates a single QCA cycle (red box) results in 4 × (L −1) number of ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ iSWAP p y gates and 8 × L number of individually-parameterized PhXZ(a, x, z) ≡ZzZaXxZ−a gates. The number of single and two-qubit layers per QCA cycle does not change as a function of system size, only total gate volume does. Nature Communications\| (2022)13 4483 2 Fig. 1 \| One-dimensional quantum cellular automata circuits. a Schematic for embedding one-dimensional chains into a subset of a two-dimensional Sycamore- class quantum processor. Gray crosses represent transmon qubits and blue rec- tangles represent couplers. Purple, green, yellow, and red paths are hypothetical example embeddings. b Generic structure of a one-dimensional quantum cellular automata (QCA) circuit where time ﬂows to the right. An initialization step is applied to a chain of L qubits, typically to place them into a classical product state with some number of bit ﬂips (∣1is). A number of unitary QCA update cycles, t, are applied to all L qubits before a measurement is performed. c The speciﬁc structure Fig. 1 \| One-dimensional quantum cellular automata circuits. a Schematic for embedding one-dimensional chains into a subset of a two-dimensional Sycamore- class quantum processor. Gray crosses represent transmon qubits and blue rec- tangles represent couplers. Small-world complex network generation on a digital quantum processor Goldilocks quantum cellular automata (QCA)12, are a class of computational models that exhibit emergent complexity despite being constructed from repeated blocks of simple local unitary operators13. They involve trade-offs in the local One of the most profound observations regarding the natural world is that, despite the simple set of physical laws that underpin it, the uni- verse displays a plethora of complex, emergent phenomena, encoun- tered in ﬁelds as diverse as biology, sociology, and physics1–3. Examples of classical systems where complexity arises as a result of many inter- acting degrees of freedom are ecosystems, the human brain, and power grids4. Certain classical cellular automata (CA) show how com- plexity can arise from simple rules without the controlling hand of a designer5. CA possess the ability to generate oscillatory, self-replicating structures and in some instances are themselves Turing complete6–10. neighborhood such as are known to be sources of complexity in classical systems and essential to self-organized criticality14. Some Goldilocks QCA have been shown to generate mutual information networks that exhibit signatures of complexity, such as large network clustering, short average path length, and broad node-strength dis- tribution, typically only observed in classical, small-world networks like social or biological networks12. In addition, QCA have been pro- posed for other applications such as lattice discretization in the simulation of strongly-correlated matter, quantum ﬁeld, and gravita- tional theories15–17. However, the categorical limitation on the ability of classical computers to simulate the time evolution of large quantum systems is a bottleneck for the discovery and exploration of QCA more generally, hampering the theoretical illumination of the class of sys- tems as a whole18. Meanwhile, the last few years have seen the creation of sizeable digital quantum processors that are already demonstrating their value as tools of scientiﬁc discovery19–25. Due to their universality, such processors are ideal platforms on which to elucidate the physics and complexity characteristics of QCA. It is known, however, that the laws constituting our best model of the universe are quantum mechanical rather than classical11. Therefore, in order to simulate the emergence of complexity more fundamen- tally, one ought to investigate computational models that are pre- dicated upon quantum mechanics. Goldilocks quantum cellular automata (QCA)12, are a class of computational models that exhibit emergent complexity despite being constructed from repeated blocks of simple local unitary operators13. They involve trade-offs in the local Nature Communications\| (2022) 13:4483 Nature Communications\| (2022) 13:4483 1 https://doi.org/10.1038/s41467-022-32056-y Mutual information network analysis At each QCA cycle depth we measure the output of the circuit in the z-basis Nc = 100, 000 times, resulting in a set of L-bit strings f∣zig and associated probabilities {Pz ≈Nz/Nc}, where Nz is the number of times bit string ∣zi is observed. The local population on each site is calculated via hnii = ð1 ∑zPzð1ÞziÞ=2 and averaged over four 1D qubit chains. Figure 2a shows the numerical emulation of such a procedure initialized with a single, central bit ﬂip on 21 qubits out to 30 QCA cycles. The two large-scale blue diamonds indicate coherent dynamics. When repeated on the Weber processor, a combination of photon loss, gate error, and state preparation and measurement (SPAM) error leads to nearly total population decoherence by t ≈10 (Fig. 2b). We therefore post-select experimental data and discard any measurements whose eigenvalues of the Ising-like operator O = ∑L i = 0 ZiZi + 1 differ from the corresponding eigenvalue of the initial state. That is, O is a dynamical invariant of the T6 rule that keeps track of the number of domain walls in the system. Figure 2c shows that post-selection results in coherent population dynamics that persist beyond t ≈10, although different observables can degrade with noise on slightly different timescales (see Fig. 3). The cycle-stamped population vignettes shown in Fig. 2d support these observations more quantitatively, with error bars representing one standard deviation on the four different chains. After t ≈15, error bars on the post-selected data become more signiﬁcant and while some qualitative features of the emulated population dynamics appear to persist, such as a larger population towards the center of the chain, it is unclear from Fig. 2 alone as to what the underlying nature of these qualitative similarities is. Moreover, our complex network analysis of the behavior of rule T6 relies on the cal- culation of two-body observables beyond the one-body observables Following the complex-network approach in neuroscience wherein functional connectivity of the brain is characterized between spatially non-adjacent regions32, we calculate the classical, Shannon mutual information between all pairs of qubits in each 1D Goldilocks QCA chain Iij ∑ 1 zi = 0 ∑ 1 zj = 0 pðzi,zjÞlog2 pðzi,zjÞ pðziÞpðzjÞ ð1Þ ð1Þ and regard it as an adjacency matrix of correlations that deﬁnes the QCA network at each cycle. Quantum cellular automata Purple, green, yellow, and red paths are hypothetical example embeddings. b Generic structure of a one-dimensional quantum cellular automata (QCA) circuit where time ﬂows to the right. An initialization step is applied to a chain of L qubits, typically to place them into a classical product state with some number of bit ﬂips (∣1is). A number of unitary QCA update cycles, t, are applied to all L qubits before a measurement is performed. c The speciﬁc structure Fig. 1 \| One-dimensional quantum cellular automata circuits. a Schematic for of a Goldilocks QCA for one QCA cycle (red box), wherein the initial state is ∣0 . . . 010 . . . 0i, the local update unitary is a controlled Hadamard gate, and mea- surement is performed in the computational basis. d After moment alignment, spin-echo insertion, and compilation down to hardware-native gates a single QCA cycle (red box) results in 4 × (L −1) number of ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ iSWAP p y gates and 8 × L number of individually-parameterized PhXZ(a, x, z) ≡ZzZaXxZ−a gates. The number of single and two-qubit layers per QCA cycle does not change as a function of system size, only total gate volume does. Nature Communications\| (2022) 13:4483 2 2 Article https://doi.org/10.1038/s41467-022-32056-y a d Raw Emulated Post-selected b c Fig. 2 \| Population dynamics and post-selection. A noise-free, numerically- emulated Goldilocks QCA out to 30 cycles, initialized with a single ∣1i in the middle of the 21-qubit chain. Coherent local population, 〈ni〉, dynamics that resist equili- bration can be observed in the form of the two blue diamond shapes. b Raw population dynamics for the same QCA circuit averaged over four 21-qubit chains embedded into the 53-qubit Weber processor. c The same experimental data as b but with post-selection applied based on domain wall conservation. d Time- stamped population vignettes show the same dynamics quantitatively for emu- lated (blue- lines for visual clarity), raw (red), and post-selected (green) data. Error bars represent one standard deviation from the mean over four different chains. Post-selected c a d Raw Emulated Post-selected b c a Emulated b d d Fig. 2 \| Population dynamics and post-selection. A noise-free, numerically- emulated Goldilocks QCA out to 30 cycles, initialized with a single ∣1i in the middle of the 21-qubit chain. Coherent local population, 〈ni〉, dynamics that resist equili- bration can be observed in the form of the two blue diamond shapes. Quantum cellular automata b Raw population dynamics for the same QCA circuit averaged over four 21-qubit chains embedded into the 53-qubit Weber processor. c The same experimental data as b but with post-selection applied based on domain wall conservation. d Time- stamped population vignettes show the same dynamics quantitatively for emu- lated (blue- lines for visual clarity), raw (red), and post-selected (green) data. Error bars represent one standard deviation from the mean over four different chains. depicted in Fig. 2. As such, we turn to a calculation of Shannon mutual information both to more deeply understand the long-time population dynamics of our QCA and to establish their complex network behavior. apply a suite of low-overhead circuit optimization, calibration, and error mitigation techniques to optimize circuit performance includ- ing moment alignment, spin-echo insertion, Floquet calibration21,24, parasitic cphase compensation, and most importantly, post- selection (see Supplementary Note 4). https://doi.org/10.1038/s41467-022-32056-y In addition, the measures have been used along with earth- quake time series-derived mutual information to model seismicity36 and with mutual information in wireless networks to explain routing efﬁciency37. Finally, complex network measures calculated on quan- tum mutual information networks in quantum Ising and Bose–Hubbard models are able to detect quantum phase transition critical points38. Hence, the use of clustering, path length, and node strength distribution in conjunction with mutual information is a well- established, quantitative procedure with predictive power. We employ this procedure in order to understand the structure of correlations in our QCA circuits and to observe the emergence of physical complexity in the presence of quantum processor noise. Figure 4a shows the coherence window, cycle-and-chain- averaged emulated (blue), raw (red), and post-selected (green) clustering coefﬁcient for L = 5–23 qubits. After the ﬁnite-size effects encountered for L ≤11, it is clear that while the raw clustering trends towards zero—that of an incoherent uniformly random state network —both the emulated and post-selected clustering stabilize towards C ≈0:3 and trend towards larger values as a function of system size, indicating substantial network transitivity beyond post-selected randomness (black dashed curve). Figure 4b shows the coherence window, cycle-and-chain-averaged weighted shortest path length, ℓ, as a function of system size, which gauges global network traversa- bility (see Supplementary Notes 6 and 7). The raw data path length (red) in Fig. 4b is large and increases as a function of system size. The post-selected (green) path length tracks the emulated (blue) path length closely, trends downward, and is always one to two orders of magnitude smaller than the raw path length. Interestingly, post- selected path length tracks the path length of post-selected ran- domness (black dashed line) nearly as well as it does emulated path length. Taken together, however, Fig. 4a, b signal the existence of small-world mutual information networks generated in the coher- ence window of the Goldilocks QCA beyond what can be obtained by post-selecting incoherent uniform randomness. The end of the coherence window (t = 12) for the largest system size simulated (L = 23) corresponds to 1056 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ iSWAP p y gates. Through decoherence, the state of a quantum processor approaches an incoherent uniformly random state with all ampli- tudes equal to 2−L/2 and for which Iij = 0 for all L ≥2. https://doi.org/10.1038/s41467-022-32056-y https://doi.org/10.1038/s41467-022-32056-y Fig. 3 \| QCA mutual information network clustering. Clustering coefﬁcient as a function of QCA cycle for three intermediate system sizes simulated on Weber, L = 15 (a), 17 (b), and 19 (c). Blue curves (lines for visual clarity) are calculated from numerical emulation, while red (green) data points are from Weber data without (with) post-selection. Error bars are one standard deviation in C over four different chains. Dashed (dotted) black lines are the clustering of the incoherent uniform random state with (without) post-selection. Fig. 3 \| QCA mutual information network clustering. Clustering coefﬁcient as a function of QCA cycle for three intermediate system sizes simulated on Weber, L = 15 (a), 17 (b), and 19 (c). Blue curves (lines for visual clarity) are calculated from numerical emulation, while red (green) data points are from Weber data without (with) post-selection. Error bars are one standard deviation in C over four different chains. Dashed (dotted) black lines are the clustering of the incoherent uniform random state with (without) post-selection. we simulated here, L = 15, 17, and 19, this proves true for all other system sizes simulated as well. Next, we note that without post-selec- tion, the clustering C calculated from raw data from Weber (red points) rises brieﬂy but then quickly decays toward zero, the incoherent uni- formly random limit (black dotted curve), at t ≈12 for all three system sizes. In contrast, the green curves in Fig. 3 show that with post- selection the experimental clustering tracks the emulated clustering closely until t ≈6 and remains larger than post-selected uniform ran- domness (black dashed curve) until t ≈12, independent of system size. There is therefore a window between t ≈4 and 12 over which we can analyze the formation of a non-random complex network in the QCA for all system sizes simulated. We provide a more detailed description of our cycle windowing process in Supplementary Note 7. distribution have seen widespread use in analyzing the structure of mutual information networks and in drawing conclusions regarding the physical complexity of the underlying system in both classical and quantum systems. For example, applying these measures to mutual information networks derived from electroencephalographic or fMRI data has been used to elucidate structure-function correlations in the brain32,35. Mutual information network analysis We choose to use classical, rather than a measure of quantum, mutual information because its calculation only requires measurements in the computational z-basis, which we have shown are amenable to post-selection. Moreover, we show in Supplementary Note 5 that the Shannon (classical) mutual information acts as a reliable proxy for von Neumann (quantum) mutual information for the T6 QCA. Complex networks are ones that are neither purely regular, such as a lattice or complete graph, nor entirely random33. The classic demonstration that a network has complex, small-world character involves showing persistently large clustering and simultaneously short path length34, with a power-law node strength distribution resulting in highly-connected nodes. By analogy with transportation networks, these features describe networks that are easily traversed both locally (clustering) and globally (path length), and exhibit hubs (broad node strength distribution). While transportation networks provide an intuitive interpretation of these complex network mea- sures, we emphasize that clustering, path length, and node strength Nature Communications\| (2022) 13:4483 3 Article Nature Communications\| (2022) 13:4483 https://doi.org/10.1038/s41467-022-32056-y Hence, the inco- herent uniformly random state is neither locally nor globally traversable and is thus not a typical random network. It is also useful to consider the effect of subjecting the incoherent uniformly random state to the same post-selection procedure as our experimental data. This can be done in three steps: (i) form an incoherent uniformly random state with all 2L amplitudes equal to 2−L/2, (ii) set any basis state amplitudes to zero if the basis state has an eigenvalue under O that differs from that of the QCA’s initial condition, and (iii) renor- malize the remaining amplitudes so that the state vector has unit norm. Upon post-selection, this decohered state is no longer uniform and the corresponding mutual information network is both non-zero and non-random, although to a much lesser extent than the states generated by Goldilocks QCA. The complexity of networks generated by Goldilocks QCA is established by computing network measures for emulated, raw processor, and post-selected processor states and then comparing each of these to post-selected incoherent uniform random states. Figure 4c further indicates the formation of small-world mutual information networks, showing that the size-normalized emulated (blue) and post-selected (green) node strength distributions (see Supplementary Note 6), P[gi/(L −1)], are relatively ﬂat between 1 × 10−2 and 2 × 10−1 compared with those of the post-selected random node strengths (black dashes), which peak between ~2.5 × 10−2 and 1.5 × 10−1, and raw (red) node strengths, which are heavily biased towards much smaller values, indicating a deﬁcit in network connectivity. Finally, Fig. 4d–g visually depicts how the mutual information for L = 23 at t = 9 differs for the raw QCA data, which approaches the network structure Clustering measures local network transitivity and is deﬁned as the ratio of the weighted number of closed triangles in the network to the weighted total number of length-2 paths in the network (i.e., the number of potentially closed triangles—see Supplementary Note 6). The ﬁrst relevant signature of network complexity is intermediate to large clustering values that do not decay with system size, in contrast to random networks. The emulated clustering (blue curves) of the QCA exhibits this signature and actually increases slightly as a function of system size, L (see Fig. 3). While we plot three of the larger system sizes Nature Communications\| (2022) 13:4483 4 4 Article https://doi.org/10.1038/s41467-022-32056-y Fig. 4 \| Small-world mutual information network behavior. Towards beyond-classical QCA In addition to their intrinsic scientiﬁc value as quantum models for emergent complexity, QCA also present intriguing prospects for establishing new inroads into the beyond-classical era. In the instance of Goldilocks rule T6, identiﬁcation of a dynamical invariant makes simulation less fragile to noise than a fully chaotic random quantum circuit (RQC). However, it also implies efﬁcient—that is, polynomial— classical emulation via direct Schrödinger evolution since it reduces the bounded size of the protected T6 Hilbert space to scale as ~0.63 L1.91 for a ﬁxed, single initial bit ﬂip at the center of the chain (see Supple- mentary Note 4). Generally, ﬁxing the number of initial ∣1is while increasing system size leads to polynomial space and time complexity. However, one can recover exponential scaling in the protected Hilbert space by simulating increasingly large chains while ﬁxing the density (rather than number) of initial ∣1is well separated by ∣0is. For a ﬁxed density, ρ∣1is, the protected Hilbert space bound scales as the binomial coefﬁcient, ~ L ρ∣1isL , which asymptotically scales exponentially in L. For context, the long-time, cycle-averaged bond entropy of rule T6 was shown to scale between a 1D area and volume law12. Although simula- tion of Goldilocks rules has shown the failure of direct matrix-product- state approaches39, given this intermediate scaling it is an open pro- blem as to whether efﬁcient classical simulatability may be achieved using a modiﬁed tensor network approach in the absence of a dyna- mical invariant12,40. Moreover, efﬁcient simulatability of area law- scaling states in two-dimensions (2D) or higher using tensor network approaches, while promising, is even less assured than in 1D. Hence, 2D QCA that exhibit area-law scaling or worse may be good candidates for beyond-classical demonstrations. Data availability The data supporting this work are available at the public Dryad repo- sitory (https://doi.org/10.5061/dryad.fbg79cnxd). The data supporting this work are available at the public Dryad repo- sitory (https://doi.org/10.5061/dryad.fbg79cnxd). https://doi.org/10.1038/s41467-022-32056-y a Coherence win- dow, cycle-and-chain-averaged clustering, C, as a function of system size, L. b Coherence window, cycle-and-chain-averaged path length, ℓ, as a function of system size, L. c Normalized node strength distribution, P[gi/(L −1)], amalgamated over all system sizes. d–g Heatmap and force-directed41 complex network visualization of mutual information network for L = 23 at t = 9 (blue: emulated; red: raw data from Weber; green: post-selected; black: post-selected incoherent uni- form randomness). Where present, error bars represent one standard deviation from the mean averaged over four different chains and cycles within the coherence window. visualization of mutual information network for L = 23 at t = 9 (blue: emulated; red: raw data from Weber; green: post-selected; black: post-selected incoherent uni- form randomness). Where present, error bars represent one standard deviation from the mean averaged over four different chains and cycles within the coherence window. Fig. 4 \| Small-world mutual information network behavior. a Coherence win- dow, cycle-and-chain-averaged clustering, C, as a function of system size, L. b Coherence window, cycle-and-chain-averaged path length, ℓ, as a function of system size, L. c Normalized node strength distribution, P[gi/(L −1)], amalgamated over all system sizes. d–g Heatmap and force-directed41 complex network of incoherent uniform randomness, and the emulated and post- selected QCA networks, which both display lattice girder-like small- world structure that resemble one another more closely than they do post-selected randomness. signatures, (ii) efﬁciently compile to low-depth sequences of hardware- native gates, and (iii) are amenable to post-selection through identiﬁ- cation of one or more dynamical invariants (For a more complete discussion of the quantum-simulational complexity of the T6 rule including post-selection overhead, please see Supplementary Note 4). 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Phys. 15, 148–153 (2019). Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-022-32056-y. 28. Brito, S., Canabarro, A., Chaves, R. & Cavalcanti, D. Statistical properties of the quantum internet. Phys. Rev. Lett. 124, 210501 (2020). Additional information 29. Bonato, C., Tomaello, A., Da Deppo, V., Naletto, G. & Villoresi, P. Feasibility of satellite quantum key distribution. N. J. Phys. 11, 045017 (2009). Correspondence and requests for materials should be addressed to Eric B. Jones, Eliot Kapit or Lincoln D. Carr. 30. Brito, S., Canabarro, A., Cavalcanti, D. & Chaves, R. Satellite-based photonic quantum networks are small-world. PRX Quantum 2, 010304 (2021). Peer review information Nature Communications thanks the anon- ymous reviewer(s) for their contribution to the peer review of this work. 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Nature Communications\| (2022) 13:4483 6 https://doi.org/10.1038/s41467-022-32056-y Article Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 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https://openalex.org/W4298073142	https://zenodo.org/records/4969862/files/%D8%AC%D9%85%D8%A7%D8%B9%D8%A7%D8%AA_%D9%86%D9%82%D8%A7%D8%B4_%D8%A7%D9%84%D9%81%D9%87%D8%B1%D8%B3%D8%A9_%D8%B9%D9%84%D9%89_%D8%B4%D8%A8%D9%83%D8%A9_%D8%A7%D9%84%D8%A5%D9%86%D8%AA%D8%B1%D9%86%D8%AA.pdf	Arabic	null	جماعات نقاش الفهرسة على شبكة الإنترنت : دراسة تحليلية تقييمية	Zenodo (CERN European Organization for Nuclear Research)	2,008	cc-by	199	جماعات نقاش الفهرسة على شبكة اإلنترنت : دراسة تحليلية تقييمية جماعات نقاش الفهرسة على شبكة اإلنترنت : دراسة تحليلية تقييمية :المستخلص يتناول هذا الفصل خدمات ،اإلنترنت مع عرض لخدمتين من خدماتها وهما خدمة البريد االلكترونى وخدمة جماعات النقاش الرتباطهما فى العمل ببعضهما البعض، ثم يعرض بشكل مفصل لخدمة جماعات النقاش على شبكة اإلنترنت، من خالل عرض المفهوم والتعريف بها ،ونبذة تاريخية عنها ،ومميزاتها وأهميتها لالتصال العلمى، وفئاتها ،وأشكالها المختلفة منذ بدايتها وحتى اآلن، وأيضًا يعرض ألهمية أرشيف جماعات النقاش كخدمة موازية لها ،واختتم الفصل بعرض ألنواع جماعات النقاش ،العامة منها والمتخصصة، مع التركيز على تخصص المكتبات .والمعلومات بفروع علومه المختلفة تقدم شبكة اإلنترنت مجموعة متنوعة من الخدمات للمستفيدين ،عن : طريق محورين أساسين ،هما أوال : سهولة االتصاالت : من خالل ربط شبكـة اإلنترنـت لعدد غير محدد من الناس عبر أنحاء العالم المختلفة ،حتى يتم تكوين مجتمع عالمى من المستفيدين، يتواصلون ويتخاطبون و يتبادلون اآلراء، سواء من فرد إلى فرد أو كمجموعـات نقاش على الخط المباشـرonline وعـبر البريد اإللكترونى، وكلها أشكال من االتصاالت القائمة بين الناس عبر اإلنترنت ثانيا: توافر المعلومات: من خالل خدمة االتصال عن بعد الذى توفره خدمات شبكة اإلنترنت لمستخدميها ،عن طريـق الوصول إلى اآلالف من ،المواقع ومصادر المعلومات عـلى شبكة اإلنترنت، وسهولة البحـث واستـقاء المعلومات.
https://openalex.org/W4200590865	https://environmentalsmoke.com.br/index.php/EnvSmoke/article/download/163/143	Spanish; Castilian	null	FEBRE MACULOSA, UM CUIDADO QUE DEVEMOS REFORÇAR	Environmental Smoke	2,021	cc-by	1,170	FEBRE MACULOSA, UM CUIDADO QUE DEVEMOS REFORÇAR SPOTTED FEVER A CARE THAT WE MUST REINFORCE José Andreey Almeida Teles1* ; Maria Luísa Souto Porto2 Resumo Farmaceutica recomenda-se procurar imediatamente o serviço médico, evitando a auto medicação. Palavras-chave: Capivara; Artrópodes; Equinos, Caninos, Marsupiais, Zoonose. Palavras-chave: Capivara; Artrópodes; Equinos, Caninos, Marsupiais, Zoonose. Palavras-chave: Capivara; Artrópodes; Equinos, Caninos, Marsupiais, Zoonose. Resumo A febre maculosa é uma doença zoonótica, infecciosa, que evolui entre 2 e 14 dias, causada por uma Rickettsia, adquirida pela picada do carrapato infectado do gênero Amblyomma. Não há relatos de transmissão pessoa a pessoa. Estes carrapatos estão dispersos no território brasileiro, sendo conhecidos como carrapato estrela e carrapato de cavalo. Equídeos, capivaras, cães e gambás, são transportadores de carrapatos potencialmente infectados, constituindo-se como reservatórios ou amplificadores de Rickettsia. Os sinais mais comuns, em humanos infectados, são febre súbita, dores musculares, cefaleia, exantema que surge entre o 2º e o 5º dias de infecção e, hemorragia. Eventualmente, sinais como fotofobia, vômitos, ânsia, dores abdominais e ainda aqueles relacionados ao sistema nervoso e urinário, com acometimento renal, podem se manifestar. A suspeita torna-se maior quando o indivíduo, portador de alguns desses sinais, tem histórico de picadas de carrapato ou exposição a área com potencial infestação por carrapatos (passeios em matas, rios, cachoeiras, trilhas, contato com animais, piqueniques, pescarias, etc.). A prevenção é mais eficiente que o tratamento, haja vista a inexistência de vacinas contra rickettsioses. A antibioticoterapia pós-exposição não é capaz de evitar a doença. Usar roupas claras, repelentes à base de DEET, vestimentas que cubram todo o corpo e a averiguação da presença de carrapatos após atividade em áreas de risco, removendo-os caso sejam encontrados, constituem-se como medidas eficazes de prevenção. Cães e cavalos e o ambiente onde vivem, devem ser submetidos ao controle de carrapatos, segundo orientações do médico veterinário. A remoção de carrapatos da pele, deve se dar pela torção até o desprendimento, evitando o esmagamento, para não ocorrer liberação de rickettsias, as quais podem penetrar no corpo a partir de microlesões. Em caso de suspeitas da doença, _________________ 1 Doutor em Ciência Veterinária. Mestre pelo Programa de Pós-graduação em Biociência Animal. Especialização em Doenças Infecto-contagiosas dos animais domésticos. Graduado em Medicina Veterinária. * telesjaa@gmail.com 2 Doutoranda em Ecologia e Saúde Ambiental. Mestrado em Ciências da Saúde. Farmaceutica 1 Doutor em Ciência Veterinária. Mestre pelo Programa de Pós-graduação em Biociência Animal. Especialização em Doenças Infecto-contagiosas dos animais domésticos. Graduado em Medicina Veterinária. * telesjaa@gmail.com 1 Doutor em Ciência Veterinária. Mestre pelo Programa de Pós-graduação em Biociência Animal. Especialização em Doenças Infecto-contagiosas dos animais domésticos. Graduado em Medicina Veterinária. * telesjaa@gmail.com 2 Doutoranda em Ecologia e Saúde Ambiental. Mestrado em Ciências da Saúde. Farmaceutica 2 Doutoranda em Ecologia e Saúde Ambiental. Mestrado em Ciências da Saúde. e-ISSN: 2595-5527 10.32435/envsmoke.20214371-72 Volume 4, Número 3 2021 e-ISSN: 2595-5527 10.32435/envsmoke.20214371-72 Volume 4, Número 3 2021 Palabras-chave: Carpincho; Artrópodos; Caballos, Caninos, Marsupiales, Zoonosis. Abstract Spotted fever is a zoonotic, infectious disease that evolves between 2 and 14 days, caused by a Rickettsia, acquired by the bite of an infected tick of the genus Amblyomma. There are no reports of person-to-person transmission. These ticks are dispersed in Brazilian territory, being known as star tick and horse tick. Equidae, capybaras, dogs and opossums are transporters of potentially infected ticks, constituting reservoirs or amplifiers of Rickettsia. The most common signs in infected humans are sudden fever, muscle pain, headache, rash that appears between the 2nd and 5th days of infection, and hemorrhage. Occasionally, signs such as photophobia, vomiting, anxiety, abdominal pain and even those related to the nervous and urinary system, with renal involvement, may manifest. Suspicion becomes greater when the individual, with some of these signs, has a history of tick bites or exposure to an area with potential infestation by ticks (walks in woods, rivers, waterfalls, trails, contact with animals, picnics, fisheries, etc.). Prevention is more efficient than treatment, given the lack of vaccines against rickettsiosis. Post-exposure antibiotic therapy is not able to prevent the disease. Wearing light clothing, DEET- based repellants, clothing that covers the entire body and checking for the presence of ticks after activity in risk areas, removing them if found, are effective prevention measures. Dogs and horses, and the environment where they live, must be subjected to tick control, as instructed by the veterinarian. The removal of ticks from the skin must be done by twisting them until they detach, avoiding crushing, so as not to release rickettsiae, which can penetrate the body through microlesions. In case of suspicion of the disease, it is recommended to seek medical attention immediately, avoiding self-medication. Keywords: Capybara. Arthropods. equine. canine, marsupial. zoonosis. Teles, J. A. A.; Porto, M. L. S. 71 Recebido em: 28 Out. 2021 Aceito em: 27 Nov. 2021 Publicado em: 30 Dez. 2021 © Copyright 2021 Recebido em: 28 Out. 2021 Aceito em: 27 Nov. 2021 Publicado em: 30 Dez. 2021 Recebido em: 28 Out. 2021 Aceito em: 27 Nov. 2021 Publicado em: 30 Dez. 2021 © Copyright 2021 71 Teles, J. A. A.; Porto, M. L. S. OFERTA DE INFORMAÇÃO SOBRE COVID-19 NO BRASIL OFERTA DE INFORMAÇÃO SOBRE COVID-19 NO BRASIL Resumen La fiebre maculosa es una enfermedad zoonótica, infecciosa que evoluciona entre 2 y 14 días, causada por una Rickettsia, adquirida por la picadura de una garrapata infectada del género Amblyomma. No hay informes de transmisión de persona a persona. Estas garrapatas se encuentran dispersas en territorio brasileño, siendo conocidas como garrapata estrella y garrapata de caballo. Los équidos, capibaras, perros y zarigüeyas son transportadores de garrapatas potencialmente infectadas, que constituyen reservorios o amplificadores de Rickettsia. Los signos más comunes en humanos infectados son fiebre repentina, dolor muscular, dolor de cabeza, sarpullido que aparece entre el segundo y quinto día de la infección y hemorragia. En ocasiones pueden manifestarse signos como fotofobia, vómitos, ansiedad, dolor abdominal e incluso los relacionados con el sistema nervioso y urinario, con afectación renal. La sospecha aumenta cuando el individuo, con alguno de estos signos, tiene antecedentes de picaduras de garrapatas o exposición a un área con potencial infestación por garrapatas (caminatas en bosques, ríos, cascadas, senderos, contacto con animales, picnics, pesquerías, etc. ). La prevención es más eficaz que el tratamiento, dada la falta de vacunas contra la rickettsiosis. La terapia con antibióticos posterior a la exposición no puede prevenir la enfermedad. El uso de ropa ligera, repelentes a base de DEET, ropa que cubra todo el cuerpo y comprobar la presencia de garrapatas después de la actividad en las zonas de riesgo, eliminándolas si se encuentran, son medidas de prevención eficaces. Los perros y caballos, y el entorno donde viven, deben ser sometidos al control de garrapatas, según las instrucciones del veterinario. La eliminación de las garrapatas de la piel se debe hacer retorciéndolas hasta que se desprendan, evitando que se aplasten, para no liberar rickettsias, que pueden penetrar en el cuerpo a través de microlesiones. En caso de sospecha de la enfermedad, se recomienda buscar atención médica de inmediato, evitando la automedicación. 72 Teles, J. A. A.; Porto, M. L. S.
https://openalex.org/W2756590219	https://bmcvetres.biomedcentral.com/track/pdf/10.1186/s12917-017-1210-5	English	null	Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi	BMC veterinary research	2,017	cc-by	6,485	© 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. Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi Sintia Almeida1, Elaine M. S. Dorneles2, Carlos Diniz1,3, Vinícius Abreu4, Cassiana Sousa1, Jorianne Alves5, Adriana Carneiro5, Priscilla Bagano1, Sharon Spier6, Debmalya Barh1,7, Andrey P. Lage2, Henrique Figueiredo8 and Vasco Azevedo1 Almeida et al. BMC Veterinary Research (2017) 13:290 DOI 10.1186/s12917-017-1210-5 Almeida et al. BMC Veterinary Research (2017) 13:290 DOI 10.1186/s12917-017-1210-5 Open Access * Correspondence: sintiaalmeida@gmail.com; vasco@icb.ufmg.br 1Instituto de Ciências Biológicas, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil Full list of author information is available at the end of the article Abstract Background: Corynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines. The aim of this study was to develop a quadruplex PCR assay that would allow simultaneous detection and biovar-typing of C. pseudotuberculosis. Methods: In the present study, genomes of C. pseudotuberculosis strains were used to identify the genes involved in the nitrate reduction pathway to improve a species identification three-primer multiplex PCR assay. The nitrate reductase gene (narG) was included in the PCR assay along with the 16S, rpoB and pld genes to enhance the diagnosis of the multiplex PCR at biovar level. Results: A novel quadruplex PCR assay for C. pseudotuberculosis species and biovar identification was developed. The results of the quadruplex PCR of 348 strains, 346 previously well-characterized clinical isolates of C. pseudotuberculosis from different hosts (goats, sheep, horse, cattle, buffalo, llamas and humans), the vaccine strain 1002 and the type strain ATCC 19410T, were compared to the results of nitrate reductase identification by biochemical test. The McNemar’s Chi-squared test used to compare the two methods used for C. pseudotuberculosis biovar identification showed no significant difference (P = 0.75) [95% CI for odds ratio (0.16–6.14)] between the quadruplex PCR and the nitrate biochemical test. Concordant results were observed for 97.13% (338 / 348) of the tested strains and the kappa value was 0.94 [95% CI (0.90–0.98)]. Conclusions: The ability of the quadruplex assay to discriminate between C. pseudotuberculosis biovar Ovis and Equi strains enhances its usefulness in the clinical microbiology laboratory. Keywords: Caseous lymphadenitis, Diagnosis, Nitrate reductase, Horse, Sheep, Goats Bacterial strains and culture conditions A total of 348 C. pseudotuberculosis strains, 346 field isolates [25–27], C. pseudotuberculosis ATCC 19410T type strain, and C. pseudotuberculosis 1002 vaccine strain, were used in this study. These strains were obtained from the repository of the Laboratório de Genética Celular e Molecular, Instituto de Ciências Biológicas and of the Laboratório de Bacteriologia Apli- cada, Escola de Veterinária of the Universidade Federal de Minas Gerais. C. pseudotuberculosis biovars Ovis and Equi were aerobically grown in brain heart infusion (BHI) (Acumedia Manufacturers, Baltimore, USA) agar plates at 37 °C for 48 h. The C. pseudotuberculosis isolates were identified by standard biochemical tests [9, 28, 29]. Nitrate reduction was assessed using nitrate broth (Merck, Billerica, USA) and further reduction beyond nitrite was tested by addition of zinc dust (Sigma-Aldrich, St Louis, USA) [28]. Disease caused by C. pseudotuberculosis biovars has different clinical manifestations in the susceptible hosts [6, 8, 17, 18], and biovar identification is import- ant for understanding the epidemiology of infection, and consequently for disease control. Moreover, biovar identification can also have clinical implications. Since cattle can be infected by strains of both biovars, which may have different tissue preferences: biovar Ovis infects chiefly the mammary gland [17] and skin [18] and biovar Equi causes ulcerative lymphangitis and coronet lesions [9, 19, 20]. A dominant genetic characteristic that differentiates the biovars of C. pseudotuberculosis is the presence of the nitrate reduction operon in the biovar Equi strains [21]. Additionally, biovar Equi strains have 15 genes that are absent in biovar Ovis strains, includ- ing the narKGHJI operon, and a gene cluster encod- ing the molybdopterins moeB, moaE, molB, molA, moeY, moaC, moeA, and moaA and two hypothetical proteins [21]. Genomic DNA extraction, primers, and quadruplex PCR Genomic DNA extraction were performed according to the previously described protocol [30]. The oligonucleo- tide primers used in this study are listed in Table 2. Primers used to target 16S rRNA, rpoB, and pld genes of C. pseudotuberculosis were previously described [14, 31, 32]. Primers targeting the narG gene were designed by aligning the narG nucleotide sequences of C. pseudotu- berculosis biovar Equi strains available from the whole genome sequenced strains (Table 1). Background two biovars, based on their ability to convert nitrate to nitrite, nitrate-positive strains are classified as biovar Equi and the nitrate negative ones as biovar Ovis [5]. In sheep and goats, CLA is predominantly caused by biovar Ovis strains, whereas horses and buffalos are mostly infected by biovar Equi strains [6–8]. Infection by C. pseudotuberculo- sis is distributed worldwide, causing significant disease in horse, sheep and goat herds [8, 9]. The main economic losses attributed to C. pseudotuberculosis infection include decreased milk production, decreased weight gain, reduced value of hides due to scarring, and the cost of the drugs and labor needed to treat disease [9]. Corynebacterium pseudotuberculosis is a facultative intra- cellular bacterium that is the causative agent of caseous lymphadenitis (CLA) in goats and sheep, abscesses and ulcerative lymphangitis in horses and oedematous skin disease (OSD) in Buffalo. It also causes sporadic infections in other species including cattle, camels, llamas, and humans [1–4]. C. pseudotuberculosis can be classified in * Correspondence: sintiaalmeida@gmail.com; vasco@icb.ufmg.br 1Instituto de Ciências Biológicas, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil Full list of author information is available at the end of the article Almeida et al. BMC Veterinary Research (2017) 13:290 Page 2 of 8 Almeida et al. BMC Veterinary Research (2017) 13:290 simultaneous detection and biovar-typing of C. pseudo- tuberculosis strains. Direct and indirect tests to detect C. pseudotuberculo- sis have already been proposed, such as complement fix- ation test [10], synergistic hemolysis inhibition test [11], microagglutination assay [12], phospholipase D (PLD) antigen-based ELISA [13] and a multiplex PCR devel- oped by our research group [14]. While these tests are useful for clinical diagnosis in diseased animals, none can differentiate the two biovars of C. pseudotuberculo- sis, which is currently only accomplished by biochemical tests. Differences between biovars are relevant for host and tissue specificity and appear to be associated with virulence [15, 16]. Nitrate reductase genes Presence or absence of nitrate reductase genes were analyzed in nineteen C. pseudotuberculosis genomes (Table 1) in our previous work [21]. aPrimers used to quadriplex PCR assay Bacterial strains and culture conditions Quadruplex PCR were carried out in a final volume of 50 μL, containing 20 ng of genomic DNA, 1 μM of each primer, 0.25 mM dNTPs, 1 units of Taq DNA polymerase (Life Tech- nologies, Carlsbad, USA), 2 mM MgCl2, and 1X buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl) (Life Tech- nologies, Carlsbad, USA). Amplification was performed using the thermal cycler (PTC-100, MJ Research, Hercules, USA) as follows: the first denaturation at 95 °C for 4 min; followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for the 30s, and extension at 72 °C for 1.5 min. The amplified products were sub- mitted to electrophoresis in 1.0% agarose gel (w/v) in Tris-borate-EDTA (TBE) buffer (89 mM Tris Base, 89 mM Boric Acid and 2 mM EDTA pH 8.0), stained with 0.5 mg / mL ethidium bromide and visualized under UV light. Currently, only nitrate reduction test distinguishes C. pseudotuberculosis biovars Ovis and Equi [5, 9]. The available phenotypic tests, performed in a labora- tory or commercially available, for C. pseudotubercu- losis identification are usually effective. However, the phenotypic tests can be expensive and unavailable for some laboratories that prefer to use molecular tech- niques. Currently, clinical microbiology laboratories are experiencing a change from classical to new diag- nostic tools as PCR, real-time PCR, sequencing and MALDI-TOF mass spectrometry [22, 23], which, due to the decrease in costs, may prefer to use molecular over biochemical tests. Moreover, the molecular tests are usually faster, easier and have less subjectivity in interpretation [24]. Our collaborative group has sequenced more than 60 C. pseudotuberculosis strains isolated from different hosts around the world, performing genomic, prote- omic and clinical studies to not only to understand the pathogen but also try to find a way to control the spread of bacteria. Thus, the aim of this study was to develop a quadruplex PCR assay that would allow Almeida et al. Bacterial strains and culture conditions BMC Veterinary Research (2017) 13:290 Page 4 of 8 Results Comparative genome analysis showed that C. pseudotu- berculosis biovar Equi strains (258, 31, 262, MB20, E19 and CCUG27541) had narKGHJI gene clusters, however strains 1/06-A, 316, 162, and CIP52.97, although show- ing positive results in the nitrate reduction test, did not exhibit narKGHJI operon in their genome. On the other hand, genomic sequence analysis identified partial genes molB, narJ, moeA, and moeB in the strains 1/06-A, 162 and CIP52.97. Since the strains 1/06-A, 316, 162, and CIP52.97 were nitrate reductase positive in biochemical test and the genes were not identified in their genomes, primers to target narKGHJI cluster and narT gene were designed (Table 2). The PCR tests (Additional file 1: Figure S1), sequencing and the optical map showed that the genes for narKGHJI and narT are present in the genomes of those strains (data not shown). Sequencing of singleplex PCR products Fig. 1 Four-primer quadruplex PCR for C. pseudotuberculosis species and biovar identification.Agarose gel 1.5% showing the PCR amplification of quadruplex PCR assay stained with ethidium bromide (0.5 mg / mL).L: GeneRuler DNA Ladder (Fermentas, Vilnius, Lithuania); Lanes 1–9:C. pseudotuberculosis biovar Equi strains C31, 258, 262, 162, 5297, 1/06A, EG-37, EG-42 and I-37; Lane 10:C. pseudotuberculosis biovar Ovis strain 1002 q g g p p In order to confirm the quadruplex PCR results, ten ran- domly chosen isolates were tested further in singleplex PCR assays with the four C. pseudotuberculosis-specific primer pairs. PCR products were purified using Agen- court AMPure XP (Beckman Coulter Company, Beverly, Massachusetts, USA) according to the manufacturer’s instructions, and each product was sequenced in both directions using primers targeting the 16S rRNA, rpoB, pld and narG gene and the Big Dye V3.1 Terminator Kit (Applied Biosystems, USA) using an ABI 3500 DNA analyzer (Applied Biosystems, California, USA). Sequences were analyzed on the Geneious suite of molecular biology (http://www.geneious.com) with 16S rRNA (GenBank accession nos X81916, X81907, and X84255), rpoB (Gen- Bank accession no. AY492239), pld (GenBank accession nos L16586 and L16587) and narG (GenBank accession no AJF93840.1) as the reference genes. Fig. 1 Four-primer quadruplex PCR for C. pseudotuberculosis species and biovar identification.Agarose gel 1.5% showing the PCR amplification of quadruplex PCR assay stained with ethidium bromide (0.5 mg / mL).L: GeneRuler DNA Ladder (Fermentas, Vilnius, Lithuania); Lanes 1–9:C. pseudotuberculosis biovar Equi strains C31, 258, 262, 162, 5297, 1/06A, EG-37, EG-42 and I-37; Lane 10:C. pseudotuberculosis biovar Ovis strain 1002 Statistical analysis Comparison between nitrate reduction test and quadru- plex PCR was performed by McNemar’s Chi-squared test, and the agreement was calculated using the kappa statistic. Statistical analysis were performed using the packages psych [33] and epibasix [34] on R software version 3.2.3 [35]. and humans) [21, 25–27] were compared to the results of nitrate reductase identification by biochemical test, and are shown in Table 3. The McNemar’s Chi-squared test used to compare the two methods employed for C. pseudotuberculosis biovar identification showed no sig- nificant difference (P = 0.75), with an odds ratio of 1 (95% CI for the odds ratio: 0.16–6.14) between the quad- ruplex PCR and the nitrate biochemical test. Concordant results were observed for 97.13% (338/348) of the strains (Table 3), and the kappa statistic value was 0.94 [95% CI (0.90–0.98)], denoting excellent concordance between biochemical and molecular tests for nitrate reductase identification. The limit of detection of the new quadru- plex PCR was 200 ng of DNA from C. pseudotuberculo- sis biovar Equi, which corresponds to approximately 100 bacteria. Bacterial strains and culture conditions BMC Veterinary Research (2017) 13:290 Page 3 of 8 Page 3 of 8 Table 2 List of oligonucleotide primers used in this study Target gene Primers Sequence (5′ →3′) Amplicom size (bp) Multiplex PCR assay Reference 16S rRNAa Forward ACCGCACTTTAGTGTGTGTG 816 Yes (25) Reverse TCTCTACGCCGATCTTGTAT rpoBa Forward CGTATGAACATCGGCCAGGT 446 Yes (26) Reverse TCCATTTCGCCGAAGCGCTG plda Forward ATAAGCGTAAGCAGGGAGCA 203 Yes (14) Reverse ATCAGCGGTGATTGTCTTCCAGG narGa Forward ACCCGTACTTGCACTCTTTC 612 Yes Present Study Reverse AGTCAGTACTTCCGCAGGTC narT Forward GCTGAAGCAAGTTCGTGTCT 202 No Present Study Reverse GTAACGGTCAGAGAACCATCC narK Forward GCTGAAGCAAGTTCGTGTCT 202 No Present Study Reverse GTAACGGTCAGAGAACCATCC narG2 Forward CAACGTGGTACCTGGTATCTG 200 No Present Study Reverse CATAGGGAGAGCGAGAACAA narH Forward GATTCTACTGACCGCCATCTC 196 No Present Study Reverse ATCAGTACCTGTCATGCCTACC narJ Forward CGTGATGGTATAGAGGTGCTG 198 No Present Study Reverse GTTGGAAGCAGTAGGGAAGGGAG narI Forward CTGTATCCACACAGGTGTTCG 215 No Present Study Reverse GTATCCTACAGGCGCTGAGA aPrimers used to quadriplex PCR assay Table 1 Corynebacterium pseudotuberculosis strains with the whole genome sequenced available in the NCBI GenBank (www.ncbi.nlm.nih.gov/genbank) in 2015 Strain Biovar Host Country Genome size (MB) Sequencing status NCBI access Reference 1002 Ovis Goat Brazil, 2.33511 Complete NC_017300.1 (37) C231 Ovis Sheep Australia 2.32821 Complete NC_017301.1 (37) FRC41 Ovis Human France 2.33791 Complete NC_014329.1 (38) I19 Ovis Cow Israel 2.33773 Complete NC_017303.1 (39) PAT10 Ovis Sheep Argentine 2.33532 Complete NC_017305.1 (40) 42/02-A Ovis Sheep Australia 2.33761 Complete NC_017306.1 (41) 3/99–5 Ovis Sheep Scotland 2.33794 Complete NC_016781.1 (41) 267 Ovis Llama USA 2.33763 Complete NC_017462.1 (6) P54B96 Ovis Antelope South Africa 2.33794 Complete NC_017031.1 (42) CIP5297 Equi Horse Kenya 2.32059 Complete NC_017307.1 (43) 1/06-A Equi Horse USA 2.27912 Complete NC_017308.1 (44) 316 Equi Horse USA 2.31041 Complete NC_016932.1 (45) 258 Equi Horse Belgium 2.36982 Complete NC_017945.1 (46) 162 Equi Camel UK 2.29346 Complete NC_018019.1 (42) 31 Equi Buffalo Egypt 2.38969 Complete NC_017730.1 (47) 262 Equi Cattle Belgium 2.32575 Complete NZ_CP012022.1 – MB20 Equi Horse USA 2.36309 Draft JPUV01 (48) E19 Equi Horse Unknow 2.36796 Complete NZ_CP012136.1 – CCUG27541 Equi Horse Unknow 2.37942 Draft JPJB01 (49) Table 1 Corynebacterium pseudotuberculosis strains with the whole genome sequenced available in the NCBI GenBank (www.ncbi.nlm.nih.gov/genbank) in 2015 Strain Biovar Host Country Genome size (MB) Sequencing status NCBI access Reference 1002 Ovis Goat Brazil, 2.33511 Complete NC_017300.1 (37) C231 Ovis Sheep Australia 2.32821 Complete NC_017301.1 (37) FRC41 Ovis Human France 2.33791 Complete NC_014329.1 (38) I19 Ovis Cow Israel 2.33773 Complete NC_017303.1 (39) PAT10 Ovis Sheep Argentine 2.33532 Complete NC_017305.1 (40) 42/02-A Ovis Sheep Australia 2.33761 Complete NC_017306.1 (41) 3/99–5 Ovis Sheep Scotland 2.33794 Complete NC_016781.1 (41) 267 Ovis Llama USA 2.33763 Complete NC_017462.1 (6) P54B96 Ovis Antelope South Africa 2.33794 Complete NC_017031.1 (42) CIP5297 Equi Horse Kenya 2.32059 Complete NC_017307.1 (43) 1/06-A Equi Horse USA 2.27912 Complete NC_017308.1 (44) 316 Equi Horse USA 2.31041 Complete NC_016932.1 (45) 258 Equi Horse Belgium 2.36982 Complete NC_017945.1 (46) 162 Equi Camel UK 2.29346 Complete NC_018019.1 (42) 31 Equi Buffalo Egypt 2.38969 Complete NC_017730.1 (47) 262 Equi Cattle Belgium 2.32575 Complete NZ_CP012022.1 – MB20 Equi Horse USA 2.36309 Draft JPUV01 (48) E19 Equi Horse Unknow 2.36796 Complete NZ_CP012136.1 – CCUG27541 Equi Horse Unknow 2.37942 Draft JPJB01 (49) Table 1 Corynebacterium pseudotuberculosis strains with the whole genome sequenced available in the NCBI GenBank (www.ncbi.nlm.nih.gov/genbank) in 2015 Table 2 List of oligonucleotide primers used in this study Target gene Primers Sequence (5′ →3′) Amplicom size (bp) Multiplex PCR assay Reference 16S rRNAa Forward ACCGCACTTTAGTGTGTGTG 816 Yes (25) Reverse TCTCTACGCCGATCTTGTAT rpoBa Forward CGTATGAACATCGGCCAGGT 446 Yes (26) Reverse TCCATTTCGCCGAAGCGCTG plda Forward ATAAGCGTAAGCAGGGAGCA 203 Yes (14) Reverse ATCAGCGGTGATTGTCTTCCAGG narGa Forward ACCCGTACTTGCACTCTTTC 612 Yes Present Study Reverse AGTCAGTACTTCCGCAGGTC narT Forward GCTGAAGCAAGTTCGTGTCT 202 No Present Study Reverse GTAACGGTCAGAGAACCATCC narK Forward GCTGAAGCAAGTTCGTGTCT 202 No Present Study Reverse GTAACGGTCAGAGAACCATCC narG2 Forward CAACGTGGTACCTGGTATCTG 200 No Present Study Reverse CATAGGGAGAGCGAGAACAA narH Forward GATTCTACTGACCGCCATCTC 196 No Present Study Reverse ATCAGTACCTGTCATGCCTACC narJ Forward CGTGATGGTATAGAGGTGCTG 198 No Present Study Reverse GTTGGAAGCAGTAGGGAAGGGAG narI Forward CTGTATCCACACAGGTGTTCG 215 No Present Study Reverse GTATCCTACAGGCGCTGAGA aPrimers used to quadriplex PCR assay Almeida et al. Discussion Previously, identification of C. pseudotuberculosis biovars was only possible only through the established procedures that included isolation and identification of the agent using biochemical tests such as the nitrate reduction test, which separates the nitrate-positive biovar Equi from ni- trate negative biovar Ovis strains [5]. Herein, we devel- oped, by the addition of a new oligonucleotide primer pair targeting the narG gene to the former multiplex PCR assay [14], a robust new assay for identification of C. pseu- dotuberculosis at species and biovar levels. The multiplex PCR assay that targets 16S rRNA, rpoB and pld genes [14] was improved by the inclusion of C. pseudotuberculosis biovar-specific primers for the narG gene (narG – Table 2), in a novel quadruplex PCR assay (Fig. 1). The assessment of our quadruplex PCR assay was performed in a double-blind fashion. The results of the quadruplex PCR of the 348 previously well-characterized strains of C. pseudotuberculosis from different hosts (goats, sheep, horse, cattle, buffalo, llamas The comparative genome analysis showed in C. pseu- dotuberculosis biovar Equi strains (258, 31, 262, MB20, E19 and CCUG27541) narKGHJI gene clusters that participate via the respiratory anaerobic process of the Page 5 of 8 Almeida et al. BMC Veterinary Research (2017) 13:290 Page 5 of 8 Page 5 of 8 Table 3 Comparison of biochemical test and a multiplex PCR assay employed for Corynebacterium pseudotuberculosis biovar identification Table 3 Comparison of biochemical test and a multiplex PCR assay employed for Corynebacterium pseudotuberculosis biovar identification Multiplex PCR assay Biochemical test TOTAL Nitrate positive Nitrate negative Nitrate positive 133 5 138 Nitrate negative 5 205 210 Total 138 210 348 McNemar’s Chi-squared test = P = 0.75, Odds Ratio: 1 (95% CI for the odds ratio: 0.16–6.14) Kappa coefficient = 0.94 (95% CI: 0.91–0.98) coefficient, which is a robust statistic that measures inter- rater agreement for qualitative items, confirms that re- gardless of the technique used for biovar classification the results obtained were very similar. It is also important to consider that kappa values range from −1 to +1, where 1 represents a perfect agreement between the raters, and 0.81 to 1.00 represents almost perfect agreement, such as the observed in our data (0.94) [28, 37]. Discussion Discordance be- tween both phenotypic and genotypic methods can be ex- plained by the effect of environmental factors on gene expression [29, 38] amino-acid substitution, the genetic background of the strain (that can vary according to geographical locations) and mutations. Considering dis- cordances due to mutations genotypic tests have proven to be more reliable and sensitive as diagnostic tool than phenotypic tests [30–32, 39–42]. Others stud- ies also showed discordant results between genotypic versus phenotypic methods [30–32, 39–41, 43, 44]. nitrate reduction similar to Escherichia coli [21, 36]. The C. pseudotuberculosis narKGHJI gene cluster showed significant similarity with the protein sequences found in other Actinomycetes, such as C. diphtheriae, C. glutami- cum, and Mycobacterium tuberculosis. All C. pseudotu- berculosis biovar Ovis strains do not present any gene of the narKGHJI operon in their genomes [21]. nitrate reduction similar to Escherichia coli [21, 36]. The C. pseudotuberculosis narKGHJI gene cluster showed significant similarity with the protein sequences found in other Actinomycetes, such as C. diphtheriae, C. glutami- cum, and Mycobacterium tuberculosis. All C. pseudotu- berculosis biovar Ovis strains do not present any gene of the narKGHJI operon in their genomes [21]. To our acknowledgement, this is the first molecular approach able to clearly differentiate between C. pseudo- tuberculosis biovar Ovis and Equi, although different re- striction patterns, ribotypes and ERIC-PCR clustering pattern have been associated to biovars [26, 36, 45–47]. The advantages of multiplex PCR assay over biochemical tests are the speed, performance and reproducibility, and the ability to test large numbers of isolates simultaneously [26, 45, 46]. Identification is based upon the number and sizes of four products amplified by PCR. Moreover, the use of molecular techniques reduces the manipulation of viable bacteria in the laboratory and consequently the risk of accidental infection, as C. pseudotuberculosis can even- tually be a zoonotic agent [2]. Moreover, this new diagnos- tic tool, the quadruplex PCR assay for identification and The nitrate locus in C. pseudotuberculosis is composed of the narKGHJI operon and by a cluster of genes encod- ing the molybdopterin moeB, moaE, molB, molA, moeY, moaC, moeA, and moaA (Fig. 2) [21]. Molybdopterin is a cofactor that is indispensable for the activity of nitrate reductase. In the narGHI complex, the narG gene is a member of a superfamily of enzymes that use a Molybdopterin-guanine-dinucleotide (Mo-bisMGD) co- factor (bisMGD) for their catalytic activity. Our results showed that among the 348 C. Funding g APL was supported by the Programa Pesquisador Mineiro – PPM (00923–15) from Fapemig and SA was supported by the Universal (481,792/2013–9) from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Competing interests h h d l h The authors declare that they have no competing interests. Discussion pseudotuber- culosis tested, only 10 strains (2.87%) showed differences between the biovar classification provided by molecular and biochemical tests (Table 3), which was observed even after repeating the assays. Furthermore, the kappa Fig. 2 Nitrate locus from C. pseudotuberculosis biovar Equi.This locus contains: the genes encoding the molybdopterin moeB, moaE, molB, molA, moeY, moaC, moeA and moaA and the genes encoding the nitrate reductase narK, narG, narH, narJ, narI. Insertion show between ansA and rpsH genes is lacking in nitrate negative C. pseudotuberculosis biovar Ovis strains. Arrows represent open reading frames and their orientations. Blue and pink: common genes shared between C. pseudotuberculosis biovar Ovis and biovar Equi strains. Pink: ribosomal proteins. Hatched: additional or different genes. Red: narKGHJI operon and grey: genes encoding the molybdopterin moeB, moaE, molB, molA, moeY, moaC, moeA and moaA Fig. 2 Nitrate locus from C. pseudotuberculosis biovar Equi.This locus contains: the genes encoding the molybdopterin moeB, moaE, molB, molA, moeY, moaC, moeA and moaA and the genes encoding the nitrate reductase narK, narG, narH, narJ, narI. Insertion show between ansA and rpsH genes is lacking in nitrate negative C. pseudotuberculosis biovar Ovis strains. Arrows represent open reading frames and their orientations. Blue and pink: common genes shared between C. pseudotuberculosis biovar Ovis and biovar Equi strains. Pink: ribosomal proteins. Hatched: additional or different genes. Red: narKGHJI operon and grey: genes encoding the molybdopterin moeB, moaE, molB, molA, moeY, moaC, moeA and moaA Page 6 of 8 Almeida et al. BMC Veterinary Research (2017) 13:290 biotyping of C. pseudotuberculosis follows the new trends on clinical microbiology laboratory that is currently in- corporating more molecular biology tools in its routine [23, 48]. In addition, despite not having been tested in the present study, due to its analytical sensitivity of 100 bac- teria. \|It is likely that this quadruplex PCR can also be applied to direct testing from clinical samples, as it has been done successfully for the three-primer (16S rRNA, rpoB, and pld) multiplex PCR [14]. diagnosis of the multiplex PCR at biovar level. There was a significant concordance between the biovar clas- sification provided by the molecular and biochemical test. The ability of the expanded quadruplex PCR assay to discriminate between C. pseudotuberculosis biovar Ovis and Equi strains enhances its value. Author details 1 1Instituto de Ciências Biológicas, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 2Escola de Veterinária, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 3Departamento de Medicina Veterinária, Federal University of Lavras, Lavras, MG, Brazil. 4Centro de Energia Nuclear na Agricultura, University of Sao Paulo, Piracicaba, SP, Brazil. 5Instituto de Ciências Biológicas, Federal University of Para, Belém, PA, Brazil. 6Department of Medicine and Epidemiology, UC Davis School of Veterinary Medicine, Davis, CA, USA. 7Centre for Genomics and Applied Gene Technology, Institute of Integrative Omics and Applied Biotechnology (IIOAB), Nonakuri, Purba Medinipur, WB, India. 8Aquacen - National Reference Laboratory for Aquatic Animal Diseases, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. Availability of data and materials The information supporting the conclusions of this article is included in the article. g y In this study, it was also observed that some C. pseu- dotuberculosis strains (1/06-A, 316, 162 and CIP52.97) were able to reduce nitrate when tested by the biochem- ical approach and were also positive in the quadruplex PCR assay, albeit did not show in their genomes genes associated with nitrate reduction. The genomic analysis of nitrate locus identified that partial genes encoding the molybdopterin and narKGHJI operon of these strains was absent [21]. These results may have been due to low overall coverage, poor capture efficiency of certain regions, genomic regions that were previously not as- sembled or poorly assembled, including unambiguously aligning repetitive regions, such as transposons, and difficulty in unambiguously aligning repetitive regions [41, 51]. Then, after resequencing of the narKGHJI op- eron region and optical mapping of these strains, it was observed that these strains have the nitrate locus in their genome and corrections on their information on GenBank are under way. Acknowledgements This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Fapemig). APL and VA thanks CNPq for their fellowships. APL is also indebted to Programa Pesquisador Mineiro (PPM 00923-15) - Fapemig. Abbreviations CLA: Caseous Lymphadenitis; Mo-bisMGD: Molybdopterin-guanine-dinucleotide; NAR: Nitrate Reductase; OSD: Oedematous Skin Disease Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file p p p The quadruplex PCR proposed in this study facilitates and deepens the level of identification of C. pseudotuber- culosis strains at clinical microbiology laboratory, and thereby improves the diagnosis of infection by providing more information for decision making. These results are especially significant considering that C. pseudotubercu- losis infects a wide range of hosts and produce different clinical manifestations. Furthermore, it was recently suggested that C. pseudotuberculosis biovars have dif- ferences at the molecular phylogenetic level, indicating an anagenesis process within the species [37, 49]. The evolutionary analysis of conserved genes (rpoB, gapA, fusA, and rsmE) suggesting a gradual anagenesis of C. pseudotuberculosis in that study [49] substantially in- creases the importance of a molecular technique cap- able of efficiently separating the biovars Ovis and Equi. In fact, a pan-genome analysis of fifteen C. pseudotu- berculosis strains showed a significant number of genes not shared by both biovars, including remarkable differ- ences in the 16 detected pathogenicity islands [50]. Additional file 1: Figure S1. A and B) 1% agarose gel containing the result of PCR performed for molecular confirmation of the amplicom narKGHJI operon and narT gene of C31, 258, 162, 5297 and 106/A strains. (DOCX 116 kb) Additional file 1: Figure S1. A and B) 1% agarose gel containing the result of PCR performed for molecular confirmation of the amplicom narKGHJI operon and narT gene of C31, 258, 162, 5297 and 106/A strains. (DOCX 116 kb) Authors’ contributions SA: drafted and wrote the manuscript, and analyzed the data. ED: drafted the manuscript and performed biochemistry tests. CD: performed biochemistry tests. VACA: development of scripts and computational analysis. CS, JA and PB performed laboratory experiments. VA, DB and AL: wrote the manuscript. VA, AC, SS, AL and HF: contributed to conception and design. All authors read and approved the final manuscript. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Conclusions A novel quadruplex PCR assay for C. pseudotuberculosis species and biovar identification was developed. The ni- trate reductase gene narG was included in the assay along with the 16S, rpoB and pld genes to improve the Page 7 of 8 Page 7 of 8 Almeida et al. BMC Veterinary Research (2017) 13:290 Almeida et al. BMC Veterinary Research (2017) 13:290 Received: 5 July 2016 Accepted: 14 September 2017 References J Am Vet Med Assoc. 2005;227:441–8. 7. Pratt SM, Spier SJ, Carroll SP, Vaughan B, Whitcomb MB, Wilson WD. Evaluation of clinical characteristics, diagnostic test results, and outcome in horses with internal infection caused by Corynebacterium pseudotuberculosis: 30 cases (1995-2003). J Am Vet Med Assoc. 2005;227:441–8. 29. Coyle MB, Lipsky BA. 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https://openalex.org/W3080349238	https://re.public.polimi.it/bitstream/11311/1145178/1/sustainability-12-06985.pdf	Latin	null	Climate Change and Social Perception: A Case Study in Southern Italy	Sustainability	2,020	cc-by	16,389	Climate Change and Social Perception: A Case Study in Southern Italy Loredana Antronico 1,, Roberto Coscarelli 1, Francesco De Pascale 1 and Dante Di Matteo 2 Loredana Antronico 1, , Roberto Coscarelli 1, Francesco De Pascale 1 and Dante Di Matteo 2 1 Research Institute for Geo‐Hydrological Protection, Italian National Research Council, 87036 Rende, CS, Italy; roberto.coscarelli@irpi.cnr.it (R.C.); francesco.depascale@irpi.cnr.it (F.D.P.) 2 Department of Architecture and Urban Studies, Polytechnic of Milan, 20133 Milan, Italy; dante.dimatteo@polimi.it 1 Research Institute for Geo‐Hydrological Protection, Italian National Research Council, 87036 Rende, CS, Italy; roberto.coscarelli@irpi.cnr.it (R.C.); francesco.depascale@irpi.cnr.it (F.D.P.) 2 Department of Architecture and Urban Studies, Polytechnic of Milan, 20133 Milan, Italy; dante.dimatteo@polimi.it d l d 1 Research Institute for Geo‐Hydrological Protection, Italian National Research Council, 87036 Rende, CS, Italy; roberto.coscarelli@irpi.cnr.it (R.C.); francesco.depascale@irpi.cnr.it (F.D.P.) 2 Department of Architecture and Urban Studies, Polytechnic of Milan, 20133 Milan, Italy; dante.dimatteo@polimi.it Correspondence: loredana.antronico@irpi.cnr.it Received: 9 July 2020; Accepted: 24 August 2020; Published: 27 August 2020 Received: 9 July 2020; Accepted: 24 August 2020; Published: 27 August 2020 Received: 9 July 2020; Accepted: 24 August 2020; Published: 27 August 2020 Received: 9 July 2020; Accepted: 24 August 2020; Published: 27 August 2020 Abstract: The consequences of climate change can involve various ambits and be very severe. For this reason, the social perception of climate change is a fundamental issue since it can influence the decisions of the policymakers, by encouraging or discouraging political, economic and social actions. In this paper, a sample of 300 interviews, collected through a standardized questionnaire and carried out among two municipalities located in southern Italy, was exploited to investigate the perception of climate change. Specific issues, regarding perceptions about climate change, concerns about its impacts, level of information, behavior and actions, exposure to extreme natural events and trust, were addressed to give answers to the research questions: (i) Is climate change perceived by the population? (ii) What is the degree of the community resilience to extreme natural events and climate change? As the main findings, this survey highlighted that the spatio‐temporal dimension affects population perception, suggesting that some issues, such as correct behavior towards the geosphere, the sustainability of anthropization processes, community resilience and disaster risk reduction policies, can be very central and useful to mitigate the effects of climate change in population and society. Climate Change and Social Perception: A Case Study in Southern Italy Moreover, climate change perception varies in relation to contextual factors, including media communication, socio‐demographic characteristics of respondents, knowledge and education, economic and institutional factors, personal values and, finally, psychological factors and experience. Keywords: climate change; social perception; resilience; Calabria (Italy) Article Article Keywords: climate change; social perception; resilience; Calabria (Italy) www.mdpi.com/journal/sustainability Sustainability 2020, 12, 6985; doi:10.3390/su12176985 1. Introduction Both dimensions arise in a circular relationship whereby greater public attention and awareness favors the activation of policies that, in turn, lead to greater awareness of the public and the encouragement of virtuous behavior. This process must, therefore, be triggered at all levels from both a top‐down and a bottom‐up perspective. With this aim, it is necessary to know the forms and contents of the current public perception of the phenomenon and be aware of the most effective communicative strategies [4]. Perception is a totally subjective assessment of a concept or sensation, influenced by whatever interests us: our being, the influence received during our personal growth, the environment in which our perception develops, etc. For this reason, the perception of a possible hazard varies from person to person, and this also explains why the victims of a disaster often provide different accounts of the same event [6]. Perceptions, feelings or beliefs shaped by socioeconomic, political and cultural factors can support or prevent the adoption of a specific behavior [7]. Risk awareness is the raising of understanding within the population of what risks exist, their potential impacts, and how they are managed [8]. Increased awareness can increase levels of worry, but this may then have a positive effect on the level of preparation. Improved preparation, in turn, reduces worry. Over a longer time scale, this may reduce awareness [9]. The personal level of information is fundamental because comprehensive information on all the dimensions of disaster risk, including hazards, exposure, vulnerability and capacity, related to persons, communities, organizations and countries and their assets, increases the preparedness and, consequently, the ability to face an extreme phenomenon. Trust is about the level of confidence actors have that they will get the input needed to perform their tasks from each other actor they are dependent on [10]. Trust in political institutions is, however, fundamental for planning communication strategies [6]. The effectiveness of communication lies in the trust in the communicator [11], because a passive communication system can also be ignored by citizens [12], thus becoming less effective than an active communication system. This study investigates the perception of climate change among the populations of two municipalities located along the Tyrrhenian side of Calabria (southern Italy). The local perception analysis of climate change consequences, assessed by means of the application of a methodology based on standardized interviews, represents a novelty for Calabria. 1. Introduction Climate change represents a great challenge for these times. The impacts of the climate tendencies can be severe in several aspects: health, agriculture, water availability, etc. One of the consequences of climate change is an increase in the occurrence of extreme events with several impacts. As an example, temperature, winter freezes and heat waves can affect the safety of communities, causing serious health problems to people and huge damages to various economic sectors, such as agriculture and energy (e.g., in [1,2]). The tendencies in rainfall patterns can cause an increase in the occurrence of drought events, severe storms and floods, with consequences on agriculture, water resources and exposure to geo‐hydrological risk with damages to population and properties. Climate change represents a necessarily interdisciplinary topic in which the traditional approach of the physical sciences can be usefully integrated with the contribution of the social sciences. Indeed, the main cause of climate change is the series of energy production and consumption actions implemented by humans. The conditions that led to it and with which the issue will have to be addressed in the near future are contained in the socio‐economic structures and value systems of Sustainability 2020, 12, 6985; doi:10.3390/su12176985 www.mdpi.com/journal/sustainability Sustainability 2020, 12, 6985 2 of 24 individuals and communities [3]. Therefore, the response to climate change requires social, political and cultural, as well as scientific, approaches [4]. Starting from this framework, the social perception of climate change is fundamental for two important reasons: first, indirectly, because it constitutes a key component of the socio‐political context within which policymakers exercise their decisions. Public opinion has the power to incentivize or disadvantage political, economic and social actions aimed at tackling the problem posed by climate change. This is in line with the position of those who argue that it is public opinion— and not the evidence brought by the scientific community—that ultimately pushes governments to take actions [5]. The second reason is more direct: the process of mitigation and adaptation to climate change requires the transformation of the behaviors of millions of individuals, who each day make individual and collective choices that have a huge impact on the amount of greenhouse gas emissions and, therefore, potentially on the planet’s climate balance. 2. Climate Change Perception: The Reference Framework The studies carried out so far on the perception of climate change present a series of paradoxes that help to explain the difficulty of the public in facing the challenge posed by climate change. Firstly, awareness of the existence of the phenomenon is growing [13]. According to a study by the European Commission [14], European respondents consider, as the most serious problems, those of “poverty, the lack of food and drinking water”, and the “global warming/climate change”. Many large‐scale public surveys have shown that climate change has been consistently perceived as a “very serious” problem in the UK, France, Australia and most continental European countries (e.g., in [15–19]). Globally, climate change is generally perceived as a much greater risk in most developing countries [20–23]. The European Investment Bank [24] launched a large‐scale survey across Europe, China and the USA. The goal of the survey was to explore the citizens’ perception about the extent of climate change. For Europeans (47%) and the Chinese (73%), climate change is the biggest challenge. Americans worry more about access to health services (45% against 39% who consider climate change as the biggest challenge). Other studies indicate growing skepticism in the late 2000s in some developed countries, fueled by economic and socio‐political factors. However, in many other countries worldwide, concern about climate change has been growing in recent years [25]. National surveys conducted in the Czech Republic in 2006 and 2007 showed that climate change was rated as a “very” or “fairly” serious global problem by 84% of the population; in 2011 this value had dropped to 64% and in 2013 to 61%. As regards Germany, other representative and quantitative surveys showed a relative decline in awareness of climate change, while in 2007 the 69% of German respondents defined climate change as one of the five environmental problems with which they were most concerned. In 2011 the percentage dropped significantly to 40% [26–28]. However, this is counterbalanced by a reduced understanding of its causes and possible solutions [29]. In particular, it is important for the study of public understanding of climate change [30], the integration in the social sciences between quantitative and qualitative methods [17]. American citizens, according to the GlobeScan survey [31], at a percentage of 77%, believe that human activities have contributed to recent climate change. In research conducted by DEFRA [32] and Bostrom et al. 1. Introduction Moreover, this specific topic, generally, still occupies a marginal position, reduced to a few case studies in the literature in Italy. In particular, in this study we consider the following two general objectives to be investigated, that represent the research questions of the survey: (i) Is climate change perceived by the population? (ii) What is the degree of the community resilience to extreme natural events and climate change? These research questions receive answers by addressing the following specific issues: (1) perception and awareness about climate change; (2) concern about impacts and effects of climate change; (3) perception of the personal level of general information about climate change based on the principal sources for each citizen (television, digital media, journals, experts, etc.); (4) personal actions and behavior taken to fight climate change; (5) perception of exposure to extreme natural events; (6) behavior if an extreme event occurs; (7) trust in actions taken by local, national and global policymakers. Sustainability 2020, 12, 6985 3 of 24 3 of 24 2. Climate Change Perception: The Reference Framework In fact, the melting of the glaciers concerns a spatial dimension far from the contexts and the daily life experience of the people interviewed. Therefore, climate change could still be perceived as a phenomenon distant in time and space, far from people’s direct life experience [4]. In the survey carried out by the European Investment Bank [24], Chinese citizens consider air pollution as the first climate change sign, while American citizens consider rising temperatures as the first sign associated with the climate change. As described later in our study, the rising temperatures are considered by the respondents in second place as a phenomenon associated with climate change, after the melting of glaciers. Other recent studies highlight the relationship between climate change and resilience. Iturriza et al. [38,39] present two studies to develop climate change awareness in the urban context; in the first study, the authors proposed a framework as a result of a systematic literature review and a co‐ creation process with the participation of a group of stakeholders through a focus group and a Delphi study. The framework concludes with the establishment of the responsibilities of each stakeholder, by defining the policies they should implement, and the effect one policy might cause on other stakeholders and among policies. As we will see in our study, citizens and stakeholders must be involved in decision‐making processes to jointly implement disaster risk reduction strategies for a community‐based approach also aimed at increasing trust in political institutions and favoring incisive territorial planning and resilience processes. In the second study the authors present a triangulation approach composed by a systematic literature review, semi‐structured interviews and case study results with the framework that defines the ideal process for developing awareness in urban areas. The resulting framework includes three main elements: awareness‐development mechanisms (experience, attention and knowledge), awareness‐development over time graphs and a learning ladder. Additionally, in our study, the development of awareness and learning also play a fundamental role in understanding a complex and problematic phenomenon, such as climate change. It requires a holistic approach, including the contribution of humanists and social scientists, which should dialogue with stakeholders, planners and policy makers to identify proposals for a sustainable, cultural and ecological regeneration of the territory. Even the experience of local communities with extreme natural phenomena influences the perception of climate change, as also emerges from our study. 2. Climate Change Perception: The Reference Framework [33], in which categories of answers were provided, most of the respondents were able to correctly identify the causes of climate change in terms of carbon dioxide emissions and deforestation. In other qualitative research with open‐ended questions, Norton and Leaman [34] found that only 30% of British citizens indicated carbon dioxide emissions as the causes of climate change. In the case of the research by Read et al. [35], only 18% of respondents cite the use of fossil fuels as a determinant of climate change. Considering, therefore, the studies on the perception of climate change, it emerges—from the public opinion—that there is an absence of opposition towards initiatives whose impact on the current lifestyle is not perceived to be relevant [15]. Lorenzoni et al. [36], starting from a series of studies carried out over the years, identify barriers perceived in the face of active commitment to climate change: lack of knowledge, uncertainty and skepticism, distrust of information sources, outsourcing of responsibilities and faults, climate change understood as a distant threat, priority to other dangers, reluctance to change own lifestyle, fatalism and feeling of powerlessness for the whole scale of the problem. Moreover, at local, national and international levels, there is an absence of trust in the responsibility taken by governments and local policymakers and in the significance of their actions. Considering the most frequent associations with climate change, as showed by Bostrom and Fischhoff [37], which will be later highlighted by our study, the main significant link is with the melting of glaciers. Even in the survey promoted by the European Investment Bank [24], European citizens consider the melting of glaciers as the first climate change sign. According to Riva [4], this relationship allows for two orders of reflection. In the first place, climate change is invisible to our eyes and everything we know about this phenomenon passes through the media rather than political or scientific communication. According to the scholar, the image of melting ice occupies an important position because, since it is not possible to see and directly understand the reality of this change, the effects are imagined by deducing and associating the presence of heat to the evidence of melting ice 4 of 24 Sustainability 2020, 12, 6985 [4]. The second reflection, however, concerns the geographical location of the image. 2. Climate Change Perception: The Reference Framework capture the differences in perceptions between young and old people to verify what the level of awareness of the phenomenon was, and by which elements it was conditioned. In Italy there are few current studies on the perception of climate change. Marincioni [6] pays particular attention to the perception of climate hazards and adaptation processes of such communities, highlighting similarities and differences related to geographical location. The results show a significant improvement in flood resilience for those communities involved in public meetings, exercises and other participatory processes. The study constitutes a strong theoretical basis useful for extending the analysis to other territories, providing the elements for a better management of climate risk in Italy. Sabato [45] carries out a didactic experimentation on the perception of climate change in adolescents in a Sicilian school. Bonati and Tononi [46] are editing a volume on the relationship between climate change and risk in the field of geographical education. De Paula Baer et al. [47] propose a study to validate a questionnaire to measure the knowledge of Italians on climate change and its consequences. Nguyen et al. [48], through qualitative and quantitative empirical research methods, carried out on a case study based upon four Mediterranean farming systems located in Oristano, to try to understand the relationship between farmers’ perceptions of climate change (i.e., increased temperature and decreased precipitation) and of present and future water availability for agriculture, as forecasted by climatic and crop models. The authors also explore asymmetries between farmers’ perceptions and present and future climate change and water scenarios as well as factors influencing perceptions. Finally, in the context of water management in agriculture, another study carried out by Vollaro et al. [49] empirically analyses the social perception of climate change and the social acceptability of potential policy reforms of water management in Italy. The results show the respondents’ awareness of climate change events and a common consensus on the need to improve efficiency in water management. These studies have several aspects in common with this study, and it is worth highlighting that community resilience and social vulnerability to climate change are two concepts closely linked to the study of social perception. In fact, investigating and understanding public perception, the collective imagination and the awareness of a community are necessary steps to promote strategies useful to enhance community resilience and decrease social vulnerability. 2. Climate Change Perception: The Reference Framework The same problems related to climate change knowledge and adapting arise with the study of Saxena et al. [40] which, using the case of Caribbean small island developing states, qualitatively analyzes in‐depth interviews with 35 climate change donors and project implementers. The authors found that most actors were aware of the 2 and 1.5 °C targets of the 2015 Paris Climate Agreement [41], but all were pessimistic about their achievements. Project implementers do not identify the ways by which these targets should be inserted into their adaptation projects. Moreover, there is no uniform understanding of concepts of “resilience” and “transformation”. Ruiz et al. [42] presented a study which quantified the relative strength of drivers of climate change perception, taking into account differences in the social, political, geographical, economic and educational identities of each considered community. The authors also analyzed the interactions among drivers, identifying, in this way, indirect influence pathways. The authors found that perception is directly influenced by the share of cultural factors and by the physical experience of weather change. Indirect influences are related to the level of community development, to its level of social interaction and to the spread of climate change information. Elshirbiny and Abrahamse [43] provided a new approach based on two mixed methods (an online survey and semi‐structured interviews) to examine climate change risk perceptions in a sample of the Egyptian population. Experiential factors were identified as the strongest predictors of climate change risk perception, while socio‐cultural factors the weakest predictors. In our study we also analyzed how these factors (socio‐cultural, experiential, political, geographical, economic and educational), mentioned in the last two cited works, influence the perception of the interviewed population. Lee et al. [44] proposed a narrative synthesis of the studies about youth perceptions of climate Lee et al. [44] proposed a narrative synthesis of the studies about youth perceptions of climate change, including research from 1993 to 2018. The analysis dealt with reported belief and concerns about climate change and perceptions of its causes and consequences, proposing viable solutions to climate change and notions of responsibility for implementing these. In our study, we also tried to Sustainability 2020, 12, 6985 5 of 24 capture the differences in perceptions between young and old people to verify what the level of awareness of the phenomenon was, and by which elements it was conditioned. 2. Climate Change Perception: The Reference Framework In this context, disasters are processes that arise from the intersection between nature and society. According to UNDRR [50], disasters result when a natural or human‐induced hazard affects a human settlement which is not appropriately resourced or organized to withstand the impact, and whose population is vulnerable because of poverty, exclusion, or social disadvantage. 3.1. The Study Area The present survey was carried out in Calabria (southern Italy) as a part of a Research Project funded under the Agreement on Scientific Cooperation between the National Research Council of Italy (CNR) and the University of Malta (UoM)—Biennial Programme 2018/2019. The Project focused on the risk perception and social vulnerability of population living in coastal areas subject to climate change in Malta and Calabria. These two Mediterranean regions show different geomorphological and climatic settings; however, although they have different exposure levels, they are both affected by extreme physical phenomena and climate change. In Calabria, the study area consists of the municipalities of Amantea and Lago (Cosenza province) with a total population of about 16,600 inhabitants (2019, Italian Census). The selected municipalities are located along the Tyrrhenian side of Calabria (Figure 1). The territory of the Amantea municipality, which develops from the coastline up to 420 m of elevation, is characterized by a hilly morphology. The coastal plain, as the whole Tyrrhenian side, is frequently hit by severe erosion phenomena on the beach, caused by both heavy storm surges and a decrease in the solid material carried down by the rivers [51,52]. In particular, in December 2019, following severe weather events, the Amantea coastal plain was affected by storm surges with consequent flooding and coastal erosion that threatened the population and caused damage to buildings and infrastructures (Figure 6 of 24 Sustainability 2020, 12, 6985 1A). The municipal area of Lago ranges from 142 m above sea level to 1155 m of elevation and it is mainly characterized by a mountain morphology and by a high frequency of both superficial and deep landslide phenomena of different types, shown in Figure 1B [53,54]. In the map of the seismic classification of Italian territory, the municipalities of Amantea and Lago fall into Zone 1—that is the most dangerous area, where major earthquakes may occur [55]. Figure 1. Location of the study area. (A) Amantea municipality; the photo shows coastline erosion due to the action of waves during the 2019 event. (B) Lago municipality; the photo shows a road partially obstructed by a landslide. Figure 1. Location of the study area. (A) Amantea municipality; the photo shows coastline erosion due to the action of waves during the 2019 event. (B) Lago municipality; the photo shows a road partially obstructed by a landslide. 3.1. The Study Area From a climatological point of view, Calabria is a region characterized by a typical subtropical Mediterranean climate. Given its position within the Mediterranean basin and its orography, strong contrasts are present between the two sea sides (Tyrrhenian and Ionian) of the region. The Tyrrhenian side, where the two study areas (Lago and Amantea) are located, is often subject air currents coming from the West, which cause mild winters and hot and dry summers, but also considerable orographic precipitation [56], especially in the areas at sea level, such as the territory of Amantea. The inner areas of the Tyrrhenian side, such as in the Lago territory, are characterized by colder winters, sometimes snowy, and fresher summers with some precipitation. The trend analysis carried out for the whole region [57] has shown for the territory, which includes the two study areas, a decreasing trend of rainfall on a yearly scale and opposite behaviors on monthly scale—negative in winter months and positive in summer months. Regarding temperature, regional studies on the series of Calabria did not show significant and uniform trends in maximum temperature data [58]. 3.2. Survey Method and Statistical Analysis This study employed a standardized questionnaire consisting of 33 close‐ended questions (yes/no, multiple‐choice, five‐point scale), divided into three sections. The first section included socio‐ demographic information of the participants, their gender, age, municipality of origin, employment status and education. Because the questionnaire was part of a larger study that focused on risk perception and social vulnerability of population in coastal areas subject to climate change, this section contained additional questions about some social characteristics that may make people in general more vulnerable. The socio‐demographic information comprised the presence of family members under 14 and over 65 years old, the presence of family members not functionally 7 of 24 Sustainability 2020, 12, 6985 independent, place of living (urban/rural), year of construction and type of dwelling, time taken to reach the work/study place and social problems present in the community where respondents live. Under the scope of this study, only the socio‐demographic variables, such as respondent gender, age, education, occupation and place of living were taken into account for the analysis. The second section of the questionnaire contained thirteen questions aimed at measuring the respondents’ perceptions of climate change. This section included questions about: (i) perception and awareness about climate change; (ii) level of information about this phenomenon; (iii) personal actions taken to fight climate change and opinion about actions taken by national and international governments; (iv) perceived concern about impacts and effects of climate change. Although most people today have at least heard of climate change, we found it useful to include in this second section, before the questions, a simple and clear description of the phenomenon. The third section of the questionnaire encompasses five questions probing the participants’ resilience to extreme natural events. This section explores: (i) perception of exposure to extreme natural events and related damages of respondent in the territory where they live; (ii) their behavior and reaction if involved by an extreme natural event; (iii) trust in the local government’s capacity. Table 1 shows an overview of the survey together with the previous studies in which the research questions have been already addressed. Table 1. Overview of the survey. Research Questions Specific Issues Example Studies Is the climate change perceived by the population? 3.2. Survey Method and Statistical Analysis Perception and awareness about climate change [22,37,59] Concern about impacts and effects of climate change [37,59] Perception of the personal level of general information about climate change based on the principal sources for each citizen (television, digital media, journals, experts, etc.) [22,36] Personal action and behavior taken to fight climate change [37,59] What is the degree of the community resilience to extreme natural events and climate change? Perception of exposure to extreme natural events [60,61] Behaviour if an extreme event occurs [62,63] Trust in actions taken by local, national and global policy makers [60,61] Table 1. Overview of the survey. In September 2019, the questionnaire was submitted to 300 citizens, of which 100 were inhabitants of Lago and 200 inhabitants of Amantea. The method for selecting participants was non‐ proportional quota sampling (based on gender and age). The interviews were conducted face‐to‐face and, as far as the data capture is concerned, the CAPI (Computer Assisted Personal Interview) technique was used. Prior to the start of the interview, participants were informed about the scientific purposes of the questionnaire and reassured that their identities were reserved. All participants gave their voluntary and informed consent before the interviews. A statistical analysis was carried out to individuate the baseline linkages between the social and demographic features of the population (first section) and the perceived climate change (second section), as well as the relationships between the same anthropological features with the resilience capacity of individuals towards extreme natural events (third section), as comes out by the sections of the questionnaire. The method of correlation was considered as preferable to reach these goals. In particular, the principle individuated by Spearman [64], in which derivation originates from the Pearson’s general correlation, as follows, was used to correlate the data: 𝑟ൌ 𝑆௫௬ ඥ𝑆௫మൈ𝑆௬మ (1) 𝑟ൌ 𝑆௫௬ ඥ𝑆௫మൈ𝑆௬మ (1) Sustainability 2020, 12, 6985 8 of 24 where r is the required correlation between a certain pair of features for which the intensity of the linkage is to be found (levels of significance), x and y are the deviation of any pair of characteristics from their mean and Sxy is their sum; Sx2 and Sy2 are the sum of the squares of all the values of x and y. 3.2. Survey Method and Statistical Analysis Given the features of the collected data, which are presented in the shape of rank (dichotomous or categorical), the (1) can be rewritten in the following form to take into account certain features that are not purely quantitatively comparable: 𝑟ൌ 1 𝑛∑ ቀ൫𝑅ሺ𝑥௜ሻെ𝑅ሺ𝑥ሻ തതതതതത൯ൈ൫𝑅ሺ𝑦௜ሻെ𝑅ሺ𝑦ሻ തതതതതത൯ቁ ௡ ௜ୀଵ ටቀ1 𝑛∑ ൫𝑅ሺ𝑥௜ሻെ𝑅ሺ𝑥ሻ തതതതതത൯ ଶ ௡ ௜ୀଵ ቁൈቀ1 𝑛∑ ൫𝑅ሺ𝑦௜ሻെ𝑅ሺ𝑦ሻ തതതതതത൯ ଶ ௡ ௜ୀଵ ቁ (2) (2) where 𝑅ሺ𝑥ሻ and 𝑅ሺ𝑦ሻ are the ranks and 𝑅ሺ𝑥ሻ തതതതതത and 𝑅ሺ𝑦ሻ തതതതതത are the mean values of the ranks [65]. The use of the Spearman correlation adjusted for the rank variables allowed us to control for the not directly comparable parameters (for instance, certain perceptions about natural events expressed on a Likert scale of evaluation are not directly linkable to the employment status or the age class, but they need smoothing) and this increases the goodness of fit of the coefficients. Moreover, a non‐ parametric and non‐linear approach, such as the Spearman’s rank correlation, is regarded as preferable because it is also suited for case studies that consider different administrative territories as part of the same correlation analysis [66]. The results reported in the following sections only consider those coefficients that are above the 95% of statistical significance (p < 0.05). 4.2.1. Perception and Awareness of Climate Change Respondents’ awareness of climate change was explored by asking “Do you believe that climate change is in effect globally?”. In total, 94% of the sample answered “yes”, while only 4% answered “I don’t know” and 2% “no”. With the aim to investigate the aspects related to knowledge about climate change, the respondents were invited to give their opinion on the main cause of this phenomenon. More than half of the respondents (59%) thought that climate change is caused by the excessive use of fossil fuels and air pollution; then they indicated deforestation (33%), I don’t know (4%), none of these (3%) and livestock farming (1%). Two multiple‐choice questions focused on which phenomena, in their opinion, were associated to climate change on both global and local scales (Figure 2). As regards the global phenomena, the most frequent answer was “melting of ice and sea level rise” (186 answers) followed by “increases of temperatures” (161 answers), “ozone hole and greenhouse effect” (134 answers), “increase in the frequency of extreme events such as hurricanes, tropical storms, floods, heat and cold waves” (83), “loss of biodiversity and loss of animal and plant species” (66), “drought and desertification” (63) and “increase of immigrations and social conflicts” (26). The results of the Spearman’s correlation show positive linkages between the age range 18–39 years and the answers “melting of ice and sea level rise” (r = 0.1334) and “loss of biodiversity and loss of animal and plant species” (r = 0.1354), while for the answer “increase of immigrations and social conflicts” a positive correlation was in both the age ranges 40–49 and more than 60 (r = 0.1241, for each of the age range). Regarding the same question, it must be highlighted that the phenomena of “drought and desertification” were mostly perceived by those who lived in “rural areas” (r = 0.2546). As concerns the local phenomena, the most frequent answer was “increase of heavy rainfall storms with impacts on persons and properties” (139 answers) followed by “shortage of water resources” (122 answers), “growth of fires” (120), “decrease in agriculture and fisheries productions” (119), “increase of immigration” (53), “economic damages and negative effects on local tourism” (52) and “diffusion of diseases” (41). 4.2.1. Perception and Awareness of Climate Change Women associated the “shortage of water resources” with climate change (r = 0.1218), while respondents aged 40–59 and over 60 years, housewives and those with low levels of education (secondary school) associated climate change with the local phenomenon of “diffusion of diseases” (r = 0.1588; r = 0.1271 and r = 0.1368, respectively). Figure 2. Questions focused on which phenomena, in respondents’ opinions, are associated with climate change on both global (A) and local (B) scales. 0 20 40 60 80 100 120 140 160 180 200 Increases of temperatures Melting of ice and sea level rise Ozone hole and greenhouse effect Increase in the frequency of extreme events such as… Loss of biodiversity and loss of animal and plant species Increase of immigrations and social conflicts Drought and desertification I don’t know Respondents A 0 20 40 60 80 100 120 140 160 180 200 Growth of fires Decrease in agriculture and fisheries productions Shortage of water resources Diffusion of diseases Increase of immigration Economic damages and negative effects on local tourism Increase of heavy rainfall storms with impacts on persons… I don’t know Respondents B Figure 2. Questions focused on which phenomena, in respondents’ opinions, are associated with climate change on both global (A) and local (B) scales. 0 20 40 60 80 100 120 140 160 180 200 Increases of temperatures Melting of ice and sea level rise Ozone hole and greenhouse effect Increase in the frequency of extreme events such as… Loss of biodiversity and loss of animal and plant species Increase of immigrations and social conflicts Drought and desertification I don’t know Respondents A 0 20 40 60 80 100 120 140 160 180 200 Growth of fires Decrease in agriculture and fisheries productions Shortage of water resources Diffusion of diseases Increase of immigration Economic damages and negative effects on local tourism Increase of heavy rainfall storms with impacts on persons… I don’t know Respondents B Figure 2. Questions focused on which phenomena, in respondents’ opinions, are associated with climate change on both global (A) and local (B) scales. 4.2.2. Level of Information on Climate Change, Personal Actions taken to Fight Climate Change In order to analyze the level of information on climate change, we firstly asked respondents if they themselves felt informed about it. About 44% felt fairly informed about climate change, while 38% felt uninformed. 4.2. Social Perception of Climate Change 4.2. Social Perception of Climate Change 4.2. Social Perception of Climate Change 4.2. Social Perception of Climate Change 4.1. Characterization of the Sample 4.1. Characterization of the Sample A total of 300 respondents took part in this study. Table 2 shows the socio‐demographic characteristics of the total sample. For the results presentation, the answers, including a 5‐point scale, from 1 (min) to 5 (max), were grouped into three categories (1 + 2 = low; 3 = medium; 4 + 5 = high). Table 2. Socio‐demographic characteristic of the respondents (n = 300). Table 2. Socio‐demographic characteristic of the respondents (n = 300 Socio‐Demographic Characteristic Percentage (%) Gender Female 52 Male 48 Age class 18–39 31 40–59 34 >60 35 Education No title 1 Primary school 3 Middle school 15 High school 49 Higher education 32 Occupation Unemployed 11 Student 7 Retired 22 Homemakers 7 Employee 40 Business owner/freelance 13 Socio‐Demographic Characteristic Percentage (%) 9 of 24 9 of 24 Sustainability 2020, 12, 6985 4.2.1. Perception and Awareness of Climate Change Regarding the multiple‐choice question concerning the sources from which they obtained climate change‐related information, the most frequent answers were “TV” (232 answers) and “internet and social network” (184). Besides, the participants also found relevant information in “newspapers, books, magazines” (125 answers) followed by “relatives and friends” 10 of 24 Sustainability 2020, 12, 6985 (40), and “university and experts” (28). As regards the respondents’ occupational statuses, the results show a positive correlation between “student” and “the use of the internet and social network” (r = 0.1374) as well as “university and experts” (r = 0.1814) as sources of information on climate change. Conversely, there is a negative correlation between retirees and the sources “internet and social network” and “university and experts” (r = −0.2470 and r = −0.1409, respectively) as they mostly receive information from “TV” (r = 0.1301). Respondents with low levels of education (primary school) catch information by “relatives and friends” (r = 0.2003), while respondents with higher education (tertiary level) are more inclined to “use internet and social networks” (r = 0.1391), “newspapers, books, magazines” (r = 0.1241), and to receive information from “university and experts” (r = 0.1457). The respondents were invited to give their opinions (personal judgment) on a number of statements linked to the problem of climate change on a 5‐level scale merged into three categories (low, medium, high; Figure 3). Around 75% of respondents considered the statement that “climate changes are linked to the economic policies of the most industrialized Countries” very true, while 85% considered the statement that “climate change is a journalistic mount” with low reliability. Regarding the assertion “climate change is linked to natural evolution of the Earth”, 45% of respondents considered this statement wrong, while 63% thought that “climate change is also tied to daily lifestyles”. The assertion that “climate change is linked to natural evolution of the Earth” is positively correlated with lower educated respondents (primary school) (r = 0.1788) and negatively correlated to those with higher education (tertiary level) (r = −0.1485). The assertion that “climate change is a journalistic mount” was negatively correlated to the youngest group of respondents (age range 18–39 years) (r = −0.1202) and with higher educated individuals (r = −0.1115). Figure 3. Opinion (personal judgment) of the respondents on a number of statements linked to the problem of climate change. Figure 3. 4.2.1. Perception and Awareness of Climate Change Opinion (personal judgment) of the respondents on a number of statements linked to the problem of climate change. In relation to public opinion over the activities of the national and international governments to tackle climate change [22], the majority of respondents (68%) thought that national and international political actions to face this problem are insufficient. According to 19% of respondents, political actions are taking place, while 13% had no opinion on this matter. We asked respondents to indicate at least two common actions taken by them (or by their family members) to face climate change (Figure 4). 11 of 24 Sustainability 2020, 12, 6985 Figure 4. Common actions taken by respondents or by their family members to face climate change. 0 20 40 60 80 100 120 140 160 180 I use heating systems based on the use of renewable resources I have improved the insulation of my house I plant trees and care for the plants in my garden I often use alternative transport options to the car I often discuss with friends, relatives and local politicians on how to reduce climate… I always carefully carry out waste separation I daily seek to limit the use of plastic material In my daily food intake I have reduced or excluded the consumption of beef I do not take any actions to reduce climate change Respondents Figure 4. Common actions taken by respondents or by their family members to face climate change. 0 20 40 60 80 100 120 140 160 180 I use heating systems based on the use of renewable resources I have improved the insulation of my house I plant trees and care for the plants in my garden I often use alternative transport options to the car I often discuss with friends, relatives and local politicians on how to reduce climate… I always carefully carry out waste separation I daily seek to limit the use of plastic material In my daily food intake I have reduced or excluded the consumption of beef I do not take any actions to reduce climate change Respondents Figure 4. Common actions taken by respondents or by their family members to face climate change. 4.2.1. Perception and Awareness of Climate Change Among the most common actions made by the respondents were “I always carefully carry out waste separation” (168), “I plant trees and care for the plants in my garden“ (101), “I daily seek to limit the use of plastic material” (72), followed by “I have improved the insulation of my house” (62), “I often use alternative transport options to the car” (44) and “I often discuss with friends, relatives and local politicians on how to reduce climate change” (30). Only eight people declared they were not acting to face climate change. In terms of respondents’ ages, the highest willingness to carry out waste separation was found among people aged 40–59 and more than 60 (r = 0.1377, for each range), while respondents aged 18–39 were those who most often using alternative transport options to the car (r = 0.1330). The answer option “I daily seek to limit the use of plastic material” was (positively) correlated with respondents holding a post‐secondary education (r = 0.1218), while the statement “I often talk with friends, relatives and local politicians on how to reduce climate change” was positively correlated with the higher educated respondents (r = 0.1259). The respondents who were “unemployed” and with “no title” were those who did not take action to face climate change (r = 0.1366 and r = 0.1913, respectively). Respondents who thought that national and international governments are not doing enough to fight climate change were generally more inclined to use heating systems based on renewable sources to face climate change (r = 0.1715). 4.2.3. Concern about Impacts and Effects of Climate Change and Psychological Attitudes towards Climate Change 4.2.3. Concern about Impacts and Effects of Climate Change and Psychological Attitudes towards Climate Change The most common answers to the multiple‐choice question “You are mainly concerned about the impacts of climate change on …” were “population of all over the world” and “nature and environment”, with 171 answers for each of the two options, followed by “myself, my family and the community where I live” (103 answers) and “my nation: Italy” (35). Differences in concern about the impacts of climate change were observed over the sample. The group of respondents aged 18–39 years and the respondents living in rural areas were most concerned with the impacts of climate change on nature and environment (r = 0.1834 and r = 0.2256, respectively). Individuals with higher education (tertiary) were concerned about the population of the world (r = 0.1254). We asked respondents when, in their opinion, the effects of climate change both globally and locally will begin to become evident. A large part of respondents (70%) thought that the effects of climate change were already happening, while for 18% the effects will be evident in next 50 years. For only 5%, after the next 50 years. Individuals in the age range 18–39 (r = 0.1357) and those with post‐secondary school level (r = 0.1281) were more inclined to think that the effects of climate change are already happening. In order to analyze some psychological attitudes (fear, trust, frustration) towards climate change, respondents were asked to give their opinions on a series of statements (Figure 5). In order to analyze some psychological attitudes (fear, trust, frustration) towards climate change, respondents were asked to give their opinions on a series of statements (Figure 5). 12 of 24 Sustainability 2020, 12, 6985 Figure 5. Questions about psychological attitudes (fear, trust, frustration) of the respondents towards climate change. 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% I am frustrated because not enough is being done about climate change I am sure that the correct strategies are being implemented to slow down climate… I am confident that we will succeed to slow down climate change I am afraid of the possible effects of climate change I am not very concerned with climate change I do not known how to respond Figure 5. Questions about psychological attitudes (fear, trust, frustration) of the respondents towards climate change. 4.2.3. Concern about Impacts and Effects of Climate Change and Psychological Attitudes towards Climate Change In total, 40% of respondents agreed with the statement “I am frustrated because not enough is being done about climate change” and 31% of them with the statement “I am afraid of the possible effects of climate change”. Only 20% agreed with the statement “I am confident that we will succeed to slow down climate change”. Respondents aged 18–39 (r = 0.1638) and those with tertiary education (r = 0.1530) were more afraid of the possible effects of climate change, unlike retirees who are less afraid of the possible effects (r = −0.1601). Respondents with post‐secondary school level were more frustrated because they thought that not enough is being done to face climate change (r = 0.1239). y g g g g The latter are, not surprisingly, more prone to think that national and international governments are not doing enough to fight climate change (r = 0.1906) than those who are confident that climate change will slow down (r = −0.1532). 4.3. Community Resilience to Extreme Natural Events 4.3. Community Resilience to Extreme Natural Events 4.3.1. Perception of Exposure to Extreme Natural Events and Related Damages 4.3.1. Perception of Exposure to Extreme Natural Events and Related Damages Figure 6 shows the answers to the question “How do you think you are personally exposed to each of these events in the territory where you live?” on a 5‐level scale merged into three categories, separately for the two sub‐samples of Lago and Amantea. Figure 6. Questions that examine to what extent the respondents perceive the level of exposure to natural events. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Earthquake Landslide Flood Coastal erosion Volcanic eruption Hurricane Tsunami Heat waves Cold wave Respondents of Lago low medium high I don't know 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Earthquake Landslide Flood Coastal erosion Volcanic eruption Hurricane Tsunami Heat waves Cold wave Respondents of Amantea low medium high I don't know 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Earthquake Landslide Flood Coastal erosion Volcanic eruption Hurricane Tsunami Heat waves Cold wave Respondents of Amantea low medium high I don't know Figure 6. Questions that examine to what extent the respondents perceive the level of exposure to natural events. Sustainability 2020, 12, 6985 13 of 24 As shown in Figure 6, the answers are not uniform between the two groups, with the exception regarding the exposure to earthquakes—indeed, 71% of Amantea’s respondents and 57% of Lago’s respondents perceived the level of exposure to earthquake as “high”. Lago’s citizens perceived higher exposure to landslides and heat waves than Amantea’s citizens, while those interviewed from Amantea perceived higher exposure to floods, coastal erosion, volcanic eruptions, hurricane, tsunami, and heat waves, compared to those interviewed from Lago. Respondents were asked (by means of a multiple‐choice question) to indicate the damage they fear most for each of the extreme events, as listed in the previous question, which could occur in their territory. For this question too, there was difference between the groups (Figure 7). The majority of Amantea’s respondents feared “physical injuries to myself or my family” and “property damage” as a consequence of extreme events, such as earthquakes, landslides, floods, coastal erosion, volcanic eruptions, hurricanes and tsunami. 4.3.2. Behavior if an Extreme Event Occurs Respondents were asked to evaluate their behavior when an extreme event occurs in the territory they live in. Around 40% of respondents provided the answer “I try to get information about the situation through TV, social networks, internet, radio, neighbors, experts”, followed by “ I trust in God” (19%), “I don’t know what I should do” (15%), “I know what I should do” (13%), “I do not know how to reply” (11%), and “I wait the intervention of the authorities” (3%). Women, housewives, and lower educated respondents (primary school) are more willing to rely on God if they are involved in an extreme natural event (r = 0.1210; r = 0.1806; r = 0.1157, respectively), while the respondents with no level of education are more inclined to not know what they should do (r = 0.1432). As regards higher educated respondents (tertiary), coefficients show a positive correlation with the answer “I try to get information about the situation through TV, social networks, internet, radio, neighbors, experts” (r = 0.1242) and a negative correlation with the answer “I trust in God” (r = −0.1666). The behavior “I don’t know what I should do”, in the case of an extreme event, is positively correlated to the answer “I do not take actions to reduce climate change” (r = 0.1593), while for the behavior “I wait the intervention of the authorities” a positive correlation (r = 0.1517) with the answer “I try to get information about the situation through TV, social networks, internet, radio, neighbors, experts” was detected. As for the multiple‐choice question about respondents’ reactions in the case of an extreme event occurring in the territory they live in, the most frequent answer was “I help first my family and others who are in difficulty” (226 answers), followed by “I rush to find an escape route” (72), “I do not panic” (61) and “I panic” (23). As regards the respondents’ occupational statuses, there was a positive correlation between “students” that “do not panic” (r = 0.1241) and between “retirees” that “help first their family and others who are in difficulty” (r = 0.1168). Respondents who “do not panic” were considerably more inclined to seek to limit the daily use of plastic material to face climate change (r = 0.1288) and they are less willing to help family and others (r = −0.1433). 4.3.2. Behavior if an Extreme Event Occurs Moreover, respondents whose reactions were “I help first my family and others who are in difficulty” (r = 0.1692) and “I do not panic” (r = 0.1226) were more inclined “to try to get information about the situation through TV, social networks, internet, radio, neighbors and experts”. Contrariwise, those who “panic”, often also said that “they would not know what to do” (r = 0.1555). Furthermore, there is a strong positive correlation between individuals who were unable to respond on the reaction that they might have if an extreme event should occur in their place of belonging and those who prefered to not respond about the behavior to be adopted (r = 0.3256). 4.3.3. Trust in Local Government Capacity In order to explore the level of safety perceived by those interviewed, individuals were asked if they would trust the local government capacity to respond to a disaster caused by an extreme natural event. The data obtained show that more than a half of the respondents did not trust local government capacity (55%), while 22% trusted and 23% did not know. Women had more trust in local government capacity to face disasters caused by extreme natural events than men (r = 0.1525). Respondents who did not trust local government capacity if an extreme event should occur in the territory they live in were more inclined to think that national and international political actions to face climate change are insufficient (r = 0.1387). Conversely, those who trusted local government capacity think that political actions are on track to face this problem (r = 0.1318). 4.3.1. Perception of Exposure to Extreme Natural Events and Related Damages Otherwise, the respondents of Lago feared “physical injuries to myself or my family” and “property damage” as a consequence of earthquakes, landslides, floods, heat wave and cold wave, while a relatively high number of respondents answered “I don’t know” for coastal erosion, volcanic eruption, tsunami and hurricanes. Figure 7. Damage that respondents fear most for each of the extreme events listed in Figure 6. 0 10 20 30 40 50 60 70 80 90 100 Physical injuries to myself or my family Property damage Damage/unusability of the workspace Psychological problems I don't know Respondents of Lago Earthquake landslide Flood Coastal erosion Volcanic eruption Hurricane Tsunami Heat wave Cold wave 0 10 20 30 40 50 60 70 80 90 100 Physical injuries to myself or my family Property damage Damage/unusability of the workspace Psychological problems I don't know Respondents of Lago Earthquake landslide Flood Coastal erosion Volcanic eruption Hurricane Tsunami Heat wave Cold wave Figure 7. Damage that respondents fear most for each of the extreme events listed in Figure 6. Figure 7. Damage that respondents fear most for each of the extreme events listed in Figure 6. Sustainability 2020, 12, 6985 14 of 24 14 of 24 5.1. Climate Change Perception Natural and social scientists have been investigating for years how people perceive climate change, what aspects they know about this phenomenon and what they ignore, what dimensions frighten them and what the behavioral responses have to be to cope with it. In this context, our study shows that respondents’ awareness of the existence of climate change, globally, is high. Various studies highlight how, in some countries, this climate change awareness 15 of 24 Sustainability 2020, 12, 6985 has not always been so high. For example, Leiserowitz [59] noted that there is a general tendency in the United States to underestimate climate change. This is because citizens, despite being aware of the problem, see it temporally distant, a long‐term event and, therefore, unworthy of great concern. This type of attitude is well explored by Bubeck et al. [67] with the formulation of the “motivational hypothesis” theory— individuals implement measures to reduce their vulnerability only when they really feel exposed to a high risk [6]. As for the causes of climate change, the majority of citizens, in our study, indicated as the first cause the excessive use of fossil fuels and air pollution and, subsequently, deforestation. The population, therefore, has attributed the responsibility for the ongoing climate change to anthropic activities. This increase in the awareness of the importance of anthropogenic factors, as influential on current climate change, is probably due to the worldwide media effect of the various events promoted by Greta Thunberg in 2019, before the arrival of Covid‐19. Indeed, these initiatives have raised public awareness and, above all, the awareness of young people, on the issue of climate change and have resulted in a social movement against climate change and human activity, including political decisions, the effects of which contribute to enhancing global warming. By examining the phenomena associated with climate change on a global scale, according to the respondents, the most frequent responses were “the melting of glaciers and rising sea levels” and “temperature rise”. This is surely due, in part, to the influence of the mass media, as we have already highlighted, which often report on the withdrawal of glaciers within the framework of climate change as first news and first images. However, as we mentioned in Section 2, this specifically depends on the influence of the space–time dimension and on the geographical concepts of relational and relative distance, later discussed. 5.1. Climate Change Perception In this sense, elastic and with “multiple rationalities” analytical models must be urged to be elaborated, rather than univocal and inflexible ones [68]. The rise in temperatures is the other global phenomenon most associated with climate change as the results of this survey supported. The weather, daily or seasonal meteorology, indeed, constitutes a second image to which common sense associates with climate change. In third place, as a phenomenon associated with climate change, there is the “ozone hole and the greenhouse effect”, also perceived among the first places in the study by Bostrom and Fischhoff [37]. Tracing climate change to the phenomenon of the destruction of the ozone layer means associating two phenomena that are not directly connected, but which are linked by the impact that human behavior has had on the environment on a global scale [4]. This association, as Riva [4] points out, although constituting an error from a scientific point of view, demonstrates the ability of common sense to give meaning to complex phenomena such as those under consideration, extrapolating parts of the problem, such as the anthropogenic effect on the environment, and leaving out others, such as the different characteristics related to the greenhouse effect and the ozone hole. On the other hand, if we consider that the results of our study showed a significant correlation between the age group 18–39 years and the answers “melting of glaciers and rising sea levels” and “loss of biodiversity and disappearance of animals and plants species”, while for the response “increase in immigration and social conflicts” a significant correlation was found with groups aged 40–59 and over 60 years, the reasons for this are closely connected with what is written above. It is plausible that those individuals with higher education perceive the melting of glaciers and the loss of biodiversity more, while elderly and lower educated people perceive the increase in immigration more, a phenomenon closer to space and time rather than the “distant phenomenon” of the melting glaciers. The rise in temperatures is the other global phenomenon most associated with climate change as the results of this survey highlighted. Those who live in rural places perceive the association with the phenomena of drought and desertification more, likewise due to a matter of greater spatio‐ temporal and cultural proximity to the problem. 5.1. Climate Change Perception This shows how important it is to spread the correct messages of information on climate change and risk communication through TV and social media, avoiding any distortions of reality which, thus, would affect citizens’ perceptions of the phenomenon. Furthermore, as regards the analysis of the discursive constructions associated with climate change, the majority of respondents consider the statement that “climate change is linked to the economic policies of the most industrialized countries” “very true”, and, the statement that “climate change is a journalistic mount” with low reliability. Those with lower education judge truthful discursive patterns that are, in fact, false as “climate change is considered to be totally natural phenomena” and “climate change is a journalistic mount”. In any case, it would be necessary to strengthen the teaching of geography in the Italian school system, for whom programs in which the study of the climate and its characteristics is foreseen. Instead, those who fall into the 18–39 age group and have a degree believe that the patterns “climate changes are linked to the natural evolution of the planet” and “climate changes are a journalistic mount” are false. The perception of retirees, who believe that the pattern “climate change is linked to the economic policies of the most industrialized countries” is strictly connected, probably, to the nostalgic sentiment of elderly people towards pre‐globalized society, which can also be found in other testimonies [76]. In relation to political actions to mitigate climate change, the majority of respondents believe that national and international political actions taken to tackle this problem are inadequate and insufficient. Lorenzoni et al. [36], with respect to the barriers perceived at a social level, first of all, in their research conducted in the United Kingdom, identify the perception of a low political commitment at a national and international level. With respect to the actions taken by the population to fight climate change, the most frequently reported answer was “I always carry out waste separation with great care”. It is easy to see the willingness to implement practices, such as the recycling of household waste, even if they are not strictly linked to the problem of climate change, as Norton and Leaman [34] point out. The second‐ most referred answer by the respondents was “I plant trees and take care of the plants in my garden”. 5.1. Climate Change Perception As for the association of local phenomena with climate change, the greater perception of the increase in heavy rains with impacts on people and property shown by the interviewees is probably due to the increase in sudden storm showers that frequently hit the Calabrian territory and the study area, causing direct and indirect losses. the association with the lack of water resources which, in winter and summer, is very frequent in the Calabrian territory is significant; the statistical correlation Sustainability 2020, 12, 6985 16 of 24 of this issue with the female sex is also of anthropological importance. In fact, Calabria has always been marked in its history by a “geography of thirst”, to which corresponds a “geography of waters” [69]. During the various pilgrimages, thirst was overcome by a perfect knowledge of the places and their waters. All places were marked, upon leaving or entering, by fountains where women went and where people, returning from work, stopped. The writer Corrado Alvaro repeatedly returns to his works on women who go in search of water at fountains outside their places, often in inaccessible and distant locations [70–72]. The association with the answer “increase in fires” could be due to the numerous forest fires which have hit the Calabrian territory in recent years. In particular, according to the report of the Calabrian Civil Protection, in the first nine months of 2017, in Calabria there were recorded more than 800 forest fires and the province most affected was Cosenza, with 413 square kilometers of area affected by fires. The perception of the spread of diseases associated with climate change was an absolutely justified and proven fear from the arrival of Covid‐19 which, although not directly connected to climate change, certainly has significant relationships with the protection of the environment, natural ecosystems, primary forests and biodiversity [73–75]. Data on the perception of information levels about climate change do not indicate a significant gap between people who feel informed and those who do not feel informed. The most frequently reported sources of information on climate change are television, internet and social networks. Those who are younger and have a university degree use newspapers, books and magazines and the internet as sources of information, and are also informed through school and university institutions. Retirees and those with lower education get more information via TV and family and friends. 5.2. Community Resilience to Extreme Natural Events and Climate Change The majority of respondents show greater concern about the impacts of climate change on the world population and on nature and the environment than the impacts on themselves, their family, the community and the country they live in. The global dimension of the phenomenon is perceived mostly by younger and higher educated people, with greater concern for the impacts on nature and the environment by those living in rural areas. The perception of the global dimension of the phenomenon, as previously mentioned, is probably related to the representation of the spatio‐ temporal diffusion of its effects, the cultural background and the media communication that has recently centralized the attention to the issue of climate change. According to the majority of respondents, the effects of climate change are already manifesting. This is due, yet again, to media communication on climate change that has been more frequent, recently, following the demonstrations organized by Greta Thunberg that have brought the theme of climate change to the attention of public opinion and politics. However, this is also due to the frequency of some extreme natural events, such as landslides and floods that have occurred locally in recent years and have affected the Calabrian territory. In addition, people aged 18–39, and those with higher education, are more afraid of the possible effects of climate change and are frustrated that not enough is being done to mitigate the effects. Retirees, on the other hand, are less afraid of the effects. Evidently, also considering the statistical correlations identified, young people are better prepared on the problem and, consequently, they are more worried because the national and international political class is not carrying out sufficient actions to slow down climate change. If we analyze the perception of exposure to extreme natural events, the perception of exposure to earthquakes by respondents from Lago and Amantea is high on both. These data, in the context of this geographical study area, confirm the high perception of risk exposure, also found in other studies carried out in different areas of Calabria—Pollino [63], Aiello Calabro [78], Costa degli Dei [60,79], Maierato [61,80]. As we have pointed out, the two municipalities are located in an area with high seismic hazard. 5.1. Climate Change Perception Additionally, also in the study of Leiserowitz [22], the latter action falls second and represents the symbol of pro‐environmental action par excellence. Even “I try to limit the use of plastic in everyday life” represents an important action, since the production, incineration and disposal of plastic 17 of 24 Sustainability 2020, 12, 6985 contributes dramatically to the increase in CO2 in the atmosphere. On this point, economic factors may have influenced the perception of respondents. Indeed, in the same period in which the interviews were carried on, the Italian government adopted the “plastic tax”, a tax on the consumption of single‐use items (MACSI) made with the use of plastic materials. The other responses given by the respondents, in order of choice, are: “I have improved the insulation of my home” and “I often use alternative transport options to the car”. In some surveys in the United Kingdom and in the United States it likewise emerges how citizens are inclined to recycle household waste more and to improve domestic energy efficiency, rather than exhibiting a willingness to change their transport habits [77]. In this respect, institutional factors, such as the absence of an efficient public transport network in Calabria, can certainly hinder the implementation of low environmental impact behaviors. Discussing the reduction in climate change with friends, family and local politicians—which is another frequently answered option—certainly helps to sensitize local communities on this issue and to find solutions on a local scale to fight climate change. Few respondents said they do not take action to address climate change. Those who are unemployed and have no qualifications, do not perform actions to reduce climate change, probably due to the lack of sensitivity and knowledge on the matter. On the other hand, young students or those who have a degree, carry out various virtuous actions compared to elderly people, who mainly deal with separate collection. 5.2. Community Resilience to Extreme Natural Events and Climate Change The perception of volcanic and tsunami risk of the Lago’s respondents is high because the study area, located along the Tyrrhenian coast of Calabria, not far from the Stromboli Volcano (Aeolian Islands), was involved in December 2002 in an emergency phase due to a tsunami risk for a landslide triggered by the eruption of the volcano [81]. The highest perception of Amantea’s respondents about the coastal erosion and the highest perception of landslide risk of the Lago’s respondents are correct, as they correspond to the main peculiarities of the two territories that are subject to these natural hazards. Sustainability 2020, 12, 6985 18 of 24 18 of 24 The behavior of the population at the occurrence of extreme natural events was examined. The majority of respondents try to obtain information on the situation via TV, social networks, internet, radio, neighbors and experts. This is followed by the response “I trust in God”. In particular, there is a significant statistical correlation of this response with the female sex, housewives and those with lower education. Several writers and scholars have argued about the relationship between women and religion in Calabria. The Calabrian population is historically linked to a religiosity, also due to all the various famines and disasters connected to extreme natural events or diseases, such as malaria, that have historically affected the territory. The feeling of devotion of women has always had a symbolic and resilient function that recalls the defense against all the calamities to which the Calabrian territory has been subjected over the centuries. In addition, various resilient behaviors are assumed by those who have adequate education, such as waiting for the authorities to intervene and looking for information on the event. In addition, we can note that some responses indicating poorly resilient behavior are significantly related between them, such as, for example, “I do not know what I should do” and “I do not take action to reduce climate change”. Similarly, responses indicating fairly resilient behaviors are likewise significantly related between them, such as, for instance, “I try to get information on the situation via TV, social networks, etc.” and “I await the intervention of the authorities”. 5.2. Community Resilience to Extreme Natural Events and Climate Change As for the reactions of the interviewees in the case of extreme events in the area they live in, the most frequently reported response was “I first help my family and others who are in difficulty”, followed by “I rush to find a way escape”. The latter reaction, particularly inadequate for some extreme events, was already found in other previous studies on the perception of seismic risk in Calabria [63]. Furthermore, the positive and significant correlation between the “students” category and the “I do not panic” response indicates resilient behavior by younger people with an adequate level of education. The positive correlation between “retirees” and the answer “in the first place I help my family and others who are in difficulty” is certainly due to the caring and protective behavior of the elderly people towards their family and loved ones. Resilient behaviors are accompanied by a positive adaptation and by the competence that refers to the ability to function effectively in the world in relation to some expectations that are based on the behavioral rules typical of the reference context [82–84]. In this study, therefore, we note how positive adaptation and competence are associated with resilient behaviors during extreme natural events. From this we understand why different positive resilient behaviors are accompanied by other equally resilient, competent and adaptive behaviors and reactions. We also note the importance of familism and education as socio‐cultural factors that influence the increase in community resilience [85,86]. Instead, the lack of trust in local administrators, emerging from this research, does not constitute a factor of community resilience [87–89]. Additionally, in another study on the perception of climate change conducted in Sweden, homeowners said their trust in local policymakers is low when it comes to managing crises [90]. However, other studies on the perception of geo‐hydrological risk in Calabria have also revealed the lack of trust of citizens towards local policymakers [60,61]. In some studies, gender difference has been found to have a considerable influence on risk perception [91]; even in this study, women have greater confidence than men in local government authorities. The behavior of Calabrian women seems consistent with their greater vulnerability, so they give greater trust to local institutions, so as to hope for greater protection in case of need [92,93]. 5.3. Practical Implications From this study, various practical implications, directly linked to the study of geographic sciences, emerge. The space–time dimension mentioned in this paper belongs to epistemology of geography. It is not possible to do geographical research without investigating the facts in their temporal evolution, without taking into account the effect of time in spatial flows and relationships and without considering the role of some historical matrices in contemporary territorial reality [94]. For instance, the first phenomenon that citizens, in this survey, associate with climate change is the melting of glaciers. As we have already highlighted, this is probably due to the images transmitted by the mass media, but it is also linked to a very important concept in geographical studies: that of space. The absolute space, in geography, does not exist. Geographical space depends on which phenomena are observed and on which tools are used to construct a geographical “representation” or “analysis”. Therefore, space is “relative”. The “relative” distance (time, cost) and the “relational” distance (cultural, linguistic, institutional, etc.) often do not coincide with the geometric distance between two places. Relative space is perceptible, but it is not always objectively determinable, and in any case, it varies over time. In this perspective, the relative space can be seen as a social product, which reflects the activities and interrelations between them [95]. Therefore, the reasons why citizens have associated the melting of glaciers with climate change refer to the relative and relational distance with this phenomenon, not to the geometric one. Moreover, from the disciplinary perspective of geography, the reflection around climate change epistemologically implies different issues, some of which appear to be central, such as the interaction between human beings and natural elements, the sustainability of anthropization processes, the possibilities of action that translate into social, political and economic choices and in the related (and possible) intervention policies [45,96]. As Sabato [45] points out, in Italy the contraction of the hours in geography teaching in most school curricula, has actually created a gap that students perceive. If it is true that some environmental issues, including climate change, can also be dealt with synergistically with other school subjects, geography plays a leading role thanks to the theoretical tools and research applications that characterize it [45,97]. 5.2. Community Resilience to Extreme Natural Events and Climate Change In this study, however, those who trust the ability of local policymakers to face an extreme event also trust the national and international political classes in the fight against climate change. On the contrary, those who have no faith in local authorities do not even believe in the ability of national and international political authorities to solve the problem. The lack of trust in the political institutions represents a perceived barrier closely linked to the lack of political action to mitigate climate change, which is classified, indeed, by Lorenzoni et al. [36] at a perceptual level of global dimension. Sustainability 2020, 12, 6985 19 of 24 19 of 24 5.3. Practical Implications The theme of climate change has returned to being central in geographic studies also thanks to the diffusion of the concept of the Anthropocene [98,99] in human and social sciences. The theory of the Anthropocene, indeed, brings geographers to confront themselves with human climate– community relations, to rethink how to interpret adaptation first, then interaction, and finally mutual transformation [100]. The climate is proving to be one of the most changing geographical factors, but also one of the most impacting on the territorializing process. Furthermore, as also emerges in our study, it seems to be increasingly considered and perceived as an element linked to the outcomes of human actions. The symptoms and perceptions of the Anthropocene, therefore, invite us to reflect on this concept, understood as a social and cultural process, in particular as a concern for the future of the planet and mankind, which has become aware of the feedback from the environment with respect to human action and of the need to redefine our relationship with the planet. In the EIB Climate Survey 2019–2020 [24] more than 6 respondents out of 10, from Europe, the U.S.A. and China, think their behavior can make a difference in tackling climate change. These results, in harmony with our study, demonstrate the importance of adopting resilient and geo‐ethical behaviors to take care of the territorial ecosystem in which we live, locally, and to safeguard our planet, globally. Political institutions, therefore, must also think about responsible, ethically correct, persuasive actions, significantly impacting on a local and global scale against climate change so that they can acquire credibility and trust citizens. It is essential that nature scientists work hand in hand with social scientists and humanists as well as politicians, policymakers, and businesses to: (i) develop an integrated and multidisciplinary approach to understand a complex phenomenon, such as climate change; (ii) promote shared and sustainable actions involving the population to reduce the disaster risk. Sustainability 2020, 12, 6985 20 of 24 20 of 24 6. Conclusions Conflicts of Interest: The authors declare no conflict of interest. 6. Conclusions The perception of climate change varies in relation to contextual factors, including media communication, socio‐demographic characteristics of respondents, knowledge and education, economic, institutional factors, personal values and, finally, psychological factors and experience. On this last aspect, from a different point of view, scientists often remember how associating individual meteorological events with climate change is imprudent and scientifically incorrect, since, as is well known, climatology refers mainly to the average characteristics of the climate [4]. In fact, on the effects of climate change, the ways of building reality, influenced by exposure to the mass media and some stereotyped images, bring the attention of public opinion to exchange climate change with daily or seasonal meteorology or to associate it with the hole in the ozone and melting glaciers [37]. Furthermore, as we have highlighted several times in this paper, the spatio‐temporal dimension, therefore—in one word, “geographic”—which affects the perception of the citizens interviewed, indicates a knowledge gap that can only be bridged through the enhancement of geographic sciences in school curricula. As can be seen, these are all issues that emerged during the discussion of this research. Therefore, with respect to the issues listed above, the perception of risk plays a fundamental role: it implies various cultural (re)productions and it constitutes a fundamental motivation for the formulation of what Landowski [101] calls “action programs”, both individual and collective. Even though the results of this study could be considered valid only for the study area, the perspectives of the obtained outputs will be compared with results of other surveys, in particular those in Malta, within the Agreement on Scientific Cooperation between CNR and the University of Malta (UoM). Author Contributions: Conceptualization, L.A., R.C., F.D.P.; data curation, R.C.; formal analysis, D.D.M.; investigation, F.D.P.; methodology, L.A. Specifically, F.D.P. and R.C. wrote Section 1 Introduction; L.A. and D.D.M. wrote Section 3 Methods; L.A. and R.C. wrote the Section 4 Results; F.D.P. wrote Section 2 Climate Change Perception: The Reference Framework and Section 5 Discussion; all authors contributed to Section 6. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: The authors would like to thank participants who gave their voluntary and informed consent before the interviews. The authors thank the anonymous reviewers for helpful and insightful comments which improved the paper. References 1. Keellings, D.; Waylen, P. 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https://openalex.org/W2617550232	https://inria.hal.science/hal-00912882/document	English	null	Integrated CHOReOS middleware - Enabling large-scale, QoS-aware adaptive choreographies	HAL (Le Centre pour la Communication Scientifique Directe)	2,013	cc-by	41,749	To cite this version: Amira Ben Hamida, Fabio Kon, Nelson Lago, Apostolos Zarras, Dionysis Athanasopoulos, et al.. Integrated CHOReOS middleware - Enabling large-scale, QoS-aware adaptive choreographies. 2013. hal-00912882 Integrated CHOReOS middleware - Enabling large-scale, QoS-aware adaptive choreographies Amira Ben Hamida, Fabio Kon, Nelson Lago, Apostolos Zarras, Dionysis Athanasopoulos, Dimitris Pilios, Panos Vassiliadis, Nikolaos Georgantas, Valérie Issarny, Georgios Mathioudakis, et al. To cite this version: Amira Ben Hamida, Fabio Kon, Nelson Lago, Apostolos Zarras, Dionysis Athanasopoulos, et al.. Integrated CHOReOS middleware - Enabling large-scale, QoS-aware adaptive choreographies. 2013. hal-00912882 Integrated CHOReOS middleware - Enabling large-scale, QoS-aware adaptive choreographies Amira Ben Hamida, Fabio Kon, Nelson Lago, Apostolos Zarras, Dionysis Athanasopoulos, Dimitris Pilios, Panos Vassiliadis, Nikolaos Georgantas, Valérie Issarny, Georgios Mathioudakis, et al. HAL Id: hal-00912882 https://inria.hal.science/hal-00912882v1 Preprint submitted on 2 Dec 2013 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. : FP7-257178 : CHOReOS Large Scale Choreographies for the Future Internet Deliverable Number : D3.3 Title of Deliverable : Integrated CHOReOS middleware - Enabling large-scale, QoS-aware adaptive choreographies Nature of Deliverable : Report + Prototype Dissemination level : Public Licence : Creative Commons Attribution 3.0 License Version : 3.3 Contractual Delivery Date : 30 September 2013 Actual Delivery Date : 30 September 2013 Contributing WP : WP3 Editor(s) : Fabio Kon (USP) Author(s) : Amira Ben Hamida (EBM), Fabio Kon (USP), Nelson Lago (USP), Apostolos Zarras (UOI), Dionysis Athanasopoulos (UOI), Dimitris Pilios (UOI), Panos Vassiliadis (UOI), Niko- laos Georgantas (Inria), Valerie Issarny (Inria), Georgios Mathioudakis (Inria), Georgios Bouloukakis (Inria), Yesid Jarma (Inria), Sara Hachem (Inria), Animesh Pathak (Inria) Reviewer(s) : Daniel Batista (USP) Integrated CHOReOS middleware - Enabling large-scale, QoS-aware adaptive choreographies http://www.choreos.eu Abstract This document describes the ﬁnal implementation and the evaluation of the CHOReOS middleware. Evaluation is achieved both via the use of the middleware on CHOReOS use-cases and via synthetic experiments and simulation. The conclusion was that the implementation of the CHOReOS middle- ware has achieved a good level of maturity for an open source project and it is ready to be used in real-world, complex choreographies. middleware, architecture, choreography, large-scale, SOA, Internet of Things, Cloud CHOReOS FP7-257178 III CHOReOS FP7-257178 IV Document History Version Changes Author(s) 1.0 Initial Commits of the deliverable on SVN Fabio Kon 1.1 Detailed descriptions of some components Leonardo Leite 1.2 Detailed descriptions of all components WP3 team 1.3 Added evaluation WP3 team 1.4 General review WP3 team 2.0 Final version after internal review WP3 team 2.1 Final version after Valerie’s additional review WP3 team Document Reviews Review Date Ver. Reviewers Comments Outline July 25th 0.1 Agreed outline by all partners na Draft August 15th 1.0 Fabio Kon Comments sent by mail QA October 9th 1.4 Daniel Batista Comments sent by mail PTC October 18th 2.1 Valerie Issarny Internal review and Valerie’s com- ments addressed and ﬁnal version ready Document History Version Changes Author(s) 1.0 Initial Commits of the deliverable on SVN Fabio Kon 1.1 Detailed descriptions of some components Leonardo Leite 1.2 Detailed descriptions of all components WP3 team 1.3 Added evaluation WP3 team 1.4 General review WP3 team 2.0 Final version after internal review WP3 team 2.1 Final version after Valerie’s additional review WP3 team Document Reviews Review Date Ver. Reviewers Comments Outline July 25th 0.1 Agreed outline by all partners na Draft August 15th 1.0 Fabio Kon Comments sent by mail QA October 9th 1.4 Daniel Batista Comments sent by mail PTC October 18th 2.1 Valerie Issarny Internal review and Valerie’s com- ments addressed and ﬁnal version ready Document Reviews CHOReOS FP7-257178 V CHOReOS FP7-257178 VI Glossary, acronyms & abbreviations Table Of Contents Table Of Contents Table Of Contents List Of Tables ............................................................................... XI List Of Figures .............................................................................. XIV 1 Introduction ............................................................................. 1 2 CHOReOS Middleware: Architecture & Implementation ............................. 3 2.1 eXecutable Service Composition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.1.1 Things Composition and Estimation C&E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.1.2 AoSBM Service Substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2 eXtensible Service Access. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.2.1 XSB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.2.2 EasyESB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table Of Contents . . . . . . . . . . . . . . . . . . . . 9 2.2.3 LSB. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.3 eXtensible Service Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.3.1 AoSBM Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.3.2 Things Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2.3.3 Plugin Manager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2.4 Cloud and Grid Middleware . . . . . . . . . . . . . . . . . . . Table Of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2.4.1 The CHOReOS Enactment Engine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 3 How to Use the CHOReOS Middleware................................................ 19 3.1 XSC – How to Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.1.1 Composition and Estimation – How to Use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.1.2 AoSBM Service Substitution – How to Use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.2 XSA – How to Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.2.1 XSB – How to Use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table Of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.2.2 EasyESB – How to Use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.2.3 LSB – How to Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.3 XSD – How to Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.3.1 AoSBM – How to Use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.3.2 Things Discovery – How to Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.3.3 Plugin Manager and Plugins – How to Use. . . . . . . . . . . . . . . . . . . . . Glossary, acronyms & abbreviations lossary, acronyms & abbreviations Item Description API Application Programming Interface BC Binding Component CA Consortium Agreement CSDL Client/Server Description Language DL Deliverable Leader DoW Description of Work DPWSDL DPWS Description Language DSB Distributed Service Bus ESB Enterprise Service Bus GA Generic Application IAC Industrial Advisory Committee IOTS Internet of Things Services JBI Java Business Integration JMSDL JMS Description Language JSDL JavaSpaces Description Language LTS Labeled Transition System MST Management Support Team QoS Quality of Service OSS Open Source Software PL Project Leader PMT Project Management Committee PO Project Ofﬁcer PTC Project Technical Committee PSDL Publish/Subscribe Description Language SL Scientiﬁc Leader SE Service Engine TDD Test Driven Development TSDL Tuple Spaces Description Language ULS Ultra-Large Scale WP Work Package WPL Work Package Leader xDL Description Language XSA eXtensible Service Access XSB eXtensible Service Bus XSC eXecutable Service Composition XSD eXtensible Service Discovery CHOReOS FP7-257178 VII CHOReOS FP7-257178 VIII Table Of Contents . . . . . . . . . . . . . . . . . . . . . 28 3.4 Cloud and Grid – How to Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 4 Evaluation ............................................................................... 33 4.1 Use-Case-Based Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.1.1 IoTS Middleware Use-Case-Based Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.1.2 XSB Use-Case-Based Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.1.3 EasyESB Evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 CHOReOS FP7-257178 IX CHOReOS FP7-257178 IX 4.1.4 AoSBM Discovery and Service Substitution Use-Case-Based Evaluation. . . . . . . . . . . . . 36 4.1.5 Cloud Enactment Engine Use-Case-Based Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.2 Experiment-Based Evaluation. . . . . . . . . . . Table Of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 4.2.1 ULS Evaluation of IoTS Middleware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 4.2.2 XSB Evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4.2.3 EasyESB Evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 4.2.4 AoSBM Discovery Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 4.2.5 AoSBM Service Substitution Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 4.2.6 Cloud Enactment Engine Evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List Of Tables List Of Tables Table 3.1: CHOReOS middleware components and associated requirements fulﬁlled . . . . . . . . . . . . . 19 Table 3.2: Choreos maven repository. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Table 3.3: Sensor access middleware REST API. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Table 3.4: Response containing the sensing value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Table 3.5: Phone services proxy REST API . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Table 3.6: Response for pending requests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Table 4.1: Use of the middleware components by the use cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Table 4.2: EasyESB evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List Of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Table 4.3: EasyESB footprint. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Table 4.4: Development effort for the application developer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Table 4.5: Development effort for the JMS binding component . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Table 4.6: Results for one-way interaction in all four scenarios with 50 concurrent clients. . . . . . . . . . 49 Table 4.7: Interaction latency on the bus for each interconnection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Table 4.8: WSBQL Query. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Table 4.9: Experimental Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table Of Contents . . 60 5 Conclusion .............................................................................. 65 Bibliography ................................................................................ 67 A Enactment Engine Listings ............................................................ 71 CHOReOS FP7-257178 X List Of Figures List Of Figures Figure 2.1: The CHOReOS middleware overall architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Figure 2.2: The CHOReOS eXtensible Service Bus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Figure 2.3: Binding component architecture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Figure 2.4: Sensor access middleware architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Figure 2.5: LSB – overall architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Figure 2.6: LSB – binding component architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Figure 2.7: Enactment Engine components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Figure 3.1: AoSBM graphical user interface. . . . . . List Of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Figure 3.2: The Enactment Engine data model for choreography speciﬁcation . . . . . . . . . . . . . . . . . . . . 30 Figure 4.1: Global IoTS evaluation environment architecture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Figure 4.2: Example deployment of the device registry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Figure 4.3: Internal operations of the NodeLauncher . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Figure 4.4: Deployment used for performing ULS evaluations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Figure 4.5: Comparison of registration response times for different input rates. . . . . . . . . . . . . . . . . . . . 44 Figure 4.6: Time analysis of probabilistic registration for different input rates. . . . . . . . . . . . . . . . . . . . . . 44 Figure 4.7: Comparison of query response times for different input rates . . . . . . . . . . . . . . . . . . . . . . . . . 44 Figure 4.8: Time analysis of query response times for different input rates. . . . . . . . . . . . . . . . . . List Of Figures . . . . . . 45 Figure 4.9: Comparison of registration and query response times for ∼15000 nodes at an input rate of 1000 requests per second. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Figure 4.10: Components of mock environment for XSB capacity testing . . . . . . . . . . . . . . . . . . . . . . . . . 49 Figure 4.11: Throughput for one-way interaction of DPWS and JMS services with or without XSB in scenarios 1, 2, 3 and 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Figure 4.12: Latency for one-way interaction of DPWS and JMS services without XSB in scenar- ios 1 and 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Figure 4.13: Latency for one-way interaction of DPWS and JMS services with XSB in scenarios 3 and 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List Of Tables . . . . . . . . 54 Table 4.10: Enactment Engine deployment scenarios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Table 4.11: Choreography enactment time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Table 4.12: Enactment Engine scalability analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Table 5.1: CHOReOS middleware components summary of achievements . . . . . . . . . . . . . . . . . . . . . . . 66 CHOReOS FP7-257178 XI CHOReOS FP7-257178 XII List Of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 CHOReOS FP7-257178 XIII Figure 4.14: Latency for one-way interaction of DPWS and JMS services with XSB in scenarios 3 and 4 for up to 50 concurrent clients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Figure 4.15: Querying service abstractions vs. concrete service descriptions – 1st set. . . . . . . . . . . . 54 Figure 4.16: Querying service abstractions vs. concrete service descriptions – 2nd set . . . . . . . . . . . 55 Figure 4.17: Time breakdown for the delegation overhead of the AbstractWeatherForecastService, when called as a Java API (numbers indicate the different phases of the translation of abstract to concrete invocations) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Figure 4.18: Time breakdown for the delegation overhead of the AbstractWeatherForecastService, when called as a Web Service (numbers indicate the different phases of the translation of abstract to concrete invocations) . . . . . . . . . . . . . . . . . . . . . . . . 58 Figure 4.19: Total execution time for the invocation of the AbstractWeatherForecastService . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Figure 4.20: Delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when called as Java APIs (numbers indicate the different phases of the translation of abstract to concrete invocations). . . . . . . . . . . . . . . . . . . . . . . . . . . List Of Figures . . . . . . . . . . 59 Figure 4.21: Delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when called as Web services (numbers indicate the different phases of the translation of abstract to concrete invocations) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Figure 4.22: Total execution time for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany 60 Figure 4.23: Time breakdown for the substituion overhead as a function of the number of services that use the substituted service. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Figure 4.24: Choreography synthetic topology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Figure 4.20: Delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when called as Java APIs (numbers indicate the different phases of the translation of abstract to concrete invocations). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Figure 4.21: Delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when called as Web services (numbers indicate the different phases of the translation of abstract to concrete invocations) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Figure 4.22: Total execution time for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany 60 Figure 4.23: Time breakdown for the substituion overhead as a function of the number of services that use the substituted service. . . . . . . . . . . . . . . . . . . . . List Of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Figure 4.24: Choreography synthetic topology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Figure CHOReOS FP7-257178 XIV 1 Introduction The CHOReOS middleware is organized as a collection of software modules, which work together to support the development, deployment, and execution of complex, large-scale Web Service choreogra- phies on a Future-Internet-like environment. It brings together technologies related to the Internet of Things, the Internet of Services, and Cloud Computing to facilitate such tasks. This is achieved by pro- viding higher-level abstractions and services that organizations can rely upon to build complex, scalable, and adaptive service compositions. This document describes the CHOReOS middleware implementation, whose open source code is available at http://www.choreos.eu/bin/view/Download/Forge and whose documentation is available at http://www.choreos.eu/bin/view/Documentation/WebHome. The document is organized as follows. Chapter 2 ﬁrst presents the middleware overall architecture and then describes the implementation of each of its major components. Chapter 3 describes how to use the CHOReOS middleware as a choreography runtime environment. Chapter 4 presents our evaluation of the middle- ware, based both on the CHOReOS use cases and on synthetic experiments and simulations, stressing the middleware capabilities. Finally, we present our conclusions in Chapter 5. CHOReOS FP7-257178 1 CHOReOS FP7-257178 2 2 CHOReOS Middleware: Architecture & Implemen- tation The CHOReOS middleware is organized in four major modules: eXecutable Service Composition (XSC), responsible for coordinating the composition of services and things, eXtensible Service Access (XSA), which provides the means to access services and things, eXtensible Service Discovery (XSD), which manages protocols and processes for discovery of services and things, and the Cloud & Grid Middleware, which manages computational resources and drives the deployment of choreographies. Figure 2.1 depicts the overall architecture of the CHOReOS middleware, presenting each middleware module, the components inside them, and the interactions among the components. The ﬁgure also shows the interactions between the CHOReOS middleware developed with WP3 and the Governance and V&V Framework developed within WP4. Application developers may choose to use all the middleware components to facilitate the construction of choreographies or, alternatively, to pick only the components that provide the functionality in which he/she might be interested. Details about the architecture and implementation of each module are described in this section. Details about how to use the middleware are provided in Section 3. CHOReOS FP7-257178 3 3 CHOReOS FP7-257178 Figure 2.1: The CHOReOS middleware overall architecture Figure 2.1: The CHOReOS middleware overall architecture Core Contributions The eXecutable Service Composition (XSC) component provides facilities for the composition of both services and things. Services are composed by devising and deploying appro- priate Coordination Delegate (CD) within the component-CD container. Basically, the component-CD container provides the communication primitives (e.g., UPDATE STATE(), WAIT(), NOTIFY()) that are used by the coordination algorithm (detailed in deliverable D2.3 [15]) to exchange what we have called additional communication (i.e., coordination information). That is, CDs mediate (in a distributed way) the business-level interaction protocols of the participant services according to the coordination logic extracted from BPMN2 choreography diagrams by the synthesis processor (also detailed in deliverable D2.3 [15]). Things are composed by employing semantic composition speciﬁcations modeled as math- ematical formulas: complex physical properties are derived from simpler ones provided by thing-based services. The XSC further comprises the abstraction-oriented service substitution facility, which allows to substitute services that provide similar functional properties. To this end, the services must be rep- resented by the same abstraction [11] that provides the necessary mappings between the interfaces of the services. The eXtensible Service Access (XSA) component provides facilities for interconnecting heteroge- neous services and things. 2 CHOReOS Middleware: Architecture & Implemen- tation More speciﬁcally, the eXtended Service Bus (XSB) component enables interconnecting services that employ heterogeneous interaction paradigms, namely, the client-server (CS), publish-subscribe (PS) or tuple space (TS) paradigms. This is based on appropriate formal mod- eling and abstraction of each service middleware protocol into a connector representing the corre- sponding paradigm. Then, the CS, PS and TS connectors are further abstracted into a single higher- layer connector, the generic application (GA) connector, which uniﬁes their features and enables their cross-connection. This formal approach is realized into the XSB service bus. XSB is implemented on top of the EasyESB component, which is a ﬂexible, dynamic-topology enterprise service bus, also implemented within CHOReOS. XSB principles have also been applied to the Light Service Bus (LSB) component, which is a lightweight interconnection solution applying to things. LSB additionally supports data streaming protocols, which are particularly important in IoT. The eXtensible Service Discovery (XSD) component provides facilities for the organization and the discovery of available services. To deal with scalability issues that concern the increasing amount of available services, the XSD employs the abstraction-oriented organization and discovery mechanisms (i.e., the mechanisms that constitute the AoSBM component) developed in WP2 [7]. Moreover, the XSD comprises a specialized Things Discovery protocol that leverages the concept of semantic abstractions for the organization of Thing-based services and offers facilities for the probabilistic registration and discovery of these services. The Cloud & Grid Middleware provides a collection of Java classes and web services that make the process of deploying complex, large-scale choreographies composed of hundreds to thousands of services very easy. It also provides elasticity features to manage the number of replicas of speciﬁc services based on the workload (e.g., amount of user requests), resource utilization (e.g., usage of CPU or memory), and of QoS attributes (e.g., response time). To achieve that, it interacts with the CHOReOS Monitoring service [16] to detect when relevant events that should trigger dynamic reconﬁguration occur. We continue by describing in more detail the internal architecture of each of these middleware com- ponents. 2.1.1. Things Composition and Estimation C&E The C&E component enables the composition of thing-based services. We exploit semantic technolo- gies for the composition speciﬁcations. Given that thing-based services are sensing/actuating services in most cases, we model our compositions as mathematical formulas. For instance, wind chill can be es- timated from temperature and wind speed measurements. Consequently, the composition speciﬁcation is: (2.1) WC = (10 √ V −V + 10.5) · (33 −T) (2.1) WC = (10 √ V −V + 10.5) · (33 −T) WC is the symbol for Windchill, V is the symbol of Windspeed, T is the symbol for Temperature. This information along with the mathematical formula are modeled in the IoT ontology. Concepts such as Temperature and Windspeed are referred to as expansion concepts. WC is the symbol for Windchill, V is the symbol of Windspeed, T is the symbol for Temperature. This information along with the mathematical formula are modeled in the IoT ontology. Concepts such as Temperature and Windspeed are referred to as expansion concepts. Application developers can use this component to extract expansion concepts (e.g., Temperature and Windspeed), or compute the formulas to have an estimation of the initial concept value (e.g., Windchill). More precisely, the component extracts the expansion concepts and the formulas through SPARQL queries as strings. They are then parsed, automatically, into Java mathematical functions using the exp4j1 jar. The component expects that services measuring the expansion concepts will be accessed for their measurements (using the QueryManager) and the formulas can then be computed using the acquired measurements as input. The details on the methods and the use of the component are presented in Section 3.3. In our current implementation, the component is internally used by the QueryManager, to seamlessly execute compositions. However, we also provide it as a stand alone component in case the user wishes to exploit its functionalities separately. 2.1. eXecutable Service Composition The eXecutable Service Composition (XSC) supports the composition of large number of services into business choreographies and large populations of things in response to user queries. Additionally, XSC supports service substitution for reconﬁguration within choreographies. The following sections provide an overview of the corresponding XSC components, namely, Composition and Estimation (Sec- tion 2.1.1), and Reconﬁguration Management for Service Substitution (Section 2.1.2). CHOReOS FP7-257178 5 5 1http://www.objecthunter.net/exp4j/ 2http://jcp.org/aboutJava/communityprocess/mrel/jsr224/index3.html 2.1.2. AoSBM Service Substitution Hereafter, we use the term AoSBM service substitution to refer to the realization of the Reconﬁguration Management for Service Substitution component (Figure 2.1). The idea of service abstractions [9] has been further exploited to facilitate service substitution. The overall design of the abstraction-oriented service substitution approach has been speciﬁed in D3.2.2 [11], while the prototype implementation of the service substitution facility has been developed during the last year of the project. Conceptually the service substitution approach belongs to XSC. However, the implementation of the approach is coupled with the AoSBM facilities that extract service abstractions. Hence, the implementation of the approach is physically packaged in the AoSBM package (Section 2.3.1). The main concepts of the AoSBM service substitution approach are functional abstraction services and impact analyzers. In particular, service substitution is performed between services that are represented by the same functional abstraction [9, 7]. Given a functional abstraction we generate a functional abstraction service that realizes the abstract interface, speciﬁed by the given functional abstraction [11]. The functional abstraction service is conﬁgured to delegate invocations, made to the abstract interface, to invocations on the interface of a concrete service that is hidden behind the functional abstraction service. To realize this delegation process, the functional abstraction service employs the mapping between the abstract interface and the interface of the hidden service instance, which is provided in the speciﬁcation of the functional abstraction that represents the hidden service instance. The core of the delegation mecha- nism is a data translation module that transforms the inputs of the abstract invocation into corresponding inputs for the concrete invocation. Moreover, the data translation module transforms the outputs of the concrete invocation into corresponding outputs of the abstract invocation. The data translation module adapts the approach for the translation of XML documents, proposed in [24], to the case of Java objects. In particular, our approach assumes that the inputs/outputs are typical Java objects that conform to the JAX-WS2 standard for XML-based Web services. The translation of a given Java object takes place in CHOReOS FP7-257178 6 three phases3: three phases3: • First, the structure of the source object is recursively traversed using Java Reﬂection and the contents of the source object are transformed to a uniﬁed data representation that is employed by the translation mechanism. The uniﬁed representation is basically a relation that consists of a set of tuples. 2.1.2. AoSBM Service Substitution • Second, the structure of the target object is recursively traversed using Java Reﬂection, and the uniﬁed data representation of the source object (produced in the ﬁrst phase) is transformed ac- cording to the structure of the target object. • Second, the structure of the target object is recursively traversed using Java Reﬂection, and the uniﬁed data representation of the source object (produced in the ﬁrst phase) is transformed ac- cording to the structure of the target object. • Third, the target object is created using Java Reﬂection and it is ﬁlled with the contents of the source object that have been transformed according to the structure of the target object. • Third, the target object is created using Java Reﬂection and it is ﬁlled with the contents of the source object that have been transformed according to the structure of the target object. Substituting the hidden service instance with another service instance that is represented by the same functional abstraction amounts to change the mapping that is employed by the functional abstrac- tion service for the delegation of the invocations. During the substitution, the service instances that depend on the substituted service instance are notiﬁed about the change. This notiﬁcation is the main responsibility of the impact analyzer that is associated with the functional abstraction service. Upon the reception of such a notiﬁcation, the dependent service instances are responsible for managing their local consistency. 2.2. eXtensible Service Access The eXtensible Service Access (XSA) represents the main runtime infrastructure for accessing both services and things. In this section, we present the eXtensible Service Bus (XSB) as well as its speciﬁc paradigms. We dedicate Section 2.2.1 to recall the main concepts of the XSB. Then in Section 2.2.2, we brieﬂy describe EasyESB, which offers the baseline middleware for the XSA framework. Moreover, it provides a suitable and scalable environment for business services to get involved into large choreogra- phies. Finally, we describe in Section 2.2.3 the Light Service Bus (LSB). It is dedicated to Things-based services and addresses Internet of Things challenges. The several components of the XSA middleware are fully integrated in the CHOReOS IDRE. 3We use the term source object to refer to the object that is transformed by the data translation module; we use the term target object to refer to the result of the transformation 2.2.1. XSB The eXtensible Service Bus (XSB) provides support for seamless integration of heterogeneous inter- action paradigms. With regard to middleware-supported interaction, the client-service (CS), publish- subscribe (PS), and tuple space (TS) paradigms are among the most widely employed ones, with nu- merous related middleware platforms, such as SOAP, REST, and Java RMI for client-server; JMS and SIENA for publish-subscribe; and JavaSpaces and LIME for Tuple space. Services relying on differ- ent interaction paradigms can be plugged into the CHOREOS XSB by employing binding components (BCs) that adapt between their native middleware and the common bus protocol. This adaptation is based on the abstractions, and in particular on the conversion between the native middleware, the cor- responding CS/PS/TS abstraction, and the Generic Application (GA) abstraction, as depicted in Figure 2.2. eXtensible Service Bus binding components (XSB BCs) are designed to be easily extensible to sup- port new middleware platforms or new interaction paradigms. Hence, each subcomponent of an XSB BC is designed with 3 levels, the higher one being the most generic and that can be further reﬁned into the two lower layers: CHOReOS FP7-257178 7 Figure 2.2: The CHOReOS eXtensible Service Bus Figure 2.2: The CHOReOS eXtensible Service Bus • The generic level provides APIs and functionalities that are shared among supporting implemen- tations of all interaction paradigms. • The generic level provides APIs and functionalities that are shared among supporting implemen- tations of all interaction paradigms. • The interaction paradigm level specializes the APIs and functionalities of the previous level for the CS, PS and TS interaction paradigms. • The interaction paradigm level specializes the APIs and functionalities of the previous level for the CS, PS and TS interaction paradigms. • The middleware platform level specializes the APIs and functionalities of the previous level for a concrete middleware. • The middleware platform level specializes the APIs and functionalities of the previous level for a concrete middleware. Besides a connector’s API, we introduce an abstract interface description language (IDL) for spec- ifying the open interfaces of systems that rely on middleware represented by the speciﬁc connector. Our IDLs are largely inspired from WSDL. We specify the IDLs conceptually, while we have also imple- mented each one of them as an XML schema document. 2.2.1. XSB Based on the ﬂexibility of XML schema, an IDL can be easily reﬁned in order to enable the description of a concrete system that is based on the connector, e.g., we can reﬁne the abstract XML elements into the precise data structures and types of the speciﬁc middleware and system. The architecture of an XSB Binding Component as provided by our architectural framework is de- picted in Figure 2.3, where the main components are the xDL Processor, Core Engine, and Envelope for Substrate Bus. Envelope for Substrate Bus The Envelope for Substrate Bus makes the binding components (BCs) deployable on top of different communication substrates. It provides the mechanisms to: The Envelope for Substrate Bus makes the binding components (BCs) deployable on top of different communication substrates. It provides the mechanisms to: 1) extract/encapsulate GA primitives from/into bus connections, before/after passing/getting them to/from the Core Engine 2) transform service primitives to paradigm primitives, then map them to GA primitives, and vice versa 3) manage the lifecycle of a service deployed on XSB, after retrieving information about this service from the xDL processor. These mechanisms are reﬁned to support a new communication bus. 2.2.2. EasyESB Core Engine The Core Engine provides all the mechanisms to: The Core Engine provides all the mechanisms to: nsform service data to paradigm XML data, then map them to GA data, and vice versa 1) transform service data to paradigm XML data, then map them to GA data, and vice 2) transform service primitives to paradigm primitives, then map them to GA primitives, and vice versa 2) transform service primitives to paradigm primitives, then map them to GA primitives, and vice versa 3) manage the connections to the service deployed on the bus. 3) manage the connections to the service deployed on the bus. The above mechanisms cooperate with each other, as well as with xDL processor for retrieving informa- tion about the service. The interaction paradigm level customizes Core Engine APIs and functionalities to the CS, PS and TS paradigms and maps them to GA APIs and functionalities. At the middleware platform level, the Core Engine performs the real interactions with the concrete middleware to establish connections, as well as to send and receive data. Same as for DL descriptions, mapping paradigm data to GA data further requires information about the semantic mapping between the constituent services that form the global application. xDL Processor The xDL processor processes the descriptions of the services deployed on the XSB. It performs both parsing of paradigm DL descriptions (CSDL, PSDL, TSDL) and mapping of them to Generic Applica- tion descriptions (GADL), where the latter relies on XSLT-based transformations. At the generic and interaction paradigm level, these functions apply to abstract descriptions, while they become concrete at the middleware platform level. We use the XML schema extensibility mechanisms to specialize these functions from one architectural level to another. In particular, mapping paradigm DL to GADL descrip- tions further requires information about the semantic mapping between the constituent services that are integrated into the global application. CHOReOS FP7-257178 8 Figure 2.3: Binding component architecture Figure 2.3: Binding component architecture 2.2.3. LSB The goal of the CHOReOS Light Service Bus (LSB) is to enable access to data sensed by smart things using thing-based services. LSB comprises the Sensor Access Middleware, Phone Services Proxy and LSB Binding Components, which are described in the following. 2.2.2. EasyESB The extensible Service Access (XSA) middleware relies on the EasyESB Enterprise Service Bus (ESB) for integrating business services. EasyESB is an open source project licensed under the CHOReOS FP7-257178 9 9 LGPL. EasyESB adopts the Model-Driven Architecture (MDA) philosophy and is available for down- load at http://research.petalslink.org/display/easyesb/EASYESB+-+Open+source+ Lightweight+Services+Bus. It is built on top of a modular and ﬂexible topology that eases the creation of new bus nodes, their update, and removal. This ability makes possible the deployment of the bus nodes on the cloud infrastructure and their adaptation in case the cloud changes, e.g., when a new physical machine is added and, thus, a new EasyESB node is created and automatically linked to the previous nodes. The creation of new nodes allows the knowledge of all the services deployed on the neighboring nodes. Within the CHOReOS project, we beneﬁt from this capability to deploy the EasyESB nodes on top of the Cloud infrastructure described in Section 2.4. EasyESB provides the backbone environment for the XSC middleware, the component-CD, by offering the needed multi-paradigm com- munication mechanisms. Finally, EasyESB eases the monitoring tasks implemented within WP4 by providing interceptors for the services bound to the bus. We implement an event-based mechanism in order to receive information captured by the interceptors. The generated information reports relevant data about the behavior of the services such as the response time, the payload, etc. During the CHOReOS project, we have been improving EasyESB by several implementations in order to make it cope with the raised challenging objectives. For instance, we beneﬁt from the cloud-aware infrastructure described in Section 2.4 and leverage the capabilities of the bus to be deployed on a scalable infrastructure. Joining both technologies represents a major step forward to the project as it enables accessing, integrating, choreographing and monitoring services from all over the world. Sensor Access Middleware The Mediator class provides the following functionality that can be used directly: The Mediator class provides the following functionality that can be used • discover all the sensors available on the device • discover the instance of a speciﬁc sensor, speciﬁed by its interface • trigger an immediate sensing by all sensors, where possible Through the Mediator, the application can perform basic discovery and sensing actions with a sin- gle method call, abstracting away all the logical details such as creating separate processes in the OS, executing inter-process communication calls, synchronization, etc. All of these lower-level issues are all handled by the Mediator and the Daemon in an OS-independent manner. In addition, all sen- sors are treated homogeneously under a common API, whether they are microphones, thermometers, accelerometers, or cameras. The MediatorListener is the abstract class that contains the functionalities to be implemented by the application developer to be able to retrieve the data from the sensors on the device. Speciﬁcally, the functionalities that the application developer should implement are: • hooks to get notiﬁed when there is a change in the state of the mediator itself, or of the sensors attached to it • hooks to get notiﬁed when there is a change in the state of the mediator itself, or of the sensors attached to it • hooks to get notiﬁed whenever there is new data available from one or more of the se • hooks to get notiﬁed whenever there is new data available from one or more of the Sensor Access Middleware The Sensor Access Middleware is deployed on thing devices, in particular smartphones, and enables exposing their data (e.g., coming from the device sensors) as thing services. Figure 2.4 depicts the overall architecture of the Sensor Access Middleware, where the two main subcomponents are the Thing Mediator and the Sensor Driver. At the level of individual devices, a thing-based service using local sensors can utilize the Thing Mediator [8] for abstracting access to different sensors attached to the device. Individual vendors can contribute Sensor Drivers to their sensors that transparently bind with this mediator and provide access to sensed data through thing-based services. The Sensor Driver and Thing Mediator are described in more detail bellow: Sensor Driver The Sensor Driver deﬁnes the API that should be implemented by the sensor driver developer. It consists of two main subcomponents: the Sensor, an Interface (API) for sensor drivers, which are the objects that hold the actual logic behind each sensor’s operation and the SensorInfo, which contains metadata about a sensor. Speciﬁcally, the functionalities that need to be implemented by each Sensor component are: • exposure of its meta-description, including the name of the interface it implements and its unique (manufacturer-speciﬁc) device type • conﬁguration of the sensor parameters (setting and getting) • access to the sensed data. This can be (i) instantaneous, when the query requests the latest sensing value of a Thing, (ii) periodic, which asynchronously returns at a constant rate (until canceled); and (iii) event-based, where a sensor replies whenever new data are available. The functionalities that need to be implemented by each SensorInfo component include: The functionalities that need to be implemented by each SensorInfo component include: CHOReOS FP7-257178 10 • providing the display name, description, and the name used for semantic matching of the sensor • providing an instance of the sensor itself, thus acting as an instance of the Factory design pattern. Thing Mediator The Thing Mediator component is meant to be accessed by the application develop- ers and it consists of two subcomponents: the (i) Mediator and the (ii) MediatorListener. Thing Mediator The Thing Mediator component is meant to be accessed by the application develop- ers and it consists of two subcomponents: the (i) Mediator and the (ii) MediatorListener. Phone Services Proxy In order to complete the LSB vision and enable access to thing-based services hosted by smart de- vices, the Phone Services Proxy component was introduced in the M24 release of the CHOReOS middleware as a solution to deal with a number of issues related to mobile communication, such as the use of NAT, transient IP addresses, enforcement of asymmetrical communication, and temporary disconnections. Clients (such as the Things Query Manager) that wish to communicate with thing- based services residing on mobile devices, send their requests and receive the corresponding results via the Phone Services Proxy (henceforth referred to as proxy), rather than directly to/from the devices themselves. Mobile devices retrieve, in a pull-based fashion, client requests that have been issued towards them via the proxy and, in turn, send replies to these requests in order for them to be forwarded to the respective clients. The introduced proxy mechanism as described here, constitutes one of the main building blocks towards the realization of the Light Service Bus (LSB), by providing a universal access solution to sensor data provided at the Things level. Clients and mobile devices communicate with the proxy using RESTful interfaces, which are de- scribed in more detailed in Section 3.2.3.2. Service requests expire after a given time period (TTL) and, in case a timeout occurs, an appropriate error message is returned to the affected client. Moreover, each mobile device has a globally unique device ID that is used to address the speciﬁc device and, ultimately, its hosted services via the proxy. When mobile devices register their hosted services to the Things Registry Manager, they also provide a unique address for each of these services. This unique address includes (a) the address of the proxy associated with the speciﬁc mobile device (b) the unique deviceID, and (c) an identiﬁer for the service registered. CHOReOS FP7-257178 11 Figure 2 4: Sensor access middleware architecture LSB Binding Components As discussed in former CHOReOS deliverables, the major issue for service access towards dealing with the FI requirements is to be able to cope with the diversity of interaction protocols involved in IoBS and IoTS and, speciﬁcally, the integration and the pair-wide adaptation of these protocols. While the XSB, described in 2.2.1, addresses effectively heterogeneity and interoperability topics in the IoBS domain, a concrete access solution is required targeted particularly to the IoTS speciﬁcs, such as dy- namic environments, resource constraints, data orientation, etc. Towards this, we introduce the binding components for LSB, being a lightweight version of the XSB for the IoTS domain. Like XSB, the LSB component is based on the CHOReOS connectors [9], which abstract the middleware-layer interaction protocols enabling Future Internet services to interact. The design process of the LSB involved the identiﬁcation of the interaction paradigms that are com- mon in the IoT domain. Towards this end, an extensive survey was conducted on the state-of-the- art protocols being used in IoT-oriented solutions. The outcomes of this study conﬁrmed the impor- tance and wide use of the interaction paradigms already identiﬁed on XSB namely (i) Client-Server, (ii) Publish-Subscribe, and (iii) Tuple space, but also underline the existence of an additional paradigm focused on continuous interaction known as (iv) Streaming. However, Streaming can be considered as an extension to any of the interaction paradigms since, from a conceptual perspective, all of them must support both discrete and continuous protocols. LSB binding components are implemented from scratch and are designed with focus on high- performance, small code footprint, compliance with all prominent IoT protocols, and easy-to-use and extend nature. Key point to the overall architectural design, as depicted in Figure 2.5, is the use of CHOReOS FP7-257178 12 REST as the common bus that handles the interactions between the several binding components and services. The REST approach provides the common integration infrastructure where all diverse inter- action solutions can be plugged. REST was selected among other WS-* solutions because of its simple architecture, lightweight na- ture, high performance in terms of message transmission speed and bandwidth consumption, and reliability, since it is based on HTTP. We chose the REST solution against DPWS, which was its main competitor, because REST provides uniform interfaces that makes its usage and debugging straightfor- ward, a fact that made REST dominate the IoT market. 2.3. eXtensible Service Discovery The eXtensible Service Discovery (XSD) supports the organization and discovery of both services and things among the vast corresponding populations available on the Future Internet. To this end, it includes a solution to service discovery based on service abstractions (Section 2.3.1), a solution to probabilistic things discovery (Section 2.3.2), and a plugin-based framework providing extensible support for any current or future discovery solution (Section 2.3.3). 2.3.1. AoSBM Discovery LSB Binding Components Figure 2.5: LSB – overall architecture Figure 2.6: LSB – binding component architecture A binding component of LSB consists of four layers/parts that are responsible for the transformation of the messages that run through the lightweight bus. These transformations are necessary to provide a cross-paradigm and cross-protocol communication solution. As it is depicted in Figure 2.6, the ﬁrst CHOReOS FP7 257178 13 Figure 2.5: LSB – overall architecture Figure 2.5: LSB – overall architecture Figure 2.6: LSB – binding component architecture A binding component of LSB consists of four layers/parts that are responsible for the transformation of the messages that run through the lightweight bus. These transformations are necessary to provide a cross-paradigm and cross-protocol communication solution. As it is depicted in Figure 2.6, the ﬁrst CHOReOS FP7-257178 13 layer represents the middleware connector of the service that initiates the interaction with the binding component and is responsible for maintaining contact with the service at any moment. The second layer involves the abstraction of the middleware-speciﬁc messages to the respective abstract primitives deﬁned for each interaction paradigm, as described in D1.4 [9]. At the third layer of the architecture lies the GA adapter, a component that transforms the paradigm-based primitives to a more abstract type, that of GA. The GA’s primitives are abstract enough to describe any type of message belonging to any of the interaction paradigms supported. The last layer of the binding component involves the mapping of a GA primitive to a speciﬁc call of the common bus, in this case, a REST call. All layers of the binding component support as well the inverse procedure of transformation e.g. from a REST call coming through the common bus to a GA primitive and ﬁnally to a middleware-speciﬁc call. 4More details concerning the WSBQL syntax and the query engine can be found at http://www.choreos.eu/bin/ Documentation/Abstraction_Oriented_Service_Base_Management 2.3.3. Plugin Manager As speciﬁed in D3.1 [8], XSD relies on a plug-in based architecture, which enables incorporating sup- port for any current or future service discovery solution applying to Business and Thing-based services. By this release, we are delivering the Plugin Manager component, the associated REST interfaces and the source code of abstract classes needed for plugin development. The Plugin Manager provides uniﬁed access to service discovery protocols present in the CHOReOS environment and essentially acts as an intermediate layer between them and the Abstraction-oriented Service Base. Via the Plu- gin Manager, the AoSBM gets populated with services discovered by these heterogeneous service discovery protocols. The Plugin Manager interfaces with these protocols via the appropriate plugins. Individual discovery protocols can be integrated in the XSD runtime system by extending the abstract AbstractPlugin class. Currently, the Java Reﬂection API is used for dynamic plugin loading. The Plugin Manager architecture was based on previous work by Inria (CONNECT Discovery Enabler [18]) and was extended by VTrip. 2.3.1. AoSBM Discovery The goal of the AoSBM component is to enable service lookup for business services in the context of the FI. To this end, we employ the concept of service abstractions, introduced in D1.3 [6] and reﬁned in D1.4 [9]. The modus operandi of the main facilities of the AoSBM have been speciﬁed in D2.1 [7], D2.2 [10], and D2.3 [15], while the design of the AoSBM software itself was provided in D3.1 [8] and D3.2.2 [11]. Intuitively, a functional abstraction represents a group of services that offer similar functional properties; it is characterized by an abstract interface and a mapping between the abstract interface and the interfaces of the represented services. A non-functional abstraction represents a group of services that offer similar non-functional properties; it is characterized by an abstract non-functional description that consists of a set of layman’s terms, one for each non-functional property of interest (e.g., response-time, availability) and a mapping between these terms to ranges of concrete values for each non-functional property of interest. In the AoSBM approach, we exploit service abstractions as a means for organizing service descrip- tions in a registry and answering lookup queries for services. A lookup query is matched against service abstractions, instead of being matched against service descriptions of concrete services. Therefore, the query execution time scales up with the number of service abstractions, instead of scaling up with the number of available service descriptions. The service organization facility of the AoSBM accepts as input service descriptions that can be gathered from multiple sources (e.g., public registries, service portals) and produces hierarchically structured abstractions. To enable the execution of lookup queries over abstraction-oriented organizations of service descrip- tions, we provide the Web Service Base Query Language (WSBQL). WSBQL is tailored to the concept of service abstractions and its syntax resembles the W3C XQuery standard. The WSBQL language comes along with a corresponding query engine which translates WSBQL queries to conventional SQL queries, which are issued to the AoSBM relational store 4. Access to the query engine is provided via a dedicated REST API that has been developed during the last year of the project [15]. CHOReOS FP7-257178 14 2.3.2. Things Discovery The Things Discovery component is aimed at handling the ultra-large scale of mobile things able to pro- vide sensing/actuating tasks, mostly mobile phones. The thing-discovery protocol relies on probabilistic computations to control the participation of mobile things as needed. In the registration phase, things hosting sensors/actuators should register their services through the executeRegistrationQuery() API. The discovery component then executes a set of probabilis- tic computations to determine whether or not the services are needed. The decision is based on the percentage of the area sensed by the already registered devices in the Registry Manager com- ponent. If the percentage is below an acceptable threshold then the new device should register its services and vice versa. The thresholds should be speciﬁed in the IoT ontologies we provide. Sim- ilarly, In the look-up phase, applications wishing to search for services need to access the methods that implement one of the three following APIs: findServices(), findSubsetofServices(), findSubsetofServicesBasedOnCoverage() . The ﬁrst API allows the discovery component to return the complete set of devices that host services matching the request. The second and third APIs trigger the discovery component to perform probabilistic computations to determine a suitable subset of devices to access for the sensing/actuating services. More details on the use of the APIs are provided in Section 3.3 2.4. Cloud and Grid Middleware The Cloud and Grid middleware addresses the scalability and distribution requirements identiﬁed in the CHOReOS project by offering three components: • The Storage Service • The Grid Service • The Enactment Engine The Storage Service simply automates the deployment of a database server on the cloud, providing its client with the credentials to access it. The current implementation deploys a traditional RDBMS, MySQL, but it is possible to extend it to handle other kinds of databases, including noSQL databases over a set of machines for load distribution. The Storage Service is actually an extension to the Enact- ment Engine, since most of it is implemented by using Enactment Engine components. The Grid service is tailored at Grid Computing, which is offered as a service to developers needing to perform high-performance computing (HPC) for their applications. In other words, CHOReOS provides CHOReOS FP7-257178 15 Grid Computing as a Service, for applications with intensive computational requirements that could be not efﬁciently handled by a single machine hosting the service, such as a service that needs to process millions of images per day. According to our experience in the CHOReOS project within the past years, we anticipate that the Cloud Computing support offered by the CHOReOS middleware will be extremely useful for a wide variety of applications in multiple contexts. The Grid support, on the other hand, is a much more specialized service that would bring beneﬁts to a more speciﬁc set of applications, e.g. that require Big Data Analytics. Speciﬁcally in the CHOReOS project, the three ﬁnal use cases demonstrated interested in using the Cloud middleware, but not the Grid middleware5. Thus, although we provide a working implementation of both approaches, our major focus during the evaluation and assessment was on the Cloud part, which has a greater potential for adoption and dissemination. The Enactment Engine is the main component of the Cloud and Grid Middleware and the focus of this section. It provides a powerful and elastic platform of hardware resources for the deployment of (1) the CHOReOS middleware, (2) business services, and (3) coordination delegates. New virtual machines are created on-the-ﬂy as needed for the enactment of choreographies, and additional virtual machines may be created or removed during run-time according to demand or more sophisticated QoS constraints, avoiding extensive upfront investments on hardware for organizations participating in the choreography. 2.4. Cloud and Grid Middleware It enables a fully-automated process for choreography deployment, which is a key feature to support Future Internet scale systems, since manually deploying large-scale choreographies is not feasible. Instances of Cloud components deployed on different organizations are able to collaborate among them to make a distributed choreography deployment across these organizations. The Cloud and Grid components export a set of easy-to-use RESTful services. The choice of REST as communication protocol and interface style is in line with the current move on the Web services world, where the large majority of new services are based on REST and not on SOAP. This is due mainly to the better performance, less overhead, more ﬂexibility, and ease of use provided by RESTful services in contexts such as the services provided by the Cloud and Grid components. 5In the beginning of the CHOReOS project, one of the use cases, Citizen journalism, planned to use the support from the Grid middleware, but the use case was dropped from the project and replaced by a new one. 2.4.1. The CHOReOS Enactment Engine The Enactment Engine deals with heterogeneity by using extensibility: although the current version can deploy only SOAP services packaged as JAR or WAR ﬁles, Enactment Engine users may extend it by writing a few new classes and conﬁguration ﬁles to support new types of services. The Enactment Engine has also an extension point to allow the creation of new node allocation policies. Regarding the extensibility to support new Cloud IaaS providers (currently only Amazon EC2 and OpenStack are supported), all the developer must do is to write a Java class implementing the CloudProvider interface from our framework. The implementations of this interface for OpenStack and Amazon EC2 have around 200 lines of code; the size tends to be similar for other IaaS implementations. Enactment Engine expects and handles faults of third-party components, which is important in large- scale distributed systems. An example of a typical failure in a cloud environment involves the virtual machine provisioning. When a new node is requested to the infrastructure provider, there is a chance that provisioning will fail. Moreover, some nodes may take much longer than average to be ready. In experiments conducted by us using the Amazon EC2 service, we veriﬁed that failures and long provisioning times affected up to 7% of the requests. Enactment Engine has a particular strategy to handle failures when invoking the IaaS provider (asking for VMs creation), that is called the reserve of idle nodes. When a request arrives, the Enactment Engine tries to create a new node. If the creation fails, or takes too long, an already created node is retrieved from a reserve of idle nodes. It avoids waiting again for another node creation. The reserve size is deﬁned by conﬁguration and it is reﬁlled every time a reserve node is requested. The reserve approach has the extra cost to keep some nodes running in an idle state. However, only a small amount of extra nodes is necessary, since they will be used only when there is some failure on the cloud provider. CHOReOS FP7-257178 16 Figure 2.7 illustrates the components that are necessary to enact a choreography. The Cloud Gate- way and the Chef Solo are third-party software used by the Enactment Engine, whereas the Deployment Manager and the Choreography Deployer are components provided by the Enactment Engine. «component» Choreography Deployer «component» Deployment Manager «component» Chef-solo «component» Cloud Gateway AWS, OpenStack, etc. 3 How to Use the CHOReOS Middleware The major goal of the CHOReOS middleware is to act as a choreography runtime. As a middleware, it acts as a software layer that sits between the underlying system (composed of OS, cloud, network, things, etc.) and the user-level application (e.g., a choreography). It raises the abstraction level pro- vided to developers so that they can design, implement, and execute complex, large-scale service compositions with ease. A developer may opt to use all of the CHOReOS middleware components as an integrated middle- ware infrastructure to support the system that he/she desires to build. Alternatively, he/she may pick and choose only the middleware components that provide the required services for a speciﬁc targeted application development. Table 3.1 lists the CHOReOS middleware components together with the re- quirements that they help to fulﬁll. Middleware Component Fulﬁlled requirement Things C&E Need for things composition, e.g., deriving complex physical properties from simpler ones provided by thing-based services Service Substitution Need for adaptation of the services used in a particular context XSB Need for seamless integration of heterogeneous interaction paradigms (client-service, publish-subscribe, and tuple space) EasyESB Need for integrating heterogeneous services Need for a runtime context enabling advanced SOA abilities such as composition monitoring, and governance LSB Need for accessing heterogeneous Things as services Service Discovery Sustain information about an ultra large amount of available services and facilitate efﬁcient service lookup Things Discovery Need for efﬁciently handling the ultra-large population of mobile things able to provide a sought service Grid as a Service Need for high-performance computing, e.g., computationally-intensive algorithms or big-data applications Enactment Engine Need to deploy and reconﬁgure tens to thousands of services with geographically dispersed users, systems, and services while offering primitives for QoS management Table 3.1: CHOReOS middleware components and associated requirements fulﬁlled Table 3.1: CHOReOS middleware components and associated requirements fulﬁlled After deciding which CHOReOS middleware components to use, the development team will need to study the programmatic interfaces of each of the components and develop software that meets their speciﬁcations. A working installation of the CHOReOS middleware is also required; so, ei- ther the developer must have access to a previously conﬁgured CHOReOS installation (e.g., each company could maintain a CHOReOS installation that is shared among several projects) or they will need to download, build, and deploy the desired middleware components. Detailed development and installation guides for the middleware can be found at http://www.choreos.eu/bin/view/ Documentation/WebHome. 2.4.1. The CHOReOS Enactment Engine 1..* 1 1 1..* 1 1..* Figure 2.7: Enactment Engine components Choreography Deployer AWS, OpenStack, etc. «component» 1 Deployment Manager Cloud Gateway Figure 2.7: Enactment Engine components Cloud Gateway creates and destroys virtual machines (also called nodes) in a cloud computing envi- ronment. This component is used by the Deployment Manager, which decides when to create or destroy the nodes. Currently, Amazon EC2 and OpenStack are supported as cloud gateways, but the Deployment Manager can be easily extended to support other platforms. Chef Solo is a conﬁguration agent installed in each node. It runs scripts for the installation of services and required middleware components. Such scripts are also called Chef recipes and are written in a Ruby-based domain-speciﬁc language. Chef Solo is a conﬁguration agent installed in each node. It runs scripts for the installation of services and required middleware components. Such scripts are also called Chef recipes and are written in a Ruby-based domain-speciﬁc language. Deployment Manager deploys services in a cloud environment. Through its services API, the De- ployment Manager receives a declarative service speciﬁcation and selects the node onto which the service will be deployed, possibly considering non-functional requirements of the service. The Deployment Manager converts the received speciﬁcation to a script that implements the service preparation and launching processes. Using the nodes API provided by the Deployment Manager, one can request the upgrade of a node, which consists of running speciﬁc Chef Solo recipes on the speciﬁed node, and thus deploying services on the node. Choreography Deployer exposes the choreographies API to provide support for the automated de- ployment of service choreographies or orchestrations (for our purposes, an orchestration here can be seen as equivalent to a simpliﬁed choreography in which coordination is centralized). The Choreography Deployer client must provide the choreography declarative speciﬁcation, which con- tains the choreography architectural description and the locations of service packages. Based on this speciﬁcation, the Choreography Deployer coordinates invocations to the multiple Deployment Managers belonging to the different participant organizations. When services are already running, the Choreography Deployer invokes consumer services, injecting on them the addresses of their dependencies. In the next chapter, we describe in more detail how the various components of the CHOReOS mid- dleware can be used by choreography developers to facilitate the coding and enactment of complex web service compositions. CHOReOS FP7-257178 17 17 CHOReOS FP7-257178 18 3.1.1. Composition and Estimation – How to Use The Composition and Estimation component is part of the XSC. It is speciﬁc to the thing- based service compositions. The source code of the project is available at (root)/trunk/ executable-service-composition/. Developers wishing to use this component should down- load the source code and run the following command: mvn clean install. The command down- loads the project’s dependencies and use the resulting jar as a library in their code. Information on the technical requirements are provided in the install ﬁles available with the source code. All compositions are speciﬁed semantically as mathematical formulas in the IoT ontologies, which are also available with the source code. This stems from the fact that all compositions are over sensing/ac- tuating services that are tightly related to the real world and rules of mathematics and physics. Developers are provided with two public APIs that allow them to request the alternatives to a missing service and compute the extracted formulas based on the measurements acquired from the alternative services. The APIs are presented below: • expandConcept(): The method requires no input as it extracts the requested at • expandConcept(): The method requires no input as it extracts the requested attribute from the request query. Using SPARQL queries the method extract the alternative concepts from the composition formulas and returns them to the user. Those concepts will them be send to the look-up component to ﬁnd the service instances that measure the new concepts. • computeQueryConcept(): The method requires no input as it uses the already extracted for- mulas from the call above. It acquires the measurements of the accessed services and sends them as input to the formulas. The result is then sent, internally to a fusion class for processing. The appropriate fusion/aggregation functions are speciﬁed in the IoT ontologies. The ﬁnal result is returned to the user as a SensorData object that can be parsed into a double. 3 How to Use the CHOReOS Middleware In the following sections of this chapter, we present an overview of how CHOReOS FP7-257178 19 to use each of the major components. to use each of the major components. to use each of the major components. The intended audience for this chapter are developers wishing to use the CHOReOS middleware to build their systems and applications, therefore, the remaining of this chapter contains low-level details of the installation and use of the middleware. Readers that are not interested in these speciﬁc details, may skip to Chapter 4 where the evaluation of the middleware is presented. 1 package org.ow2.choreos.abstractionGenerator.abstractionImpl.weatherforecastservice; 2 import org.ow2.choreos.abstractionGenerator.InputOutputDataTranslator; 3 import javax.jws.WebMethod; 4 import org.ow2.choreos.abstractionGenerator.Serialiazer; 5 import javax.jws.WebService; 6 import org.ow2.choreos.serviceRepresentation.functional.ServiceInterface; 7 import org.ow2.choreos.abstractionGenerator.BaseAbstractionImplService; 8 import org.ow2.choreos.abstractionGenerator.MappingsManipulation; 9 import org.ow2.choreos.abstractionGenerator.ClassCreation; 10 1 package org.ow2.choreos.abstractionGenerator.abstractionImpl.weatherforecastservice; 2 import org.ow2.choreos.abstractionGenerator.InputOutputDataTranslator; 3 import javax.jws.WebMethod; 4 import org.ow2.choreos.abstractionGenerator.Serialiazer; 5 import javax.jws.WebService; 6 import org.ow2.choreos.serviceRepresentation.functional.ServiceInterface; 7 import org.ow2.choreos.abstractionGenerator.BaseAbstractionImplService; 8 import org.ow2.choreos.abstractionGenerator.MappingsManipulation; 9 import org.ow2.choreos.abstractionGenerator.ClassCreation; 10 3.1.2. AoSBM Service Substitution – How to Use The AoSBM service substitution facility can be used to generate automatically a functional abstraction service that realizes the abstract interface, speciﬁed by a given functional abstraction. This task is performed via the AoSBM GUI (Figure 3.1). Speciﬁcally, the AoSBM curator must create a functional abstraction that represents a set of similar services, using the AoSBM abstraction-oriented organiza- tion facility [15]. After this step, the generation of the functional abstraction service can take place. The generation process takes, as input, the AoSBM representation of the abstract interface that is speciﬁed by the functional abstraction and produces a JAX-WS-compliant Java interface that provides the oper- ations, speciﬁed in the abstract interface. Listing 3.1 gives an example of the JAX-WS-compliant Java interface that is automatically generated for a functional abstraction that represents weather forecast services (more details regarding this example can be found in [11]). CHOReOS FP7-257178 20 Figure 3.1: AoSBM graphical user interface Figure 3.1: AoSBM graphical user interface 11 @WebService 12 public interface WeatherForecastServiceInterface{ 13 14 @WebMethod public GetDailyWeatherForecastResponseOut getDailyWeatherForecast(GetDailyWeatherForecastIn input); 15 16 @WebMethod public GetDailyWeatherForecastOnUpdateResponseOut getDailyWeatherForecastOnUpdate (GetDailyWeatherForecastOnUpdateIn input); 17 18 } Listing 3.1: JAX-WS-compliant Java interface for a functional abstraction that represents weather forecast services The AoSBM service substitution facility further generates the Java implementation of the JAX-WS- compliant Java interface. As shown in Listing 3.2, the Java implementation of the JAX-WS-compliant Java interface provides the implementation for each operation, speciﬁed in the abstract interface. The interface implementation extends the BaseAbstractionImplService class, which declares certain attributes that are used for the delegation of invocations that are made to the functional abstraction service to the concrete service that is hidden behind the functional abstraction service. Speciﬁcally, these attributes include the endpoint address of the hidden concrete service, and the mapping from the interface of the functional abstraction service to the interface of the hidden concrete service. 1 package org.ow2.choreos.abstractionGenerator.abstractionImpl.weatherforecastservice; 2 i 2 h b i G I O D T l Figure 3.1: AoSBM graphical user interface 11 @WebService 12 public interface WeatherForecastServiceInterface{ 13 14 @WebMethod public GetDailyWeatherForecastResponseOut getDailyWeatherForecast(GetDailyWeatherForecastIn input); 15 16 @WebMethod public GetDailyWeatherForecastOnUpdateResponseOut getDailyWeatherForecastOnUpdate (GetDailyWeatherForecastOnUpdateIn input); 17 18 } p y p g y ( y p ) @WebMethod public GetDailyWeatherForecastOnUpdateResponseOut getDailyWeatherForecastOnUpdate (GetDailyWeatherForecastOnUpdateIn input) Listing 3.2: Implementation of the JAX-WS-compliant Java interface The implementation of each operation follows the same pattern: • It has, as a parameter, an input object that encapsulates the input data for the operation, as speci- ﬁed by the given functional abstraction, and returns, as a result, an output object that encapsulates the input data, as speciﬁed by the given functional abstraction. • The operation loads the values of the attributes that are used for the delegation of invo- cations, from disk, by calling the LoadVariables() method, which is inherited from the BaseAbstractionImplService class; the values are stored in the form of a .ser ﬁle. • Then, the operation of the concrete service is invoked, by calling invokeConcreteService(). This method is also inherited from the BaseAbstractionImplService class; its major respon- sibilities are (1) to translate the data of the input object to the input parameters that are actually used for the call to the concrete service and (2) to perform the actual call. • Finally, the data that are returned from the call to the concrete service are translated, to produce the output object of the operation. This is done by calling the setOutput() method. • Finally, the data that are returned from the call to the concrete service are translated, to produce the output object of the operation. This is done by calling the setOutput() method. • As discussed in Section 2.1.2, the input/output data translation process is based on the mapping information that is speciﬁed by the given functional abstraction and it is done using the Java reﬂection framework. • As discussed in Section 2.1.2, the input/output data translation process is based on the mapping information that is speciﬁed by the given functional abstraction and it is done using the Java reﬂection framework. To substitute the concrete service that is hidden behind the functional abstraction service, the en- tity that is in charge of the substitution must call the adapt() operation that is inherited from the BaseAbstractionImplService class. The adapt() operation sets the interface name and the endpoint address of the service that is going to substitute the hidden concrete service. Moreover, it notiﬁes the impact analyzers that have previously registered to the functional abstraction service [11]. Listing 3.1: JAX-WS-compliant Java interface for a functional abstraction that represents weather forecast services 1 package org.ow2.choreos.abstractionGenerator.abstractionImpl.weatherforecastservice; 2 import org.ow2.choreos.abstractionGenerator.InputOutputDataTranslator; 3 import javax.jws.WebMethod; 4 import org.ow2.choreos.abstractionGenerator.Serialiazer; 5 import javax.jws.WebService; 6 import org.ow2.choreos.serviceRepresentation.functional.ServiceInterface; 7 import org.ow2.choreos.abstractionGenerator.BaseAbstractionImplService; 8 import org.ow2.choreos.abstractionGenerator.MappingsManipulation; 9 import org.ow2.choreos.abstractionGenerator.ClassCreation; 10 11 @WebService() 12 public class WeatherForecastServiceImpl extends BaseAbstractionImplService implements WeatherForecastServiceInterface{ 13 14 public WeatherForecastServiceImpl (){ 15 super(”WeatherForecastService”); 16 } 17 18 public GetDailyWeatherForecastResponseOut getDailyWeatherForecast(GetDailyWeatherForecastIn input){ 19 20 GetDailyWeatherForecastResponseOut result = new GetDailyWeatherForecastResponseOut(); 21 22 LoadVariables(); 23 24 ServiceInterface abstractionServiceInterface = interfaceMapping.getServiceInterface(abstractionName); 25 26 Object output = invokeConcreteService(input,”getDailyWeatherForecast”, abstractionServiceInterface); 1 package org.ow2.choreos.abstractionGenerator.abstractionImpl.weatherforecastservice; 2 import org.ow2.choreos.abstractionGenerator.InputOutputDataTranslator; 3 import javax.jws.WebMethod; 4 import org.ow2.choreos.abstractionGenerator.Serialiazer; 5 import javax.jws.WebService; 6 import org.ow2.choreos.serviceRepresentation.functional.ServiceInterface; 7 import org.ow2.choreos.abstractionGenerator.BaseAbstractionImplService; 8 import org.ow2.choreos.abstractionGenerator.MappingsManipulation; 9 import org.ow2.choreos.abstractionGenerator.ClassCreation; 10 11 @WebService() 12 public class WeatherForecastServiceImpl extends BaseAbstractionImplService implements WeatherForecastServiceInterface{ 13 14 public WeatherForecastServiceImpl (){ 15 super(”WeatherForecastService”); 16 } 17 18 public GetDailyWeatherForecastResponseOut getDailyWeatherForecast(GetDailyWeatherForecastIn input){ 19 20 GetDailyWeatherForecastResponseOut result = new GetDailyWeatherForecastResponseOut(); 21 22 LoadVariables(); 23 24 ServiceInterface abstractionServiceInterface = interfaceMapping.getServiceInterface(abstractionName); 25 26 Object output = invokeConcreteService(input,”getDailyWeatherForecast”, abstractionServiceInterface); 1 package org.ow2.choreos.abstractionGenerator.abstractionImpl.weatherforecastservice; 2 import org.ow2.choreos.abstractionGenerator.InputOutputDataTranslator; 3 import javax.jws.WebMethod; 4 import org.ow2.choreos.abstractionGenerator.Serialiazer; 5 import javax.jws.WebService; 6 import org.ow2.choreos.serviceRepresentation.functional.ServiceInterface; 7 import org.ow2.choreos.abstractionGenerator.BaseAbstractionImplService; 8 import org.ow2.choreos.abstractionGenerator.MappingsManipulation; 9 import org.ow2.choreos.abstractionGenerator.ClassCreation; 10 11 @WebService() 12 public class WeatherForecastServiceImpl extends BaseAbstractionImplService implements WeatherForecastServiceInterface{ 13 14 public WeatherForecastServiceImpl (){ 15 super(”WeatherForecastService”); 16 } 17 18 public GetDailyWeatherForecastResponseOut getDailyWeatherForecast(GetDailyWeatherForecastIn input){ 19 20 GetDailyWeatherForecastResponseOut result = new GetDailyWeatherForecastResponseOut(); 21 22 LoadVariables(); 23 24 ServiceInterface abstractionServiceInterface = interfaceMapping.getServiceInterface(abstractionName); 25 26 Object output = invokeConcreteService(input,”getDailyWeatherForecast”, abstractionServiceInterface); CHOReOS FP7-257178 21 27 28 setOutPut (output, abstractionServiceInterface, ”getDailyWeatherForecast” , result); 29 30 return result; 31 32 } 33 34 public GetDailyWeatherForecastOnUpdateResponseOut getDailyWeatherForecastOnUpdate(GetDailyWeatherForecastOnUpdateIn input){ 35 36 GetDailyWeatherForecastOnUpdateResponseOut result = new GetDailyWeatherForecastOnUpdateResponseOut(); 37 38 LoadVariables(); 39 40 ServiceInterface abstractionServiceInterface = interfaceMapping.getServiceInterface(abstractionName); 41 42 Object output = invokeConcreteService(input,”getDailyWeatherForecastOnUpdate”, abstractionServiceInterface); 43 44 setOutPut (output, abstractionServiceInterface, ”getDailyWeatherForecastOnUpdate” , result); 45 46 return result; 47 48 } 49 } Listing 3.1: JAX-WS-compliant Java interface for a functional abstraction that represents weather forecast services The AoSBM service substitution facility further generates the Java implementation of the JAX-WS- compliant Java interface. As shown in Listing 3.2, the Java implementation of the JAX-WS-compliant Java interface provides the implementation for each operation, speciﬁed in the abstract interface. The interface implementation extends the BaseAbstractionImplService class, which declares certain attributes that are used for the delegation of invocations that are made to the functional abstraction service to the concrete service that is hidden behind the functional abstraction service. Speciﬁcally, these attributes include the endpoint address of the hidden concrete service, and the mapping from the interface of the functional abstraction service to the interface of the hidden concrete service. 3.2. XSA – How to Use 3.2.1. XSB – How to Use This section provides detailed instructions on setting-up and using the eXtensible Service Bus. Addi- tional information are provided on-line available on: http://choreos.eu/bin/Documentation/ Extensible_Service_Bus and in deliverable [14]. Technical Requirements To execute the XSB Binding Components with EasyESB the main require- ments are: 1) Download Java JDK 1.6 2) Download maven 2.2.1 3) Download and unzip the Rio ( http://www.rio-project.org ) tarball 3) Download and unzip the Rio ( http://www.rio-project.org ) tarball Setting-up the Development Environment The setup of the XSB Binding Components on a develop- ment environment like Eclipse, requires some steps. For example, the XSB DPWS Binding Component requires the following: 1) Download the sources of the corresponding B.C.: connectors/clientServer/WebServices/DPWSBindingComponent/easyesbjmeds 2) Add this repository to your settings.xml ﬁle. (Table 3.2): 2) Add this repository to your settings.xml ﬁle. (Table 3.2): <repository> <id>choreos-petalsLink</id> <name>choreos maven repository</name> <url>http://maven.petalslink.com/repo</url> </repository> <repository> <id>choreos-petalsLink</id> <name>choreos maven repository</name> <url>http://maven.petalslink.com/repo</url> </repository> 1) Start the application that receive the one-way invocation: java -jar Easyesbjmeds-1.0-SNAPSHOT-OneWaySystem.jar Listing 3.2: Implementation of the JAX-WS-compliant Java interface A simpler alternative that does not involve the notiﬁcation of impact analyzers amounts to calling the setServiceInterface() and the setServiceInstance() operations, which set, respectively, the name and the the endpoint address of the service that is going to substitute the hidden concrete service. The generation of functional abstraction services makes use of the JAX-WS wsimport tool. Specif- ically, the tool is used for the creation of the JAX-WS stub classes, needed for the invocation of the concrete service that is hidden behind the functional abstraction service. Detailed examples of functional abstraction services can be found in the CHOReOS AoSBM dis- tribution ((root)/trunk/extensible-service-discovery/AoSBM/src/main/resources/ generatedAbstractionImplementations/). CHOReOS FP7-257178 22 Moreover, examples of clients that use and adapt the generated functional abstraction ser- vices can be found in ((root)/trunk/extensible-service-discovery/AoSBM/src/main/ resources/clientForInvokingGeneratedAbstractions/). Table 3.2: Choreos maven repository 3) Execute mvn install to obtain the binding, or mvn install -Pdistrib to obtain a easyESB distribution with the DPWS Binding Component. 4) Run target/easyesbjmeds/bin/startup.bat to start the easyESB distribution with the DPWS Binding Component. 4) Run target/easyesbjmeds/bin/startup.bat to start the easyESB distribution with the DPWS Binding Component. Using the Application You can verify the binding component by running two scenarios. In the ﬁrst scenario, two DPWS applications have a one-way exchange through the XSB Binding Components. The ﬁrst application sends a notiﬁcation that is received by the second application as a one-way invo- cation. You can run the above scenario by following the following steps: CHOReOS FP7-257178 23 2) In the menu, choose option 1 to start the system. 3) Start an easy ESB node with the DPWS Binding Component. 4) Deploy the corresponding service unit: src/test/resources/oneWay/UnitOneWay.xml 5) Start a second easy ESB node with a DPWS Binding Component. 5) Start a second easy ESB node with a DPWS Binding Component. 6) Deploy the service unit of the system that sends the notiﬁcation: src/test/resources/notification/UnitNotification.xml 6) Deploy the service unit of the system that sends the notiﬁcation: src/test/resources/notification/UnitNotification.xml 7) Start the application that sends the notiﬁcation: 7) Start the application that sends the notiﬁcation: 7) Start the application that sends the notiﬁcation: java -jar Easyesbjmeds-1.0-SNAPSHOT-NotificationSystem.jar java -jar Easyesbjmeds-1.0-SNAPSHOT-NotificationSystem.jar 3.2.2. EasyESB – How to Use The EasyESB user manual is fully described in the deliverable D5.3.2 [14]. Additional relevant user guides and requirements of the EasyESB Bus are provided at http://choreos.eu/bin/ Documentation/easyesb. The bus is open source, both source code and binaries are available. Both Linux and Windows distributions are provided. A list of speciﬁc user guides about administrating the bus, connecting it to new nodes, etc., is available at https://research.petalslink.org/ display/easyesb/Specific+How-tos. Sensor Access Middleware – How to Use Response JSON message { "sensorDataResponse":{ "timestamp":1378975233604, "value":"75.878", "dataType":"org.ow2.choreos.sensordata.double.noiselevel" } } Table 3.4: Response containing the sensing value Sensor Access Middleware – How to Use This section describes the requirements of the Sensor Access Middleware component and provides de- tailed instructions on setting-up and using the project. Additional information are provided online and is available at http://choreos.eu/bin/view/Documentation/Sensor_Access_Middleware. Technical Requirements The Sensor Access Middleware component is written in Java and imple- mented as an Android application. The Android operating system is chosen among others for its open- source nature and the ﬂexibility it provides as a development platform. For running the application on a mobile device, the lowest supported Android version is the 2.3 Gingerbread. According to Google, over 97.6% of the enabled Android devices use an Android version higher than 2.3. Setting-up the Development Environment The set-up of the application project in a development environment does not require any special procedure. The project just needs be imported to the active workspace, for Eclipse use File →Import →Existing Projects into Workspace. A JDK 1.6 or higher and the Android SDK are required to achieve a successful set-up. Using the Application To run the application, a compatible Android device or an emulator is needed. However, since the Android emulator introduces some issues regarding sensors, the use of a real device is encouraged. Prior to starting the Things services on the Phone, the remote addresses (URLs) of the Phone Services Proxy and the Registry Manager should be conﬁgured through the settings window in the application. Communication with the proxy is required to guarantee the access to the Things services as discussed in Section 2.2.3.2. The Registry Manager is contacted by the application to execute the registering and un-registering procedures. After starting the Things services (by clicking the enable button in the app), the onboard REST server starts automatically and exposes the deﬁned API. This API is summarized on Table 3.3, where the IP address and port are the ones assigned to the device when connected to a local network: CHOReOS FP7-257178 24 URI Method Parameters http://128.0.0.192:8080/_servicename_ GET getnoiselevel, getlocation, gettemperature, etc. Table 3.3: Sensor access middleware REST API Table 3.3: Sensor access middleware REST API Responses to sensing requests are sent back to requesters as JSON messages. Each response message contains (i) a timestamp deﬁning the time of the sensing, (ii) the sensing data type, e.g., the CHOReOS custom datatype of noise level, and (iii) the value itself. An example response is shown in Table 3.4. Table 3.6: Response for pending requests In the case that any services are requested from a device, a sensing session starts in order to gather new data from the sensors. When ready, the data are sent back to the proxy using a POST call (sendresponse). The body of an example call of this type is shown in Table 3.4. The newly received data are forwarded back to the requester as a response to the initial request. If the service requested is unavailable or takes too long to respond, a timeout occurs on the requester. Phone Services Proxy – How to Use Phone Services Proxy – How to Use As described in Section 2.2.3.2, the Phone Services Proxy is designed to provide a robust and complete access solution to remote Thing services. Therefore, by deﬁnition, it acts as an intermediate between the devices providing the services and the requesters, being clients or applications. To guar- antee the ﬂexibility and interoperability of these communications the proxy is based on a deﬁned REST API. Additional information are provided online and is available at http://choreos.eu/bin/view/ Documentation/Phone_Services_Proxy. Technical Requirements The Phone Services Proxy is a Web application project written in Java. It is packaged as a war ﬁle and can be deployed on a Tomcat server (version 6 or higher). Setting-up the Development Environment The proxy server is developed based on the Maven project management tool. Hence, the setup of the web application on a development environment like Eclipse, requires a working installation of Maven (version 2 or higher). After the typical importing procedure to the workspace of the environment, the execution of the mvn clean install command is required for the download of the dependencies and the building of the project. Using the Proxy After the deployment of the Proxy server, it is ready to start serving requests for Things services access. A requester uses the corresponding URI deﬁned in the API (Table 3.5) to issue a request. Included in this request are the device’s unique ID and the name of the service (e.g., getnoiselevel). A pending requests data structure inside the proxy is used for keeping the requests for all devices and their services. Each device that supports the Proxy contacts it periodically and checks if any new requests are avail- able concerning any of the Thing services offered. This is done with the use of the pendingrequest CHOReOS FP7-257178 25 Table 3.5: Phone services proxy REST API method of the proxy’s deﬁned API. When the pendingrequests call is received on the proxy, the re- spective data structure is checked and, if any requested services are found for this device, a response is constructed containing a JSON message that includes them. An example of such a response is shown in Table 3.6. Response for pending requests { "pendingRequests":[ { "serviceName":"getnoiselevel" } ] } Table 3.6: Response for pending requests Table 3.6: Response for pending requests 3.3.1. AoSBM – How to Use 3.3.1. AoSBM – How to Use 3.3.1. AoSBM – How to Use At a glance, the usage of the AoSBM concerns two key actors: (1) the AoSBM curator, who is respon- sible for the registration of information about available services and the organization of the services with respect to abstractions and (2) the AoSBM user, who uses the AoSBM to perform service lookup queries. In the ﬁrst place, the AoSBM curator is supposed to install and conﬁgure the AoSBM in one or more nodes. This task is fairly easy; detailed instructions can be found in the CHOReOS instal- lation and user guides [14], in the CHOReOS courseware [17], and at the CHOReOS documenta- tion Web site (http://choreos.eu/bin/Documentation/Abstraction_Oriented_Service_ In the ﬁrst place, the AoSBM curator is supposed to install and conﬁgure the AoSBM in one or more nodes. This task is fairly easy; detailed instructions can be found in the CHOReOS instal- lation and user guides [14], in the CHOReOS courseware [17], and at the CHOReOS documenta- tion Web site (http://choreos.eu/bin/Documentation/Abstraction_Oriented_Service_ Base_Management). Following, the AoSBM curator must populate the AoSBM with information about available service descriptions and organize this information with respect to abstractions. The ser- vice registration and organization tasks can be performed via the AoSBM GUI; again detailed instruc- tions concerning these tasks can be found in the CHOReOS installation and user guides [14] in the tion Web site (http://choreos.eu/bin/Documentation/Abstraction_Oriented_Service_ Base_Management). Following, the AoSBM curator must populate the AoSBM with information about available service descriptions and organize this information with respect to abstractions. The ser- vice registration and organization tasks can be performed via the AoSBM GUI; again detailed instruc- tions concerning these tasks can be found in the CHOReOS installation and user guides [14], in the CHOReOS courseware [17], and at the CHOReOS documentation Web site. Once the AoSBM is setup, there are two main usage options for the AoSBM users who wish to execute service lookup queries. The ﬁrst option is to write down a WSBQL query, load the query via the AoSBM GUI and execute it. As before, these tasks are demonstrated in detail in the CHOReOS installation and user guides [14] and in the CHOReOS courseware [17]. Moreover, a document that refers to the syntax and semantics of WSBQL is available at the CHOReOS documentation Web site. LSB Binding Components – How to Use An LSB binding component (BC) acts as an intermediate between the service it is binded to and the outside world. Any outgoing calls from the service will go through the binding component and the same occurs for incoming connections. By doing so, the messages are abstracted from the speciﬁc protocol and interaction paradigm to the generic type and then transmitted using a common bus, in this case REST. A BC can be attached to a service just by changing the addresses of the calls. The initial step is to conﬁgure the destination address on the service that initiates the call (e.g. Application A in Figure 2.5) and then the BC itself should be conﬁgured to include the ﬁnal destination address of the messages. Additional information are provided online and is available at http://choreos.eu/bin/ view/Documentation/Binding_Components_LSB. Technical Requirements A binding component reserves two TCP ports (8090, 8092), used for re- ceiving the incoming requests from the common bus and the service it is attached to. Setting-up the Development Environment The project does not require special conﬁguration in any development environment. After the typical import, the command mvn clean install should be executed to download the external libraries required. The binding components for LSB are written in Java, thus a working JVM is needed for the execution. Extending the Binding Components As described in section 2.2.3.3 the ﬁrst layer of a binding com- ponent for LSB is the middleware connector, which is responsible for handling the communication with CHOReOS FP7-257178 26 the service connected. In its current implementation the binding components support some of the dom- inant protocols in the IoT domain, which are used to provide the proof of concept. However, a developer could easily extend the support of any type of service to the binding components. This can be done by conﬁguring the middleware connector so as to map the outgoing requests and the incoming calls between the speciﬁc protocol of the service and the paradigm adapter layer. The rest of the procedure is handled automatically by LSB. the service connected. In its current implementation the binding components support some of the dom- inant protocols in the IoT domain, which are used to provide the proof of concept. However, a developer could easily extend the support of any type of service to the binding components. 3.3.1. AoSBM – How to Use The second option for using the AoSBM query facilities is via the QueryEngineService REST API that we developed for this speciﬁc purpose. The modus operandi of the API is explained in depth in [15]. Moreover, a Java doc that explains the functionalities that are provided can be found in the CHOReOS AoSBM distribution ((root)/trunk/extensible-service-discovery/AoSBM/doc/). LSB Binding Components – How to Use This can be done by conﬁguring the middleware connector so as to map the outgoing requests and the incoming calls between the speciﬁc protocol of the service and the paradigm adapter layer. The rest of the procedure is handled automatically by LSB. 3.3.2. Things Discovery – How to Use The Things Discovery component enables the registration and look up for thing-based services, mostly sensors/actuators services. The component is to be used by developers wishing to advertise thing- based services to the Registry Manager and developers wishing to provide applications that consume the registered services. Users are required to integrate the TD components, namely the RegistrationManager and the QueryManager with their application’s code. The source code is available at the following address: (root)/trunk/extensible-service-discovery/. For each of the projects, developers should run a mvn clean install to acquire all the project’s dependencies. Developers can then add the resulting jar ﬁles as libraries in their code (More information is provided in the install ﬁles provided with the source code of the RegistrationManager and QueryManager). Finally, the address of the RegistryManager should be provided by the user. Users who wish to advertise their services can do so through the executeRegistrationQuery() method by providing the following: the deviceId, the name of the service, the type of the service, the type of the sensor/actuator, the concept it measures/acts on, the type of the data the service produces, the current location of the device, its expected mobility path and the unit of the provided measurement. CHOReOS FP7-257178 27 We are currently working on decreasing the set of parameters by extracting some of them directly from the IoT ontologies. It is possible that the component declines the developer’s request to register its services for a duration of time. This is due to our probabilistic computations that allow service instances to register if needed only. The decision is valid for the request time and location only. As such, the developer can retry at later periods. The method returns true if registration was successful and false otherwise. Users developing applications that consume registered services have three alternatives to discover- ing those services: • findServices() method. The method takes the attribute to measure/act upon and the location of interest as input. It returns the addresses of all services the satisfy the request. • findServices() method. The method takes the attribute to measure/act upon and the location of interest as input. It returns the addresses of all services the satisfy the request. • findSubsetOfServices() method. The method takes, as input, the attribute to measure/act upon, the location of interest and the desired spatial probability distribution of devices hosting the services and the number n of the required services. 3.3.2. Things Discovery – How to Use It returns the addresses of n services that satisfy the request. For the current version of the work, we support two distribution types: Normal distribution if the location of interest is a point in space (e.g., an intersection on the road), Uniform distribution if the location of interest is an area (e.g., Paris city). • findSubsetOfServicesBasedOnCoverage() method. The method takes the attribute to measure/act upon, the location of interest and the spatial probabilisty distribution of devices host- ing the services and the minimum required coverage percentage, i.e., the minimum acceptable percentage of the area to be covered/sensed by the selected services. It returns the addresses of a number of services, unknown a priori, that satisfy the request. The Registry Manager is a Web Service that developers should deploy in order to be able to reg- ister their sensing/actuating services. To deploy and run the Registry Manager, users need to down- load the source code available at (root)/trunk/extensible-service-discovery/registry_ manager/ and run the mvn clean install command. This will generate a war ﬁle that should be deployed in a Tomcat server. 3.4. Cloud and Grid – How to Use In this section, we focus on describing brieﬂy the required steps to use the CHOReOS Cloud Enact- ment Engine to deploy a web service choreography. The use of the Grid middleware, as explained in Section 2.4, was not the focus of the last year of the CHOReOS project; readers interested in its details can refer to deliverable D3.2.1 and the online documentation at http://choreos.eu/bin/view/ Documentation/grid_doc. This section is not aimed to make a developer fully capable of using the Enactment Engine, but rather to provide an overall idea about the technical work necessary to setup and use the Enactment Engine. Detailed and comprehensive instructions are provided by the Enactment Engine User Guide, avail- able at http://choreos.eu/bin/Documentation/enactment_engine_doc; for the interested reader, we highly recommend downloading the User Guide from this page. In the current document, we call deployer the Enactment Engine user, i.e., the one who wants to deploy a choreography, and the target environment the machines where services are deployed. Such terminology is in accordance to the OMG’s component-based systems deployment standard [26]. Note that if you use CHOReOS full capabilities, the deployer may be the CHOReOS system itself. After downloading and compiling the Enactment Engine source code, it is necessary to conﬁgure the Enactment Engine, providing it some information about the node allocation policy (how services must be distributed among different nodes) and the used cloud gateway. A cloud gateway here is just any service capable of providing new virtual machines on demand. In this step, one can enable or not some extra features of the Enactment Engine, such as automatically destroying inactive nodes, monitoring nodes with Ganglia, monitoring services with the EasyESB, linking an EasierBSM instance to the EasyESB nodes to be created, and so on. All of this conﬁguration is performed by editing text properties ﬁles. Once the Enactment Engine is running, the deployer must prepare the Enactment Engine input. Such input describes all the necessary information for choreography deployment, and it encompass mainly: in which kind of package each service is distributed (JAR, WAR, etc.), the package URL, which services depend on which services within the choreography, and in which cloud each service must be deployed. 3.3.3. Plugin Manager and Plugins – How to Use Plugins enable access to diverse service discovery protocols. All plugins must extend the AbstractPlu- gin class, thus deﬁning a new plugin type. The Plugin Manager can create multiple instances of the same plugin type. For example, for N UDDI registries the Plugin Manager would create N instances of the ”UDDI” plugin type. Each plugin instance has the responsibility to deliver service-related information from the underlying service registry to the Plugin Manager. Based on how the communication between the plugin and the service registry takes place, the plugins can be categorized by their supported dis- covery mode: Active, Passive, or both. Plugins that operate in ”passive discovery mode” are passive receivers of messages from the under- lying service registry. These messages indicate that a service has been added/removed/updated in the registry. As soon as a registry message is received, it is up to the plugin to inform the Plugin Manager accordingly. In the event of a service addition or update, the plugin must also provide the respective service description in a uniﬁed format. This ”uniﬁed format” is currently being deﬁned. On the other hand, plugins that operate in ”active discovery mode” must explicitly request service descriptions from the underlying registry. This mode is needed for registries that do not provide an eventing mechanism. Plugins that support this mode implement the retrieveServices() method, which is invoked by the Plugin Manager. The retrieveServices() method call results in the (indirect and asynchronous) retrieval of all services that match a speciﬁc ”ServiceFilter”, which is passed as a parameter. The exact format of the ”ServiceFilter” has yet to be formally deﬁned and will probably take into account numerous parameters like service semantics, underlying transport protocol, request timeout, upper/lower limit to the number of services returned etc. CHOReOS FP7-257178 28 In addition, a plugin could support both modes (e.g. DPWS/WS-Discovery) Finally, in order to evaluate the current Plugin Manager API, a plugin for a ”dummy” registry was developed. This ”dummy” registry acts as a daemon that reads service descriptions from a speciﬁc folder and informs its clients for new, updated and deleted services. Hence, the associated plugin operates in passive-discovery mode. 1The Java API actually uses the REST API under the hood. 3.4. Cloud and Grid – How to Use This description must adhere to the choreography speciﬁcation data model deﬁned by the Enactment Engine (Figure 3.2); it can be eother provided in XML format through the Enactment Engine REST API, or one can invoke the Enactment Engine using the Java API and build the choreography speciﬁcation using Java objects. After the choreography speciﬁcation is ready, the deployer must invoke the Enactment Engine by using the REST API or the Java API1. The Enactment Engine will then deploy the services on the target environment and bind the services, so they can call each other. Service binding is performed by invoking the setInvocationAddress operation provide by services. After the deployment is completed, the Enactment Engine returns a choreography description (Figure 3.2), which speciﬁes in which node each service was deployed and how one can access each one of the deployed services. This response is also returned in the XML format or as Java objects, depending on the deployer’s choice. While the deployed choreography is active, it is possible to perform modiﬁcations to it. For instance, one may decide to switch from using a service offered by one provider to a compatible service by a different provider, or to increase/decrease the number of deployed replicas of a given service in order 1The Java API actually uses the REST API under the hood. CHOReOS FP7-257178 29 -instanceId : String -nativeUri : String -easyEsbNodeAdminEndpoint : String ServiceInstance -packageUri : String -packageType : PackageType -endpointName : String -port : Integer -owner : String -group : String -numberOfInstances : Integer -version : String -resourceImpact DeployableServiceSpec -name : String -serviceType : ServiceType -roles : String ServiceSpec -serviceSpecName : String -serviceSpecRole : String ServiceDependency -serviceType : ServiceType -uri : String BusUri -URIs : String LegacyServiceSpec ChoreographySpec DeployableService -URIs : String LegacyService -id : String Choreography -nodeId : String -ip : String -hostname : String CloudNode COMMAND_LINE EASY_ESB TOMCAT PackageType «enumeration» SOAP REST «enumeration» ServiceType -uuid : String Service 1 1 0..* 0..* 0..* selected nodes 0..* 0..* 0..* 0..* 0..* 1 1 Figure 3.2: The Enactment Engine data model for choreography speciﬁcation ServiceSpec ServiceType PackageType -name : String -serviceType : ServiceType -roles : String BusUri ChoreographySpec CloudNode DeployableService Choreography d : String Figure 3.2: The Enactment Engine data model for choreography speciﬁcation to adapt to ﬂuctuations in usage load. 3.4. Cloud and Grid – How to Use The deployer simply uses the API again to submit an updated version of the choreography speciﬁcation to the Enactment Engine and requests the reenactment of the choreography. The Enactment Engine, in turn, detects the modiﬁcations made to the choreography and performs the requested modiﬁcations, by deploying new versions of services, removing service replicas etc. to adapt to ﬂuctuations in usage load. The deployer simply uses the API again to submit an updated version of the choreography speciﬁcation to the Enactment Engine and requests the reenactment of the choreography. The Enactment Engine, in turn, detects the modiﬁcations made to the choreography and performs the requested modiﬁcations, by deploying new versions of services, removing service replicas etc. to adapt to ﬂuctuations in usage load. The deployer simply uses the API again to submit an updated version of the choreography speciﬁcation to the Enactment Engine and requests the reenactment of the choreography. The Enactment Engine, in turn, detects the modiﬁcations made to the choreography and performs the requested modiﬁcations, by deploying new versions of services, removing service replicas etc. This capability, together with the ﬂexibility offered by the CHOReOS monitoring subsystem, presents the user with the framework necessary to adjust the run-time environment of the choreography accord- ing to QoS parameters and constraints, such as response time or cost. The monitoring subsystem is able to collect low-level data from virtual machines, such as disk and network bandwidth utilization or memory and cpu consumption, as well as higher-level data from the EasyESB bus, speciﬁcally several aspects related to service response time. This collected data is forwarded to the Glimpse Complex Event Processor [12, 13] for further processing. The CEP is a rule-based system which allows the user to deﬁne, at run-time, the event detection rules he is interested in together with the action to be taken whenever one such rule is triggered. These rules not only are able to identify QoS violations but also may correlate them with the collected low-level data, empowering the user to create different speciﬁc actions to be taken according to the context of each QoS violation. Such actions tipically consist of requests for the Enactment Engine to modify the running choreography (add or remove service repli- cas, migrate a service to a more or less powerful virtual node, etc.) to maintain the required QoS. An example is discussed in the already mentioned Enactment Engine User Guide. 4.1. Use-Case-Based Evaluation The three CHOReOS use cases served as a test-bed to demonstrate the use of the CHOReOS mid- dleware. Table 4.1 shows the use cases in which each middleware component or group of components was validated. Middleware UC-WP6 UC-WP7 UC-WP8 Component Airport ACRB DynaRoute AoSBM Service Discovery × × AoSBM Service Substitution × Cloud Enactment Engine × × × IoTS middleware × × × XSB × × EasyESB × × × Table 4.1: Use of the middleware components by the use cases Table 4.1: Use of the middleware components by the use cases 3.4. Cloud and Grid – How to Use CHOReOS FP7-257178 30 For each kind of service package, there are some rules to be followed. For example, JAR ﬁles must be runnable JARs, WAR ﬁles providing SOAP services must contain the sun-jaxws.xml ﬁle, and so on. Service implementation should also adhere to some minor requirements, such as logging on ﬁles, rather than on console, not relying on absolute paths of resources, not depending on run-time objects living on other services, and so on. Moreover, each service must properly implement the setInvocationAddress operation, so it can receive the addresses of its dependencies, that are other services within the choreography. The complete set of requirements and guidelines for using the Enactment Engine are available at http://choreos.eu/bin/Documentation/enactment_ engine_doc. CHOReOS FP7-257178 31 CHOReOS FP7-257178 32 4 Evaluation The evaluation of middleware components was based not only on use cases, but also on experiments and simulations. Note that when using the middleware components in the use cases, but also for experimental evaluation purposes, certain components are employed together as a group across the major modules (XSC, XSA, XSD, Cloud & Grid) of the CHOReOS middleware architecture. Thus, in the following sections: AoSBM Discovery and AoSBM Service Substitution are evaluated together; Things Composition & Estimation, Things Discovery, and LSB constitute the IoTS middleware, and are assessed as a whole; XSB and EasyESB are evaluated independently, but also, for part of its evaluation, XSB executes on top of EasyESB. Hence, we describe, next, AoSBM, Cloud Enactment Engine, IoTS middleware, XSB, and EasyESB evaluations, beginning with use-case-based ones and ﬁnally presenting experiment-based evaluations. 4.1.1. IoTS Middleware Use-Case-Based Evaluation The IoTS Middleware, including the Things Composition & estimation, Things Discovery and LSB, has been widely used by the three CHOReOS use-cases (UC-WP6, UC-WP7, UC-WP8) to facilitate the discovery and access of the Things-based services that take place in the deﬁned scenarios. This qualitative assessment targets to underline the contribution of the IoTS middleware in real-world envi- ronments that are exposed through the use-cases. The UC-WP6 Passenger Friendly Airport use-case concentrates to services provided to air trans- portation customers. It describes the collection and exploitation of information that appear in an airport environment towards adapting the services offered to travelers and hence, offering a better quality on traveling. The IoTS Middleware is used in this use-case to enable access of sensors appearing both in the environment and the travelers’ devices. For example, sound level sensors are used to indicate CHOReOS FP7-257178 33 the noise level at any time in the airport. That supports the automatic adjustment of the sound level during the security announcements at the airport (e.g. louder announcements when the airport is very crowded). Furthermore, location services that are deployed on the passengers’ devices are used by the airport authorities to provide directional information, making this way the navigation in the airport spaces more convenient. the noise level at any time in the airport. That supports the automatic adjustment of the sound level during the security announcements at the airport (e.g. louder announcements when the airport is very crowded). Furthermore, location services that are deployed on the passengers’ devices are used by the airport authorities to provide directional information, making this way the navigation in the airport spaces more convenient. The UC-WP7 use-case, named Adaptive Customer Relationship Booster (ACRB), addresses market- ing by leveraging the CHOReOS technologies. The IoTS Middleware was integrated to this use-case targeted to provide contextual information of the users (potential customers) to the marketing applica- tions. Speciﬁcally, the applications developed in the context of ACRB can use the IoTS Middleware to discover mobile devices of users appearing close to points of interest e.g., a store, and then request information from or take actions on them e.g., send promotional material. Example Things services that are supported are the location and the message delivery service. 4.1.1. IoTS Middleware Use-Case-Based Evaluation The ﬁrst of them is used to expose the current location of the mobile devices and the latter is used by the marketing applications for delivering promotional material to users that might be interested. The UC-WP8 DynaRoute use-case targets on assisting citizens in the transportation domain. Thing- based services are used in this context to provide contextual information about the stakeholders that take part in the scenario, such as the taxis, the taxi companies and the citizens. Speciﬁcally, location services are deployed on the taxi ﬂeet to expose at any time the current location of the taxis to the taxi company and the users. By doing so, the citizens can discover close-by taxis and furthermore, this way the taxi company can manage the ﬂeet more effectively. Additionally, in the same context of use, DynaRoute supports social interactions among users. Location services are deployed on users’ mobile devices enabling the DynaRoute application to identify physical proximity between friends. The ﬂexibility of the IoTS middleware facilitated the discovery and access of the Things services in the aforementioned implementations with minimal effort.Its lightweight nature managed the resource constraints implicated by the mobile devices, which have a signiﬁcant role in the use-cases, while keep- ing the performance at a high level. More details on the IoTS Middleware integration in the CHOReOS use-cases can be found in the deliverables related to them, namely, D6.5, D7.5 and D8.5 Despite the fact that the deﬁned use-cases offer a large number of services that could be the required context to perform a quantitative assessment, we concluded that an ultra-large-scale (ULS) evaluation based on simulation, would provide better insights on the performance and scalability of the IoTS Mid- dleware. The procedure taken and the results of this ULS evaluation are presented in Section 4.2.1. 4.1.2. XSB Use-Case-Based Evaluation The eXtended Service Bus (XSB) has been assessed as part of the CHOReOS runtime environment in supporting the development of the use cases UC-WP6 and UC-WP8. This is a qualitative evaluation and shows the capacity of XSB in enabling the interconnection of heterogeneous services, namely, services that employ middleware platforms which apply different interaction paradigms among the widely used client-server, publish-subscribe and tuple space. In particular in the UC-WP6 Passenger Friendly Airport use case, XSB is used for including, into the designed choreography of – in their majority – Web services following the client-server paradigm, ser- vices following other paradigms. More concretely, an air trafﬁc management notiﬁcation service using JMS publish-subscribe middleware for signaling a non-anticipated landing of a ﬂight is interconnected with the airport local management Web service. Additionally, an amenities (speciﬁcally, in terms of ho- tel accommodation) reservation service employing a JavaSpaces tuple space middleware for managing accommodation resources is interconnected with the airport local arrangements Web service, which books accommodation for the incoming passengers that have to spend the night. In the UC-WP8 DynaRoute use case, XSB is used for cross-connecting an airline ﬂight delay noti- ﬁcation service, which again employs a JMS publish-subscribe middleware, with the rest of the Web services choreography. The above implementations were facilitated by the high extensibility of the XSB framework, which CHOReOS FP7-257178 34 enabled developing binding components for all the interconnected services and their heterogeneous middleware platforms with minimal effort. Additionally, putting in place all the necessary adaptations at the business interface and data level of the interconnected services was signiﬁcantly facilitated by the application development support offered by the XSB framework, which again reduces considerably the developer’s effort. Finally, the good performance of the XSB running on top of the EasyESB allowed executing the global choreographies with low latencies that are compatible with the overall system requirements and the user’s perception of interactivity whenever the user is involved. More details about how XSB was used in each of the two use cases can be found in the CHOReOS deliverables related to the ﬁnal evaluation of the use-cases, namely, D6.5 and D8.5. Moreover, we have carried out a precise quantitative evaluation of XSB in terms of application development support, extensibility, and performance (including testing under stress conditions). The details concerning this evaluation and our results are given in Section 4.2.2. 4.1.3. EasyESB Evaluation EasyESB is the cornerstone component of the CHOReOS Middleware, consisting in the elementary brick deployed by the Enactment Engine. It is the basis of the EasyESB and XSB distributions and is fully integrated with the Cloud and Grid Middleware. More precisely, EasyESB provides a suitable sub- strate for the Interaction Binding Components of the XSB middleware. The EasyESB architecture has been reﬁned in order to support the building of Interaction Binding Components atop of it. Furthermore, we have provided interaction paradigms at the levels for Client Service, Publish Subscribe and Tuple Space. Finally, the interaction within Interaction Binding is supported by Generic Application on top of the EasyESB bus protocol. Consequently, evaluating both components on top of use case and simulation-based conditions in- volves evaluating EasyESB as well. Thus, we refer to sections4.2.2 and 4.2.6 for a general understand- ing of the evaluation test bed. Further to these, we have set the following tests based on both use case (WP7) and simulation evaluations. The following Table 4.2 shows the tests, the hardware conditions, and the behavior of EasyESB in such environments. The tests illustrate EasyESB’ capabilities at de- ployment time, and its capacity to scale up in hosting a large number of services on top of each single node. Consequently, connected EasyESB nodes are able to support an ultra large scale number of services without saturating the underlying hardware infrastructure. Topology Tests on a single pc including: EasyESB, the services, the GUI and the Proﬁler. Initial CPU CPU (Intel U7300 @ 1.30GHz) Used CPU CPU after all the services has been proxiﬁed 60% (30% per core) Initial RAM 4GB RAM, 3GB available for applications, 1 used by the OS+Desktop Used RAM 376M when started, 1625M after wrapping all the services Deployment Parameters ESB launched with params ”-Xmx2048M -Xss128K”. Use Case-based Simulation 60 services for Choreography 7 and 16 services for Choreography 4 (near 80 services). Simulation-based test 1100 services deployed on a single EasyESB node. Table 4.2: EasyESB evaluation We note also that EasyESB is a standalone bus that does not require the installation of further soft- ware such as for IBM and Oracle Solutions. Furthermore, EasyESB nodes can be deployed indepen- dently and at different times, forming a dynamic distributed topology. New nodes can join the current topology and nodes can be removed at runtime. 4.1.4. AoSBM Discovery and Service Substitution Use-Case-Based Evaluation The abstraction-oriented service organization and discovery facilities of the AoSBM have been as- sessed as part of the overall CHOReOS development process to facilitate the development of UC-WP6 and UC-WP7. The way that it has been used is discussed in D2.3 [15]. Brieﬂy, descriptions of services that have been developed in the context of UC-WP6 and UC-WP7 have been registered in the AoSBM. The service descriptions have been organized with respect to abstractions, using the AoSBM clustering facilities. The AoSBM querying facilities have been used in the synthesis process to retrieve information about available services that correspond to the participant roles of the choreographies involved in the use cases. As detailed in [15], the experience from the use of AoSBM in UC-WP6 and UC-WP7 further allowed to assess the AoSBM from a qualitative perspective. In particular, the precision and recall of the AoSBM facilities have been evaluated. Overall, the results showed that the AoSBM is able to mine abstractions that are relevant to the different participant roles, speciﬁed in UC-WP6 and UC-WP7, and that the mined abstractions can be used for the discovery of concrete services that are relevant to the different participant roles. The starting point for the assessment of the AoSBM service substitution sup- port was services developed in the context of UC-WP6. We have worked with functional abstraction services, inspired by roles speciﬁed in the Passenger Friendly Airport use case. The details concerning this study and our ﬁndings are given in Section 4.2.5. 4.1.3. EasyESB Evaluation By calling the function addNeighborNode(), nodes be- come aware of each other and can update their list of known services deployed on neighboring nodes. In Table 4.3 we list the memory usage of several ESBs for the storage and when started with a minimal conﬁguration. The table shows that EasyESB has a small footprint that enables its deployment on top We note also that EasyESB is a standalone bus that does not require the installation of further soft- ware such as for IBM and Oracle Solutions. Furthermore, EasyESB nodes can be deployed indepen- dently and at different times, forming a dynamic distributed topology. New nodes can join the current topology and nodes can be removed at runtime. By calling the function addNeighborNode(), nodes be- come aware of each other and can update their list of known services deployed on neighboring nodes. In Table 4.3 we list the memory usage of several ESBs for the storage and when started with a minimal conﬁguration. The table shows that EasyESB has a small footprint that enables its deployment on top CHOReOS FP7-257178 35 of restricted hardware devices. The modularity of the architecture of EasyESB confers a lightweight footprint that further allows holding only the needed functionalities in a speciﬁc distribution. In terms of memory usage, EasyESB can be stored in 50 MB of memory, in comparison with existing solutions, such as Mule ESB with 75 MB, IBM Websphere with 550-600 MB, and 1.1 GB for Oracle Service Bus. When started in its minimal version, EasyESB uses 300 MB, while Mule ESB consumes 768 MB, IBM Websphere uses 1GB to 2GB, and Oracle recommends 4GB. EasyESB Mule ESB IBM Websphere Oracle Service Bus Memory Usage 50MB 75MB 550-600MB 1.1GB Runtime Memory Usage (minimal conﬁguration) 300MB 768MB 1to 2 GB 4GB Table 4.3: EasyESB footprint 4.1.5. Cloud Enactment Engine Use-Case-Based Evaluation The Enactment Engine has been used, at least partially, by all three CHOReOS use cases. This usage has substantially helped in improving it, since this actual use aided us in ﬁnding bugs, in improving documentation (consolidated on the Enactment Engine User Guide available at the CHOReOS web site), and in prioritizing features to be developed, such as, for instance, the automatic proxiﬁcation of legacy services through the EasyESB bus, which is an ongoing implementation project to satisfy DynaRoute demands. The use case with more concrete results up until now in using the Enactment Engine is the Airport use case. The Enactment Engine has enabled the deployment of airport services on the Amazon cloud in an automated way. The Java code produced to enable the enactment of this choreography, by invoking Enactment Engine, encompasses both the choreography speciﬁcation and the enactment launch. The choreography speciﬁcation (Appendix, Listing A.1) is assembled with POJOs that follow the Enactment Engine data model, and it has 254 lines describing the choreography, which corresponds to 15 services. If we remove empty lines and method declarations (they exist just to better organize the speciﬁcation code) we will have in average 11 lines per service. The enactment launch (Appendix, Listing A.2) uses the Java API to invoke the corresponding Enactment Engine operation and is pretty straightforward, counting only around 30 lines. CHOReOS FP7-257178 36 The very reduced amount of code lines necessary to automate the deployment of each choreogra- phy gives an idea of how much work by the deployment team can be saved. Although some work is necessary to properly set up the Enactment Engine, this needs to be done just once; after this step, several choreographies may be deployed taking advantage of the Enactment Engine instance already conﬁgured. The Enactment Engine itself may also be conﬁgured by third parties and delivered as a service. 4.2. Experiment-Based Evaluation In addition to the evaluation based on the use cases mentioned above, some key middleware compo- nents were also validated based on stress testing and on simulations. These studies are described in the following subsections. 4.2.1. ULS Evaluation of IoTS Middleware The technical evaluation of the Internet of Things Services (IoTS) middleware proves to be challenging since it is necessary to ﬁnd an environment which is able to provide the tools for evaluating the scala- bility, heterogeneity, and mobility support aspects of the middleware. Speciﬁcally, it is imperative to ﬁnd an evaluation environment that is able to fulﬁll the following requirements: • Ultra-Large scale: The environment must provide the means for deploying the IoTS middleware in at least 10.000 real or simulated things. • Ultra-Large scale: The environment must provide the means for deploying the IoTS middleware in at least 10.000 real or simulated things. • Realistic deployment conditions: The environment must provide the means to deploy the mid- dleware components such as the Registry Manager under realistic conditions. • Time constraints: Ultra-Large Scale evaluations must be completed within reasonable time frames, ranging from a few hours up to a few days. • Mobility: The environment must provide mobility, or the means to simulate mobility accurately (e.g. Using mobility traces as input). In the following, we present the efforts towards the technical assessment of the Internet of Things Services (IoTS) middleware. The remainder of this section is structured as follows. In Section 4.2.1.1 we present a summary of the current assessment methodologies for technical assessment, as well as a short discussion on their applicability to the technical assessment of CHOReOS’ IoTS middleware. Section 4.2.1.2 provides a detail description of the chosen assessment strategy and its individual com- ponents, before presenting our evaluation results in Section 4.2.1.3, and our conclusions in Section 5. Background Throughout the years, the Networking community has developed different tools to provide realistic evaluation through simulation. Perhaps the most well-known is ns-2 [23], which is a discrete event sim- ulator aimed at networking research. Written in OTcl and C++, it is a widely used tool in the networking community with a great number of modules available for it. Another well-known discrete event simulator is OMNeT++ [33], which is entirely C++ based and also provides a large number of modules, with some good wireless sensor network simulators built on top of it. Nevertheless, these two tools are unﬁt for evaluating Inria’s IoTS middleware, since they only allow the deployment of applications written in C++. A less well-known tool is JiST (Java in Simulation Time) [1], developed at Cornell University. It was speciﬁcally designed for running unmodiﬁed Java network applications, and for providing a scalable simulation environment able to run on commodity hardware. Furthermore, as part of the same project, SWANS, a wireless ad-hoc network simulator was built on top of it. These simulation frameworks include basic mobility models, such as Random Waypoint [3], for allow- ing users validate their solutions in dynamic environments. They also allow the use of mobility datasets as input. Unfortunately, publicly available large-scale mobility datasets are scarce, even in well-known dataset sources such as CRAWDAD [29]. Because of the limitations of these simple models, and the limited availability of large-scale mobility traces, some research efforts from the Trafﬁc engineering and vehicular communications communities point towards developing tools for simulating macroscopic and microscopic mobility in both urban and rural areas. Such is the case of SUMO [2] and VanetMo- biSim [22], which not only allow the simulation of moving vehicles, but also pedestrians. Moreover, they allow importing actual maps for easily building large-scale mobility scenarios in real urban areas. Furthermore, the output of these simulations may be used as input for the simulators described above. Discussion As discussed in Page 37, testbed-only evaluations are not inline with CHOReOS’ needs, because of scale or time limitations. On the other hand, scalable simulation environments such as JiST, are good candidates for the ULS evaluation of CHOReOS IoTS middleware. In such an environment, the middleware could be evaluated in networks with millions of communicating devices. Background Traditionally, two main methodologies can be used to perform a thorough technical assessment of a solution: Testbed-based evaluation and Simulation-based evaluation. In the following we detail the strengths and drawbacks of both methods, before presenting a short discussion on their application for the technical assessment of the CHOReOS IoTS middleware. Testbed-Based Evaluation Over the years, testbed-based evaluation has proven to be a powerful tool for assessment in different domains. Perhaps its main advantage is that it provides the means for performing tests in real-time. In the particular ﬁeld of IoT, it also enables an easy evaluation of mobility and heterogeneity aspects. On the other hand, when owned, testbeds are expensive to build in both time and money. Moreover, because of these characteristics, scalability is limited. CHOReOS FP7-257178 37 However, because of the growing number of worldwide research projects oriented towards building the Future Internet, there are a number of publicly accessible testbeds. The largest one deployed so far is SmartSantander [27], which provides a city-scale testbed, currently composed of around 2000 ZigBee sensor nodes. Other large scale deployments include SensLAB [19] with around 1024 sensor nodes, both wired and wireless, across 4 different sites; KanseiGenie [30] with around 700 wired and wireless sensor nodes; and WISEBED [5] with 500 wireless and wired sensor across 5 federated testbeds. Even though some of these projects already provide large-scale numbers, they are still far from the minimum objective of 10,000 nodes. Other research efforts attempt at lowering the deployment costs by involving both government and industrial partners. Such is the case of PhoneLab at the University of Buffalo [25], where 200 Smart- phones where handed to students, staff, and faculty with the support of the NSF and Sprint. A different strategy is adopted by LifeMap at Yonsei University [31], where researchers made available a mobility monitoring application on the Android Market in an attempt to obtain crowdsourced mobility data. Even though this projects propose some interesting ideas for achieving ultra-large scales, they can only do so in the longterm. Simulation-Based Evaluation Often neglected in the Computer Science ﬁeld, simulation-based eval- uation is a ﬂexible tool that allows faster modeling and validation at lower costs. Moreover, it provides good scalability depending on the simulation tool and the CPU resources of the hardware it is deployed on. However, the main challenge is to ﬁnd both tools and models that yield sufﬁciently realistic results. Background However, the ULS aspect of the evaluation does not only concern simulating large numbers of smart things, but also the scalability of crucial components such as the Registry Manager. Along those lines, question on whether or not the simulator can “fairly” simulate these components appears naturally. Inside a sim- ulation environment, there is no actual way of ensuring resource allocation to simulated entities as it would be in the real world, where the Registry Manager would be a powerful machine with dedicated CHOReOS FP7-257178 38 Figure 4.1: Global IoTS evaluation environment architecture Figure 4.1: Global IoTS evaluation environment architecture resources. Moreover, a “hybrid” approach where simulating entities residing inside the simulation en- vironment communicate with a real-life entity, is not possible since simulated entities would be running in “simulated time”, whereas real-life entities would be running in “real time”. In other words, time will be “slower” for simulated entities than for real-life entities, thus providing incoherent results. Therefore, simulation-only evaluations are not ﬁt either for the assessment of the IoTS middleware. resources. Moreover, a “hybrid” approach where simulating entities residing inside the simulation en- vironment communicate with a real-life entity, is not possible since simulated entities would be running in “simulated time”, whereas real-life entities would be running in “real time”. In other words, time will be “slower” for simulated entities than for real-life entities, thus providing incoherent results. Therefore, simulation-only evaluations are not ﬁt either for the assessment of the IoTS middleware. Assessment Strategy To tackle the issues raised by testbed-only and simulation-only evaluation approaches, we propose an assessment strategy in which we combine ofﬂine mobility simulation and network trafﬁc generation with online real-time testbed evaluation. We propose the use of synthetic or real mobility traces to obtain mobility data and generate network trafﬁc. This information is then used to create emulated devices that will interact in real-time with other middleware components deployed on a testbed. In the following, we ﬁrst present the overall description of such an environment. The details of each component follow thereafter. Global System Architecture The global architecture of the proposed evaluation environment is de- picted in Figure 4.1. There are ﬁve main elements composing the system that can be divided into two categories: Assessment Harness Elements, and CHOReOS IoTS middleware elements. • Assessment Harness Elements: • TrafﬁcGenerator: It’s the main element of the Assessment Harness. It generates network trafﬁc based on data from a mobility trace, and it calculates the mobility of each device, represented by the path (list of times and coordinates) the device will follow over time. It generates a Launch Trace to be used by the NodeLauncher, as well as Query Trace to be used by the QueryLauncher. • NodeLauncher: Creates, launches, and stops instances of IoTNodes based on the Launch Trace generated by the TrafﬁcGenerator. • NodeLauncher: Creates, launches, and stops instances of IoTNodes based on the Launch Trace generated by the TrafﬁcGenerator. • QueryLauncher: Generates lookup and access queries based on the Query Trace gen- erated by the TrafﬁcGenerator. It is able to launch queries concurrently using the Query Manager component and measure their response time. CHOReOS FP7-257178 39 Figure 4.2: Example deployment of the device registry Figure 4.2: Example deployment of the device registry • CHOReOS IoTS Middleware Elements: Timestamp,Device ID,Longitude,Latitude Usually, mobility traces only contain the positions of a set of IDs over time. Therefore, no information on network trafﬁc that can be obtained directly from them. Thus, to generate trafﬁc, the TrafﬁcGenerator parses the mobility trace to obtain a time map, where, for each timestamp on the trace, there is a list of device IDs and their coordinates. Based on information from this map, the TrafﬁcGenerator will analyze the trace in order to ﬁnd the time(s) in the trace at which a user-inputed trafﬁc rate (lambda) is satisﬁed. Based on this information, the TrafﬁcGenerator calculates a Poisson arrival process to simulate Internet session trafﬁc [4]. At each Poisson arrival, represented by a timestamp, the TrafﬁcGenerator will look in the map for devices with a matching timestamp, and their coordinates at the time. If the coordinates of a node at a particular timestamp are not available, the TrafﬁcGenerator will look for the latest timestamp at which the node’s coordinates where known, and calculate the position of the node at the desired timestamp. Then, for each device, a list of subsequent timestamps and coordinates is obtained. by linear interpolation. This list represents the path the device will follow. The length of this list is deﬁned by the user at the start of the evaluation. Finally the arrival and path information is placed in a Launch Trace ﬁle that is used by the NodeLauncher to create each individual device, and which has the following format: • CHOReOS IoTS Middleware Elements: It obtains mobility information from either synthetic or real mobility CHOReOS FP7-257178 40 Figure 4.3: Internal operations of the NodeLauncher Figure 4.3: Internal operations of the NodeLauncher traces, and it generates network trafﬁc. In order to be used by our system, input mobility traces are required to have the following format: traces, and it generates network trafﬁc. In order to be used by our system, input mobility traces are required to have the following format: Timestamp,Device ID,Longitude,Latitude,Path The TrafﬁcGenerator also provides the input for the QueryLauncher. Based on the generated Launch Trace, particularly in the number of individual Device IDs in it, and the lowest and highest timestamps, the TrafﬁcGenerator will generate a Query Trace that will serve as an input for the QueryLauncher. As for the Launch Trace, the Query Trace follows a Poisson process, and is written with the following format: • CHOReOS IoTS Middleware Elements: • Device Registry: Deploys the Registry Manager component under realistic conditions. It stores information on the services provided by the devices, including a network address for each service and relevant metadata. It is also able to provide service metadata on-demand. • Device Registry: Deploys the Registry Manager component under realistic conditions. It stores information on the services provided by the devices, including a network address for each service and relevant metadata. It is also able to provide service metadata on-demand. • IoTNodes: These elements emulate devices running the Registration Manager component. They are created by the NodeLauncher. Each device performs a service registration query and measures its response time. Moreover, each registered device will execute a service corresponding to the one it has registered. In our assessment environment, each element runs as an independent process. This allows distribut- ing the elements in a computer cluster, thus yielding its large computing resources, and enables our environment to perform ultra-large scale evaluations. The execution of the environment is controlled by different shell scripts that allow launching and stopping the different elements, and ﬁne tuning system parameters, such as the memory used by the processes. In the following, we describe in detail each element composing our assessment environment. Device Registry: Storing Service Information One of the primary concerns raised by a simulation- only evaluation approach, is its ability to provide a fair evaluation of the Registry Manager, a component which, under real life deployment conditions, would be allocated to dedicated computing resources. Therefore, the ﬁrst step we undertook was to deploy the component as it would be deployed in a production system, thus ensuring adequate resource provisioning. To closely resemble production systems, the Device Registry is setup as a three-tier architecture such as the one depicted in Figure 4.2. In this setup, incoming requests are received by an HTTP web server (e.g. Apache HTTP) that performs simple load balancing through Round Robin Scheduling, and forwarded to a cluster of application servers (e.g. Apache Tomcat) hosting the Registry Manager. Once the registration request is processed, the registration information and metadata are stored in a networked database (e.g. Apache Derby). TrafﬁcGenerator: Mobility and Network Trafﬁc Generation The TrafﬁcGenerator is the main ele- ment of the assessment harness. Timestamp,Concept,Location • A registration ﬁle containing its Device ID, and the timestamps at which the device contacts the register in order to register itself. At the end of the simulation, each group of ﬁles will be merged into a single one according to their type for facilitating their analysis. At the end of the simulation, each group of ﬁles will be merged into a single one according to their type for facilitating their analysis. QueryLauncher: Service Lookup and Access The ﬁnal element in our environment is the Query- Launcher. This element will generate sensing queries to obtain information of a particular physical concept on a desired geographical location. As in the case of device generation, sensing queries follow a Poisson arrival process, which is provided as input in the form of a Query Trace. QueryLauncher: Service Lookup and Access The ﬁnal element in our environment is the Query- Launcher. This element will generate sensing queries to obtain information of a particular physical concept on a desired geographical location. As in the case of device generation, sensing queries follow a Poisson arrival process, which is provided as input in the form of a Query Trace. At each arrival extracted from the Query Trace, the QueryLauncher will create a thread. In each thread, a query is generated with the concept and location information from the trace, for then being passed to the Query Manager component. Creating threads allows the QueryLauncher to perform concurrent queries. The Query Manager component will then look in the Device Registry for devices hosting the requested service, and access them to obtain a numerical answer to the query. Each Query thread will generate as output several different CSV formatted ﬁles: • A main ﬁle containing the requested physical concept, the number of services to access, two timestamps when the query is launched and a response is received, and the result from the query. • A lookup ﬁle containing a thread number, and the timestamps at which the thread contacts the registry to ﬁnd out the information of the services providing the requested concept. • An access ﬁle containing a thread number, and the timestamps at which the thread attempts to access each IoTNode to retrieve data. At the end of the simulation, each group of ﬁles will be merged into a single one according to their type for facilitating their analysis. Timestamp,Concept,Location Both of these procedures are depicted in Figure 4.3. Once the TrafﬁcGenerator provides its output traces, both devices and queries will be launched by the control scripts. NodeLauncher and IoTNodes: Service Registration Once the mobility and network trafﬁc is gener- ated, devices are created. In our environment, each device is an independent lightweight process that we call an IoTNode. This allows distributing the execution of a large number of devices across multiple CHOReOS FP7-257178 41 machines, thus facilitating the scalability of the simulations. Each IoTNode process is launched by the NodeLauncher based on the Launch Trace generated by the TrafﬁcGenerator. When the IoTNode is launched, it will attempt to register its service(s) via the instance of the Regis- tration Manager component deployed on it. To this end, it will use the information on the type of sensor it possesses, its current coordinates, the path it will follow, and a coverage threshold deﬁned at the start of the simulation. The Registration Manager will decide whether the emulated device should register or not based on the coverage threshold, and the macroscopic mobility model deﬁned in the IoTS Mid- dleware (e.g. Truncated Levy Walk). If the registration decision is positive, the Registration Manager will register the device for a duration calculated using the device’s path, and the IoTNode sets up a REST server to handle requests for accessing its service(s). The address of each service is made up by the address of the host machine, a port number assigned by the NodeLauncher, and the name of the service. Once the REST server is setup, the IoTNode will calculate its lifetime based on its path. At the end of its lifetime the IoTNode will stop its REST server and the lightweight process will stop. Each generated device will generate as output several different CSV formatted ﬁles: • A main ﬁle containing its Device ID, the result from the registration query, a timestamp for when the registration request was made, and a timestamp for when the registration response was received. • A ﬁnd ﬁle containing its Device ID, and the timestamps at which the device contacts the registry to ﬁnd out how many devices are already registered. • A calculation ﬁle containing its Device ID, and the timestamps at which the registration decision calculations. Timestamp,Concept,Location At the end of the simulation, each group of ﬁles will be merged into a single one according to their type for facilitating their analysis. CHOReOS FP7-257178 42 Figure 4.4: Deployment used for performing ULS evaluations Figure 4.4: Deployment used for performing ULS evaluations Known Limitations The current version of our evaluation environment presents a number of limita- tions introduced by the hardware and operating system on top of which the evaluation is performed. Known Limitations The current version of our evaluation environment presents a number of limita- tions introduced by the hardware and operating system on top of which the evaluation is performed. The main limitation introduced by the hardware that supports the evaluation environment is memory availability. The memory available in the machine will limit the number of IoTNode processes that can run concurrently. Similarly, the conﬁguration of the OS on which the evaluations are run may impose limits on the number of concurrent processes a user might run. A different limitation comes from the maximum number of TCP ports available at an IP address. Even if the machine the evaluations are run on had unlimited memory resources, and the OS was conﬁgured to support unlimited processes for a user, each IoTNode needs to bind to a TCP port to provide access to its service. Therefore, the maximum number of available TCP ports imposes a limit on the number of IoTNodes that can be run concurrently on a single machine. Evaluation Results As input, we used our trace analysis tool to generate trafﬁc CHOReOS FP7-257178 43 200 400 600 800 1000 0 1000 3000 5000 Deterministic Probabilistic Input (req/s) Time (ms) Figure 4.5: Comparison of registration re- sponse times for different input rates 100 300 500 700 900 0 1000 2000 3000 4000 5000 Find Calculation Registration Input (req/s) Response Time (ms) Figure 4.6: Time analysis of probabilistic reg- istration for different input rates 200 400 600 800 1000 0 1000 3000 5000 Deterministic Probabilistic Input (req/s) Response Time (ms) Figure 4.7: Comparison of query response times for different input rates 100 300 500 700 900 0 1000 2000 3000 4000 5000 Find Calculation Registration Input (req/s) Response Time (ms) Figure 4.6: Time analysis of probabilistic reg- istration for different input rates 200 400 600 800 1000 0 1000 3000 5000 Deterministic Probabilistic Input (req/s) Time (ms) Figure 4.6: Time analysis of probabilistic reg- istration for different input rates Figure 4.5: Comparison of registration re- sponse times for different input rates 200 400 600 800 1000 0 1000 3000 5000 Deterministic Probabilistic Input (req/s) Response Time (ms) Figure 4.7: Comparison of query response times for different input rates 200 400 600 800 1000 0 1000 3000 5000 Deterministic Probabilistic Input (req/s) Response Time (ms) Figure 4.7: Comparison of query response times for different input rates 200 400 600 800 1000 0 1000 3000 5000 Deterministic Probabilistic Input (req/s) Response Time (ms) Figure 4.7: Comparison of query response times for different input rates from most populated sections of the original Cologne trace [32]. We ﬁrst measured the response times using both deterministic registration and Probabilistic registra- tion with a coverage threshold of 80%. Figure 4.5 presents these results. Clearly, when the system uses deterministic registration, response times are lower than when using probabilistic registration since no calculations for reaching a registration decision are involved. Nevertheless, probabilistic registration still delivers reasonably fast response times. In order to better understand the impact of the operations involved in probabilistic registration on the total response time, we calculated the response time of each one of the operations. Evaluation Results This section presents the evaluation results obtained by our evaluation environment. The main objective of our evaluations is assessing the ability of the system of handling large loads of registration and lookup, and access queries. To this end we measure the response times of both types of queries for both deterministic and probabilistic registration, and deterministic and probabilistic lookup. We performed these evaluations using a synthetic mobility trace generated with SUMO [2], which uses real mobility data for generating the individual mobility of over 120.000 vehicles during a period of two hours in the city of Cologne [32]. To our knowledge, this is the largest realistic mobility trace freely available at the time of writing. We deployed our system in Inria’s computer cluster. The system’s elements were distributed inside the cluster to maximize resource utilization. The deployed architecture is depicted in Figure 4.4. For our experiments we conducted two sets of results. The ﬁrst set aims at comparing the perfor- mances of a system using deterministic registration only, with a system where the CHOReOS IoTS middleware is deployed, and therefore probabilistic methods are used for smartly reducing the number of devices to handle. For this set of results, we varied the input rate to the system from 100 requests per second up to 1000 requests per second. The results show averages from 20 runs and we have calculated the 95% conﬁdence intervals. Evaluation Results These are the Find operation, which retrieves the number of already devices offering the same service that a device is attempting to register; the Calculation operation, which conducts the necessary calculations to generate a registration decision; and the Registration operation, which effectively attempts to register a device that has reached a positive registration decision. The results are depicted in Figure 4.6. As seen in the ﬁgure, the response time of both Calculation and Registration operations, is somewhat stable with respect to the input rate, taking around 200 ms in both cases. Nevertheless, the response time of the Find operation grows as the input grows. This behavior is due mainly due to two different reasons. First, as the input grows, the number of devices concurrently accessing the Device Registry grows as well, thus having a negative impact on the response times. Second, as the number of already registered devices grows, the time needed for retrieving this information from the Device Registry database is larger. However, this delay is dependent solely on the search algorithms implemented on the database. We next proceeded to measure the response times using both deterministic Lookup and Access and Probabilistic Lookup and Access with a coverage threshold of 80%. Figure 4.7 clearly shows the impact of the smart probabilistic methods employed by the CHOReOS IoTS middleware on query response CHOReOS FP7-257178 44 100 300 500 700 900 0 1000 2000 3000 4000 5000 Lookup Access Input (req/s) Response Time (ms) (a) Query response times using Deterministic Lookup and Access 100 300 500 700 900 0 1000 2000 3000 4000 5000 Lookup Access Input (req/s) Response Time (ms) (b) Query response times using Probabilistic Lookup and Access Figure 4.8: Time analysis of query response times for different input rates 100 300 500 700 900 0 1000 2000 3000 4000 5000 Lookup Access Input (req/s) Response Time (ms) (b) Query response times using Probabilistic Lookup and Access (a) Query response times using Deterministic Lookup and Access (b) Query response times using Probabilistic Lookup and Access Figure 4.8: Time analysis of query response times for different input rates Registration Lookup+Access 0 10 20 30 40 Deterministic Prob. Registration + Det. Evaluation Results Access Probabilistic Response Time (s) Figure 4.9: Comparison of registration and query response times for ∼15000 nodes at an input rate of 1000 requests per second Figure 4.9: Comparison of registration and query response times for ∼15000 nodes at an input rate of 1000 requests per second times. When using deterministic lookup and access, the system retrieves the full set of registered devices at each query request and attempts to access them all in order to provide a result. Therefore, response times grow larger as the number of registered devices, and the number of query request increases. On the other hand, the beneﬁts of using probabilistic methodologies is clearly shown. In this case, response times are lower and are kept rather stable with respect to number of registered devices and the number of query requests, since the system retrieves only a small subset of registered devices based on the desired coverage threshold. This is further conﬁrmed by studying the individual behaviors of Lookup and Access for both the deterministic and probabilistic methods, as depicted in Figure 4.8. In both cases, the Lookup response time is very low, and is rather stable for all input rates. In the case of deterministic Lookup and Access, the Access response times grows as the number of registered devices grows, as the system attempts to access the full set of registered devices. In contrast, by using probabilistic Lookup and Access, the Access response times are stable, and are lower than the results of deterministic Lookup and Access specially for high input rates, when a higher number of devices is registered. Note that, the registration strategy has an impact on query response times, since it will indirectly determine the number of devices the query must look for and access. The second set of results aims at measuring the performance of a the system under ULS trafﬁc for a longer period of time. To this end, our trace analysis tool generated trafﬁc at a rate of 1000 requests per second during 15 seconds based on the information from the Cologne mobility trace [32]. For this set of results, we evaluated a system using only deterministic registration, lookup and access, a system using CHOReOS FP7-257178 45 probabilistic registration and deterministic lookup and access, and a system using the full CHOReOS solution (probabilistic registration, lookup and access). Evaluation Results In Figure 4.9, the response times of both registration and service query operations for the three described systems are shown. In the case of registration, as expected the response times of a full deterministic strategy are lower than the systems using probabilistic registration only. Moreover, the additional overhead introduced by the operations involved in probabilistic registration is clearly shown. In the case of Lookup and Access, as expected, the response times of the systems using deterministic access is higher than the system using probabilistic only. Furthermore, the impact of the registration strategy on service query response times is shown, as the system using probabilistic registration and deterministic access presents a lower response time than the system using deterministic registration only. CONCLUSION: Overall, these results show the ability of our proposed assessment strategy to deploy the IoTS middleware under realistic conditions, and its capacity to provide meaningful results for the assessment of the solution. Moreover, the obtained results show the ability of the IoTS middleware to smartly tackle the ULS challenge. 1http://www.jini.org/wiki/JavaSpaces Speciﬁcation 2http://ws4d.e-technik.uni-rostock.de/jmeds/ 3http://activemq.apache.org/ 1http://www.jini.org/wiki/JavaSpaces Speciﬁcation 2 3http://activemq.apache.org/ 4http://metrics.sourceforge.net 4.2.2. XSB Evaluation Based on this implementation, we evaluate the XSB with respect to the two criteria introduced above. We present our evaluation results in the following. and to send commands. We provide support for the three mentioned middleware platforms by producing appropriate binding components. Based on this implementation, we evaluate the XSB with respect to the two criteria introduced above. We present our evaluation results in the following. Support to Developers The XSB framework can be used by both application and middleware developers. We evaluate ﬁrst the effort for the application developer and accordingly the provided support by our solution for developing complex applications from the integration of services that employ heterogeneous interaction paradigms. Second, given that we have designed our architectural framework with particular consideration for its extensibility, we evaluate the easiness for the middleware developer in integrating new middleware platforms, in particular with regard to building related binding components (BCs). Application Developer – Effort for Application Design Table 4.4 summarizes our measurements of the development effort required for the S&R scenario. Essentially, this effort includes writing an xDL description for each constituent service, and providing mapping directives between the data exchanged among the services. GA-IDL service descriptions are then generated automatically by using the tools provided by our platform. We see that application development effort is considerably low, since our platform takes care of resolving the interaction paradigm and middleware heterogeneity among the constituent services. xDL description Generated desc. Mapping directives (XML lines) (XML lines) (XML lines) Java Spaces system 148 98 72 DPWS system 50 61 76 JMS system 209 90 78 Total 407 249 226 Table 4.4: Development effort for the application developer Table 4.4: Development effort for the application developer Middleware Developer – Extensibility Referring to the architectural framework of Figure 2.3, we measure the effort for building a BC for the JMS Apache ActiveMQ middleware platform. Table 4.5 summarizes this effort, in terms of implemented numbers of: (1) Lines of code, (2) XML schema lines regarding the xDL descriptions, and (3) XML lines of conﬁguration ﬁles for the architectural framework. We have performed our measurements with the Metrics 1.3.6 Eclipse plugin4. We provide measure- ments for each one of the three components of the framework, as well as the ratio of the effort speciﬁc to the JMS platform (reﬁnement of subcomponents) over the total effort (i.e., including the generic code written once and reusable each time). We see that considerably small effort, no more than 6% of the total effort, is required for the integration of a new middleware platform. This points out the signiﬁcant support offered, resulting in considerable easiness for integrating new middleware platforms and related high extensibility of our approach. 4.2.2. XSB Evaluation We evaluate the eXtended Service Bus (XSB) framework and runtime with respect to two criteria: First, the support that the XSB framework offers to developers – both application developers and middleware developers – when developing a new complex application that is composed as a choreography of a set of heterogeneous services, or when deploying a service, which requires development of an appro- priate binding component for the middleware technology that the service is based upon. Second, the performance of the XSB runtime in terms of latency and throughput, under both low trafﬁc and stress conditions. For our evaluations, we rely upon the Search and Rescue scenario that we quote below from [21]. This scenario prescribes interconnections between all three interaction paradigms of interest, i.e., client-service (CS), publish-subscribe (PS) and tuple space (TS), within a service choreography that precisely follows CHOReOS principles. Search and Rescue (S&R) operations after a disaster, such as a ﬂood or earthquake, are carried out in hazardous environments and require personnel from multiple agencies (e.g., ﬁre-ﬁghters, police) to coordinate. To detect survivors, sensors are installed at various places of the hazardous area. Such sensors communicate their location. S&R personnel also notify at short intervals of their current positions via their PDAs. Upon sensing some life sign, sensor nodes send out notiﬁcations. At the same time, nearby light-emitting actuators start lighting the place to facilitate the rescuing effort. Sensors, PDAs, and actuators interact among them and with external actors via a tuple space. Tuple space location and life sign data are sent via client-service invocations to a planning service that recommends at real time the optimal deployment of rescue forces. This output is notiﬁed via a publish-subscribe system to the coordinator of the operation on her smartphone and also to a number of con- trol/monitoring centers. The coordinator may approve and command S&R personnel via the publish-subscribe system and the tuple space system to rush into the spot. We have implemented the S&R scenario on top of the XSB framework. Our scenario implementation integrates: (1) sensors, actuators and personnel equipment communicating over a Jini JavaSpaces TS1; (2) the planning service implemented as a JMEDS DPWS Web Service2; and (3) a JMS PS system based on Apache ActiveMQ3 that the coordinator of the operation uses to receive recommendations CHOReOS FP7-257178 46 and to send commands. We provide support for the three mentioned middleware platforms by producing appropriate binding components. Performance The XSB runtime introduces a number of extensions to the typical ESB infrastructure, such as transfer of GA primitives as payload of substrate bus (EasyESB) communication primitives, and, more importantly, runtime model transformations inside the BC; these enable cross-connection and adaptation among heterogeneous interaction paradigms. Hence, we need to evaluate the performance of our solution and the time overhead introduced. CHOReOS FP7-257178 47 Lines of code XML schema Conﬁguration (lines) (XML lines) xDL Processor 7520 2617 111 Core Engine 9993 219 137 Envelope for Substrate Bus 508 0 0 Total 18021 2836 248 Written by the developer 1162 191 12 Effort 6% 6% 4% Table 4.5: Development effort for the JMS binding component Table 4.5: Development effort for the JMS binding component We evaluate the performance of the XSB runtime with two experimental setups. In the ﬁrst one, we employ the XSB with a mock substrate bus, which enables us to assess the pure XSB functionality and to test the XSB under stress conditions without having to internally tune at the same time the EasyESB conﬁguration. We interconnect services employing the same or different interaction paradigms, and measure end-to-end latency and throughput, under both low trafﬁc and stress conditions. In the second experiment, we employ the XSB on top of the EasyESB and evaluate the latency overhead introduced by the EasyESB and by the XSB for various combinations of interconnected interaction paradigms, under low trafﬁc conditions. End-to-End Performance under Stress Testing We evaluate the performance of the XSB Bind- ing Components under stress conditions relying on [28] and [20]. Our approach enables setting a lightweight testing environment around the XSB with practical hardware resources and making sure that the XSB employs its maximum capacity. In particular, to evaluate the performance capacity of an XSB, the ESB has to be under high load, i.e., we have to saturate the ESB system to determine its maximum performance. To this end, service requestors and service providers need to send and receive high request rate through the ESB. Test Scenario: We set up our XSB Binding Components with a mock substrate bus and mock services (requestors, providers). In order to test the connectivity of heterogeneous services under stress condi- tions, we utilize the imported middleware services of the XSB Framework (DPWS, JMS and JavaSpaces services). 1) C - S: Using the DPWS middleware, we create service mock clients and a mock web service running on M1 and M3, correspondingly. 2) P - b - Sb: Using the JMS middleware, we create mock publishers, a broker, and a mock subscriber 1) C - S: Using the DPWS middleware, we create service mock clients and a mock web service running on M1 and M3, correspondingly. 2) P - b - Sb: Using the JMS middleware, we create mock publishers, a broker, and a mock subscriber running on M1, M2 and M3, correspondingly. Performance We make sure to remove any potential bottlenecks from the services (clients/providers) and we create a bottleneck at the service bus; our purpose is to measure throughput and one-way end-to- end latency. More speciﬁcally, we use middleware clients (DPWS or JMS) and threads to create many mock service clients. Then, the mock substrate bus that we conﬁgure for the evaluation can handle their service requests as messages. We also need to have a service provider that can receive thousands of messages per second in order to overload the XSB. The CPU usage of the machine hosting the XSB should be close to 100% to reach the maximum performance of the XSB. In addition, it is important that both the service clients and providers are not highly loaded. Test Setup: We used the following software and hardware for our experiments. The setup consisted of four machines, connected via Inria’s 100 Mb/s Ethernet network. The ﬁrst and second machine (M1, M2) have each an Intel core i5-2540M x 2.6 GHz (6 GB RAM), the third (M3) has an Intel Xeon W3550e 3.08 GHz x 4 (7.8 RAM), and the last machine (M4) has an Intel core T7200 2.00 GHz x 2 (2.0 GB RAM). Running tests on powerful machines allows simulating a large number of clients more accurately, as opposed to simple core machines. We provide four test scenarios: CHOReOS FP7-257178 48 3) C - XSB - S: Using the DPWS middleware and the XSB over the mock substrate bus, we cre- ate service mock clients, an XSB service bus, and a Web service running on M1, M2 and M3, correspondingly. 4) P - b - XSB - S: Using the DPWS and JMS middleware, as well as the XSB over the mock substrate bus, we create mock publishers, a broker, an XSB service bus, and a web service running on M1, M4, M2 and M3, correspondingly. 4) P - b - XSB - S: Using the DPWS and JMS middleware, as well as the XSB over the mock substrate bus, we create mock publishers, a broker, an XSB service bus, and a web service running on M1, M4, M2 and M3, correspondingly. The ﬁrst scenario doesn’t have any service bus, service clients are simply forwarding messages to the web service. Performance The second scenario uses the XSB, and messages pass through it performing a transformation from CS to GA and back to CS primitives. The third scenario doesn’t have any service bus, publishers are simply forwarding messages to the broker, and the broker to the subscriber. Finally, the forth scenario uses the XSB and messages are passed through it performing a transformation from CS to GA and from GA to PS primitives (Figure 4.10). Note that, in all four cases, clients make requests every second. In our measurements, we discard the ﬁrst 4000 messages allowing machines to reach a steady state. Services generally don’t exchange very large quantities of data, so messages usually tend to be of average size. Hence, we set the size of messages to 1 KB. Figure 4.10: Components of mock environment for XSB capacity testing Results: Table 4.6 presents some detailed data for a test run with 50 concurrent clients. Showing data for each performed test run in this table would be impractical, as it would require several pages. Scenario Latency Throughput Avg. Clients (ms.) (req./s.) C-S 2,13 62,78 50 P-b-Sb 1,16 49,92 50 C-XSB-S 17,85 53,9 50 P-b-XSB-S 16,52 56,04 50 Table 4.6: Results for one-way interaction in all four scenarios with 50 concurrent clients Table 4.6: Results for one-way interaction in all four scenarios with 50 concurrent clients Figure 4.11 shows the measured throughput when sending one-way messages to the services, in function of the number of concurrent clients for each of the above scenarios. The procedure we ap- plied to execute this experiment is the following: i) In all four cases, after the XSB reaches the steady state, service providers are counting incoming messages for a duration of time, and ii) we repeat the same experiment by increasing the service clients. Thus, JMS and DPWS service providers receive CHOReOS FP7-257178 49 an increasing number of messages according to the concurrent clients. We observed that the number of messages passing via the XSB per second (throughput) remains unchanged after 90 concurrent clients. We veriﬁed at the same time that the CPU usage of the machine hosting the XSB had reached its maximum, while the CPU for clients/providers is about 15% - 20%. In scenario 1 using the DPWS middleware, the maximum throughput is 220,89 requests per second, while in scenario 2 using the JMS middleware, the maximum throughput is 243,47 requests per second. For communication through the XSB, in scenario 3 the maximum throughput is 82,29 requests per second, while in scenario 4 it is 90.8 requests/sec. 0 50 100 150 200 250 0 50 100 150 200 250 Throughput Simultaneous clients requests/sec C − XSB − S P − b − XSB − S P − b − Sb C − S Figure 4.11: Throughput for one-way interaction of DPWS and JMS services with or without XSB in scenarios 1, 2, 3 and 4 Figure 4.11: Throughput for one-way interaction of DPWS and JMS services with or without XSB in scenarios 1, 2, 3 and 4 Figures 4.12 and 4.13 show the measured one-way latency when making calls on the services, in function of the number of concurrent clients for each of the above scenarios. Figure 4.10: Components of mock environment for XSB capacity testing The procedure we applied to execute this experiment is the following: i) In all four cases, after the XSB reaches the steady state, service clients are sending requests continuously for a duration of time, ii) service providers are active to receive all messages, and iii) we repeat the same experiment by increasing the service clients. We observed that messages passing via the XSB have high latency after 50 concurrent clients. When using the DPWS middleware, the latency is 3 milliseconds, while, when using the JMS middleware, it is 1,8 milliseconds, both for 240 simultaneous clients and without passing messages through the XSB, as shown in Figure 4.12. On the other hand, for communication through the XSB, the latency reaches quite high values (Figure 4.13). In Figure 4.13, latency values for a low numbers of clients are not clear due to the scale used, thus we depict in Figure 4.14 the measured latency for up to 50 clients. The latency for 50 clients is 17,85 milliseconds when using the DPWS middleware and 16,2 milliseconds when using the JMS middleware. Considering the fact that the CPU usage of the XSB reached its maximum in both experiments, results show that after 90 concurrent clients, the XSB cannot manage the reached number of clients. In our future work, we intend to optimize the XSB for improving its throughput and latency values in the case of many concurrent clients. Latency Overhead on the Bus We measure execution times for a number of layouts: (i) one-way and two-way interaction inside our implemented CS system; (ii) end-to-end interaction between a publisher and a subscriber inside our implemented PS system; (iii) end-to-end interaction between a writer and a reader inside our implemented TS system; (iv) one-way and two-way interaction between two CS peers via EasyESB; and (v) interaction between all pair combinations of CS, PS and TS peers via XSB. We repeat each measurement a 100 times and calculate mean values. Figure 4.10: Components of mock environment for XSB capacity testing Based on these experiments, we CHOReOS FP7-257178 50 0 50 100 150 200 250 1 1.5 2 2.5 3 3.5 4 One−way Latency Simultaneous clients ms C − S P − b − Sb Figure 4.12: Latency for one-way interaction of DPWS and JMS services without XSB in scenar- ios 1 and 2 0 50 100 150 200 250 1 1.5 2 2.5 3 3.5 4 One−way Latency Simultaneous clients ms C − S P − b − Sb Figure 4.12: Latency for one-way interaction of DPWS and JMS services without XSB in scenar- ios 1 and 2 evaluate the latency overhead introduced by the EasyESB for an one-way CS-CS communication, and the latency overhead introduced by the XSB for an one-way CS-CS as well as all other pair combina- tions of communication. Our results are summarized in Table 4.7. We see that the latency overhead introduced by the XSB for a CS-CS interconnection is only 1% greater than the latency overhead in- troduced by the EasyESB itself. When conversion between heterogeneous interaction paradigms is involved, the XSB latency overhead ranges from 7% to 15,5%, where we note that we always compare with the EasyESB CS-CS homogeneous interconnection, since EasyESB support for other interac- tion paradigms is not available. We see that the performance cost introduced by the XSB remains at reasonable levels. evaluate the latency overhead introduced by the EasyESB for an one-way CS-CS communication, and the latency overhead introduced by the XSB for an one-way CS-CS as well as all other pair combina- tions of communication. Our results are summarized in Table 4.7. We see that the latency overhead introduced by the XSB for a CS-CS interconnection is only 1% greater than the latency overhead in- troduced by the EasyESB itself. When conversion between heterogeneous interaction paradigms is involved, the XSB latency overhead ranges from 7% to 15,5%, where we note that we always compare with the EasyESB CS-CS homogeneous interconnection, since EasyESB support for other interac- tion paradigms is not available. We see that the performance cost introduced by the XSB remains at reasonable levels. Figure 4.10: Components of mock environment for XSB capacity testing CHOReOS FP7-257178 51 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 x 10 4 Simultaneous clients ms C − XSB − S P − b − XSB − S Figure 4.13: Latency for one-way interaction of DPWS and JMS services with XSB in scenarios 3 and 4 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 x 10 4 Simultaneous clients ms C − XSB − S P − b − XSB − S Latency for one-way interaction of DPWS and JMS services with XSB in scenarios Figure 4.13: Latency for one-way interaction of DPWS and JMS services with XSB in scenarios 3 and 4 5 10 15 20 25 30 35 40 45 50 12 13 14 15 16 17 18 19 One−way Latency Simultaneous clients ms C − XSB − S P − b − XSB − S Figure 4.14: Latency for one-way interaction of DPWS and JMS services with XSB in scenarios 3 and 4 for up to 50 concurrent clients 5 10 15 20 25 30 35 40 45 50 12 13 14 15 16 17 18 19 One−way Latency Simultaneous clients ms C − XSB − S P − b − XSB − S Figure 4.14: Latency for one-way interaction of DPWS and JMS services with XSB in scenarios 3 and 4 for up to 50 concurrent clients ms 25 30 Simultaneous clients Figure 4.14: Latency for one-way interaction of DPWS and JMS services with XSB in scenarios 3 and 4 for up to 50 concurrent clients Figure 4.10: Components of mock environment for XSB capacity testing Interconnection Latency (ms) one-way CS - CS via EasyESB 258 one-way CS - CS via XSB 261,5 CS - PS via XSB 283 CS - TS via XSB 276 PS - TS via XSB 298 Table 4.7: Interaction latency on the bus for each interconnection Table 4.7: Interaction latency on the bus for each interconnection Table 4.7: Interaction latency on the bus for each interconnection CONCLUSION: Our evaluation of the XSB framework and runtime showed quite good results in terms of both developer support and performance. We note that our software engineering support evaluation is based only on counting lines of code for a single developer. A more comprehensive empirical evalua- tion would require as well a subjective evaluation of the framework facilities by a number of developers. We also point out that stress testing requires very well-thought experimental setups when such a big number of factors intervene, both in hardware and in software in which there are many indirection layers and dependencies on external components. 4.2.3. EasyESB Evaluation Please refer to section 4.1.3. Experimental Setup To address this question, we compared the time it takes to execute WSBQL queries for weather forecast services (Table 4.8) over service abstractions, stored in the AoSBM relational store, with the time it takes to execute corresponding SQL queries, over concrete service descriptions, which were also stored in the AoSBM relational store. Table 4.8: WSBQL Query Table 4.8: WSBQL Query let $aosbm = db(‘‘localhost/mySB’’) for $c in $aosbm/servicecollections for $sa in $c/hierarchies/abstractions for $if in $sa/representativeinterfaces for $o1 in $if/representativeoperations for $o2 in $if/representativeoperations for $m1 in $o1/representativemessages for $m2 in $o2/representativemessages for $p1 in $m1/representativemessagetypes for $p2 in $m1/representativemessagetypes for $p3 in $m2/representativemessagetypes where $if/rsi name like %Weather% and $op1/rop name like %Temperature% and $p1/rmt name like %Location% and $p1/rmt type = ‘String’ and $p2/rmt name like %Date% and $p2/rmt type = ‘String’ and $p3/rmt name like %UnitSystem% return Abstractions.fullObject Our WSBQL queries consist of three main parts: Our WSBQL queries consist of three main parts: • The ﬁrst part consists of variable deﬁnitions. The variables deﬁne constraints that should be satisﬁed by retrieved abstractions. In particular, we have the following variables: $sa stands for a service abstraction; $if corresponds to the abstract interface of $sa; $o1 and $o2 represent two operations that should be provided by the required abstract interface; ﬁnally, $p1 and $p2 are two parameters of $o1, while $p3 is a parameter of $o2. • The constraints that should be met by the retrieved service abstractions are speciﬁed in the where part of the query. Speciﬁcally, for operation $o1, we have the following constraints: the name of $o1 should include the term Temperature; the name of parameter $p1 should include the term Location and the type of $p1 should be String; the name of parameter $p2 should include the term Date and the type of $p2 should also be String. For the operation $o2, there is only one constraint that concerns the name of parameter $p3, which should include the term UnitSystem. • The return part of the query dictates the information that should be included in the result: with the fullObject option, the result contains full information about the retrieved service abstrac- tions and the represented services. • The return part of the query dictates the information that should be included in the result: with the fullObject option, the result contains full information about the retrieved service abstrac- tions and the represented services. 4.2.4. AoSBM Discovery Evaluation In the CHOReOS deliverable D2.3, we provided a qualitative assessment of the usefulness of AoSBM in the CHOReOS use cases. Here, we provide a quantitative assessment that focuses on the performance of AoSBM in a ULS setting. Overall, AoSBM is driven by the hypothesis that service abstractions allow for efﬁcient execution of service lookup queries in the context of the Future Internet, where the amount of available service descriptions is expected to dramatically grow. Therefore, the AoSBM evaluation focuses on the following research question: • Is querying based on abstractions faster than conventional querying, based on concrete service descriptions? ng based on abstractions faster than conventional querying, based on concrete service ? • Is querying based on abstractions faster than conventional querying, based on concrete service descriptions? CHOReOS FP7-257178 52 Experimental Setup The queries were executed over various AoSBM instances, populated with different synthetic data. More speciﬁcally, we organized our experiments in two sets (Table 4.9). In the ﬁrst set of experiments, we varied the number of abstractions from 5 ∗103 to 106 and the number of service descriptions from 5 ∗104 to 107; hence, each abstraction represented 10 services. In the second set of experiments, the CHOReOS FP7-257178 53 Table 4.9: Experimental Setup data set properties AoSBM instances # service abstractions 5 ∗103 104 5 ∗104 105 5 ∗105 106 # concrete services 5 ∗104 105 5 ∗105 106 5 ∗106 107 # represented services per abstraction 10 # operations per service 3 # in parameters per operation 2 # out parameters per operation 2 overall disk space (MB) 159 325 1700 3451 17920 37478 (a) 1st set of experiments data set properties AoSBM instances # service abstractions 104 # concrete services 5 ∗104 105 5 ∗105 106 5 ∗106 107 # represented services per abstraction 5 10 50 100 500 1000 # operations per service 3 # in parameters per operation 2 # out parameters per operation 2 overall disk space (MB) 171 325 1587 3205 16486 33382 (a) 2nd set of experiments Table 4.9: Experimental Setup number of stored abstractions was 104, while the number of service descriptions ranged from 5 ∗104 to 107; thus, the number of represented services per abstraction varied from 5 to 1000. Further details concerning the experiment setup for each set of experiments (operations per service, in/out parameters per operation, required disk space) are given in Table 4.9. We executed our experiments on a typical Intel Core 2, 2.2GHz, 3GB RAM; for the AoSBM relational store we employed MySQL server 5.5. We performed each experiment 10 times. Figure 4.16: Querying service abstractions vs. concrete service descriptions – 2nd set Figure 4.16: Querying service abstractions vs. concrete service descriptions – 2nd set Our ﬁndings are summarized in the following points: Our ﬁndings are summarized in the following points: • In both sets of experiments (Figure 4.15 and 4.16), we observe that the SQL query execution time increases with the number of concrete service descriptions stored in the AoSBM relational store. • In both sets of experiments (Figure 4.15 and 4.16), we observe that the SQL query execution time increases with the number of concrete service descriptions stored in the AoSBM relational store. • Concerning the WSBQL query, in the ﬁrst set of experiments, the execution time increases with the number of stored service abstractions. On the other hand, in the second set of experiments, the WSBQL query execution time increases with the size of the result; the number of represented services for the service abstractions that are returned by the query varies from 5 to 1000. • In the ﬁrst set of experiments, querying over service abstractions is 88% to 99% faster than querying over concrete service descriptions. Similarly, in the second set of experiments querying over service abstractions is 90% to 99% faster than querying over concrete service descriptions. • In the ﬁrst set of experiments, querying over service abstractions is 88% to 99% faster than querying over concrete service descriptions. Similarly, in the second set of experiments querying over service abstractions is 90% to 99% faster than querying over concrete service descriptions. CONCLUSION: To summarize, the main take-away message from the AoSBM evaluation is that querying over service abstractions is much faster than querying over concrete service descriptions; while conventional querying increases the number of available service descriptions, abstractions- oriented querying increases with the number of available abstractions. Results & Findings Figures 4.15 and 4.16 gives the results that we obtained; the reported numbers are the average values. Figure 4.15: Querying service abstractions vs. concrete service descriptions – 1st set Figures 4.15 and 4.16 gives the results that we obtained; the reported numbers are the average values. ure 4.15: Querying service abstractions vs. concrete service descriptions – 1st se Figure 4.15: Querying service abstractions vs. concrete service descriptions – 1st set Figure 4.15: Querying service abstractions vs. concrete service descriptions – 1st set CHOReOS FP7-257178 54 Figure 4.16: Querying service abstractions vs. concrete service descriptions – 2nd set Experimental Setup RQ1 - Assessing the Delegation Overhead To evaluate the delegation overhead introduced by our approach, we have worked with 6 functional abstraction services, inspired by roles performed within the WP6 Passenger Friendly Airport use case: RQ1 - Assessing the Delegation Overhead To evaluate the delegation overhead introduced by our approach, we have worked with 6 functional abstraction services, inspired by roles performed within the WP6 Passenger Friendly Airport use case: • AbstractWeatherForecastService: serves for the substitution of weather forecasting ser- vice instances. • AbstractHotel: deals with the substitution of hotel service instances. • AbstractSecurityCompany: allows the substitution of security company service in • AbstractAirplane: serves for the substitution of airplane service instances. • AbstractDisplaysManagement: enables the substitution of airport notiﬁcation service in- stances. • AbstractAirportBusCompany: deals with the substitution of services for the transport of pas- sengers within the airport. • AbstractAirportBusCompany: deals with the substitution of services for the transport of pas- sengers within the airport. The assessment of the delegation overhead is performed by invoking each functional abstraction service both as a Web service and as a simple Java class. We investigate the delegation overhead and its breakdown in the three different parts of our method for the transformation of abstract to concrete invocations. In the case of the AbstractWeatherForecastService, we vary the size of the input (and accordingly, the output) to see its effect over the different parts of the execution. In particular, the input (respectively, the output) parameter is a list and we consider invocations for which the size of the list is 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 elements. The rest of the functional abstraction services do not offer us the luxury of modifying their input size (and accordingly, their output). Therefore, in these cases we simply measure the delegation overhead, without assessing the input/output size effect on this overhead. For each measurement, we execute it 10 times and take the average execution time. The deployment for the experimental assessment is as follows: the concrete service instances as well as the functional abstraction services are deployed on a server workstation and the client that invokes the services is located in another PC. RQ2 - Assessing the Substitution Overhead For the assessment of the substitution overhead, without loss of generality, we focus on the case of the AbstractWeatherForecastService. 4.2.5. AoSBM Service Substitution Evaluation We have experimentally assessed our AoSBM service substitution approach with different web services and conﬁgurations of their invocation. Our evaluation concerns two main aspects. The ﬁrst aspect is the time overhead that is introduced by the AoSBM service substitution approach for the translation of invocations made on a functional abstraction service, to invocations on the concrete services that are hidden behind the functional abstraction service (Section 2.1.2) [11]. The second aspect is the time overhead that is introduced by the approach for the actual substitution of one concrete service with another (Section 2.1.2) [11]. Regarding these two aspects, we focus on the following research questions: RQ1: What is the time breakdown for the translation of an abstract invocation to a concrete invocation, with respect to the three different phases of the functional abstraction service delegation mecha- nism? What is the effect of the input/output size to the translation time? CHOReOS FP7-257178 55 RQ2: What is the time breakdown for service substitution? How much time is spent to notify the services that use the substituted service about the beginning/end of the substitution and how much time is required to perform the change of the mapping that is used by the functional abstraction service? RQ2: What is the time breakdown for service substitution? How much time is spent to notify the services that use the substituted service about the beginning/end of the substitution and how much time is required to perform the change of the mapping that is used by the functional abstraction service? In the rest of this subsection we shed some light to the above questions. Before proceeding, however, we detail our experimental setup. Experimental Setup To study the time breakdown for substituting the concrete service instance that is hidden be- hind AbstractWeatherForecastService, with another one we consider different conﬁgura- tions, where the number of services that use the hidden substituted service instance, via AbstractWeatherForecastService, varies from 100 to 1000. Therefore, the number of services that are notiﬁed, also varies from 100 to 1000. CHOReOS FP7-257178 56 Results & Findings RQ1 - Assessing the Delegation Overhead Figure 4.17, gives the delegation overhead break- down for the AbstractWeatherForecastService, when invoked as a simple Java class, while Figure 4.18 gives the delegation overhead breakdown for the AbstractWeatherForecastService, when invoked as a Web service. Figure 4.19, gives the overall invocation time in the aforementioned two cases. The results are relatively similar and we can summarize our ﬁndings as follows. RQ1 - Assessing the Delegation Overhead Figure 4.17, gives the delegation overhead break- down for the AbstractWeatherForecastService, when invoked as a simple Java class, while Figure 4.18 gives the delegation overhead breakdown for the AbstractWeatherForecastService, when invoked as a Web service. Figure 4.19, gives the overall invocation time in the aforementioned two cases. The results are relatively similar and we can summarize our ﬁndings as follows. RQ1 - Assessing the Delegation Overhead Figure 4.17, gives the delegation overhead break- down for the AbstractWeatherForecastService, when invoked as a simple Java class, while down for the AbstractWeatherForecastService, when invoked as a simple Java class, while Figure 4.18 gives the delegation overhead breakdown for the AbstractWeatherForecastService, when invoked as a Web service. Figure 4.19, gives the overall invocation time in the aforementioned two cases. The results are relatively similar and we can summarize our ﬁndings as follows. • As the size of the input increases (respectively, the output), the total time for the completion of a invocation increases, too. • As the size of the input increases (respectively, the output), the total time for the completion of a invocation increases, too. • When the functional abstraction service is invoked as a Web Service, there is an extra cost in- curred compared to its invocation as a Java API, due to the extra dereference level that the Web service incurs. Experimental Setup • The execution time of the two ﬁrst phases of the translation of abstract to concrete invocations, that require a conversion to the uniﬁed intermediate representation and a customization towards the particularities of the actually invoked concrete service, are affected by the size of the input/output data that are translated. The larger the lists we have to manage, the more time we spend for their management and manipulation. • The second phase of the data translation, both concerning the input and the output takes the lion’s share in the delegation overhead breakdown, been found several orders of magnitude higher than any phase of the translation process. This is due to the fact that (a) our mechanism traverses the input or output object using the Java reﬂection mechanism and (b) it searches in the mappings that have been created with the use of lists, iterating over all the objects in the list until the appropriate object is found, suffering thus from the necessary time penalty that the iteration over large lists incurs. • The time needed to initialize the input or output objects increases as the input or output object increases. • The time needed to initialize the input or output objects increases as the input or output object increases. Figure 4.20 gives the delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when invoked as a simple Java classes, while Figure 4.21 gives the delegation overhead breakdown, when invoked as a Web services. Figure 4.22 gives the overall invocation time in the aforementioned two cases. Figure 4.20 gives the delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when invoked as a simple Java classes, while Figure 4.21 gives the delegation overhead breakdown, when invoked as a Web services. Figure 4.22 gives the overall invocation time in the aforementioned two cases. The results are relatively similar and we can summarize our ﬁndings as follows. The results are relatively similar and we can summarize our ﬁndings as follows. • Once again, we pay a small extra price for the invocation of a functional abstraction service as a Web service, as compared to its invocation via a Java API. • Once again, we pay a small extra price for the invocation of a functional abstraction service as a Web service, as compared to its invocation via a Java API. Experimental Setup CHOReOS FP7-257178 57 Figure 4.17: Time breakdown for the delegation overhead of the AbstractWeatherForecastService, when called as a Java API (numbers indicate the different phases of the translation of abstract to concrete invocations) Figure 4.17: Time breakdown for the delegation overhead of the AbstractWeatherForecastService, when called as a Java API (numbers indicate the different phases of the translation of abstract to concrete invocations) breakdown Figure 4.17: Time breakdown for the delegation overhead of the AbstractWeatherForecastService, when called as a Java API (numbers indicate the different phases of the translation of abstract to concrete invocations) Figure 4.18: Time breakdown for the delegation overhead of the AbstractWeatherForecastService, when called as a Web Service (numbers indicate the different phases of the translation of abstract to concrete invocations) Figure 4.18: Time breakdown for the delegation overhead of the AbstractWeatherForecastService, when called as a Web Service (numbers indicate the different phases of the translation of abstract to concrete invocations) CHOReOS FP7-257178 58 Figure 4.19: Total execution time for the invocation of the AbstractWeatherForecastService Figure 4.19: Total execution time for the invocation of the AbstractWeatherForecastService Figure 4.20: Delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when called as Java APIs (numbers indicate the different phases of the translation of abstract to concrete invocations) Figure 4.20: Delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when called as Java APIs (numbers indicate the different phases of the translation of abstract to concrete invocations) Figure 4.20: Delegation overhead breakdown for AbstractHotel, AbstractSec Figure 4.20: Delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when called as Java APIs (numbers indicate the different phases of the translation of abstract to concrete invocations) Figure 4.20: Delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when called as Java APIs (numbers indicate the different phases of the translation of abstract to concrete invocations) • The time needed for the notiﬁcation of the services that use the substituted service for the comple- tion of the substitution process, increases with the number of these services, for reasons similar to the above observation. • The time needed for changing the mapping that is used by the functional abstraction service is independent of the number of services that use the substituted service. • The total time needed for the service substitution increases in accordance with the number of services that use the substituted service. Experimental Setup • The second phase of the input data translation is typically (but not always) slower than the rest of the data translation steps. The output restructuring behaves similarly. However, we do not observe differences that are orders of magnitude higher, although there are three out of six times where some restructuring is three times higher than the rest of the parts. Clearly, the size and simplicity of the input and output objects are the responsible reasons for this behavior. RQ2 - Assessing the Substitution Overhead Figure 4.23, gives the results of the experimental assessment of the substitution overhead. Our observations are summarized below: RQ2 - Assessing the Substitution Overhead Figure 4.23, gives the results of the experimental assessment of the substitution overhead. Our observations are summarized below: • The number of the services that use the substituted service, affects the time needed for their notiﬁcation from the impact analyzer that the substitution starts. Naturally, this is due to the fact that an increased number of such services requires more invocations, for the commencement of the substitution process. • The number of the services that use the substituted service, affects the time needed for their notiﬁcation from the impact analyzer that the substitution starts. Naturally, this is due to the fact that an increased number of such services requires more invocations, for the commencement of the substitution process. Experimental Setup This is due to the two notiﬁcation phases, as already explained. CONCLUSION: To sum up, there are two main take-away messages from the experimental evaluation of the abstraction-oriented service substitution: (1) service substitution introduces an execution over- head that is proportional to the amount of data that is transferred via functional abstraction services; (2) supporting consistent substitution by notifying affected services further introduces an execution over- head that is proportional to the number of affected services. Overall, as anticipated extensibility and adaptability have a negative effect on performance. The use of middleware that allows to achieve these properties should be conservative, i.e., it should be used if these properties are really necessary. CHOReOS FP7-257178 59 Figure 4.21: Delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when called as Web services (numbers indicate the different phases of the translation of abstract to concrete invocations) Figure 4.21: Delegation overhead breakdown for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany, when called as Web services (numbers indicate the different phases of the translation of abstract to concrete invocations) Figure 4.22: Total execution time for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany Figure 4.22: Total execution time for AbstractHotel, AbstractSecurityCompany, AbstractAirplane,AbstractDisplaysManagement, and AbstractAirportBusCompany 4.2.6. Cloud Enactment Engine Evaluation Experimental Setup The Enactment Engine was executed on a machine with 8 GB of RAM, an Intel Core i7 CPU with 2.7 GHz and GNU/Linux kernel 3.6.7. ditional service. The idle nodes reserve size was ﬁve and the node creation timeout was 300 seconds. We used Amazon EC2 as the cloud computing service and the virtual machines were EC2 small in- stances, each one with 1.7 GiB of RAM, one vCPU with processing power equivalent to 1.0–1.2 GHz, and Ubuntu GNU/Linux 12.04. The Enactment Engine was executed on a machine with 8 GB of RAM, an Intel Core i7 CPU with 2.7 GHz and GNU/Linux kernel 3.6.7. ditional service. The idle nodes reserve size was ﬁve and the node creation timeout was 300 seconds. We used Amazon EC2 as the cloud computing service and the virtual machines were EC2 small in- stances, each one with 1.7 GiB of RAM, one vCPU with processing power equivalent to 1.0–1.2 GHz, and Ubuntu GNU/Linux 12.04. The Enactment Engine was executed on a machine with 8 GB of RAM, an Intel Core i7 CPU with 2.7 GHz and GNU/Linux kernel 3.6.7. Each scenario was executed 30 times and all the values in Table 4.11 presents the average execution time with a conﬁdence interval of 95%. It presents, for each scenario, the time necessary to deploy all the compositions plus the time to invoke them. It also shows how many compositions, as well as services, were successfully deployed for each scenario, for all executions. We have also conducted a scalability experiment by varying in 4 steps the deployed choreography size and the amount of nodes available on the cloud environment, whereas keeping constant the num- ber of deployed choreographies (only one) and the ratio of 10 deployed services per virtual machine. Each one of the 4 steps were executed 5 times. The environment used to run the Enactment Engine was a virtual machine (8GB RAM and 4 VCPU) running on top of a Open Stack installation. As in the previous experiments, the created nodes were Amazon EC2 instances. The results of this experiment are shown on Table 4.12, where for each scenario we have the average deployment time and the aver- age number of successfully deployed services, both followed by theirs respective standard deviations. Experimental Setup We conducted experiments to evaluate the performance and scalability of the proposed Enactment Engine in terms of its capability to deploy a signiﬁcant number of compositions onto a real-world cloud computing platform. Our experiments use a synthetic choreography workload modeled as shown in Figure 4.24. The arrow direction is from the requester to the requested service. Although replies are not drawn for simplicity reasons, they are always sent back in a synchronous manner. This topology was chosen because (1) it contains most types of conﬁgurations found in choreographies, including branches and subsequent joints and (2) it follows a repetitive pattern that can be used to smoothly increase the size of the composition to analyze how the performance of the Enactment Engine behaves as its workload increases. Initially, we conducted a multi-variable analysis of Enactment Engine by deploying service composi- tions in the following scenarios: 1) a small set of small compositions; 2) a small set of large composi- tions; 3) a large set of small compositions; 4) a larger ratio of services per node. Table 4.10 quantiﬁes these scenarios. In our experiments, the node allocation policy was the “limited round robin”, in which services are distributed across the available nodes, and the quantity of nodes is conﬁgured before each experiment. Experimental Setup If the amount of services is not divisible by the number of nodes, some of the nodes will host one ad- CHOReOS FP7-257178 60 Figure 4.23: Time breakdown for the substituion overhead as a function of the number of ser- vices that use the substituted service Figure 4.23: Time breakdown for the substituion overhead as a function of the number of ser- vices that use the substituted service Table 4.10: Enactment Engine deployment scenarios Scenario # of Size of each # of Cloud # of Services # Compositions Composition Nodes per Node 1 10 10 9 11 or 12 2 10 100 90 11 or 12 3 100 10 90 11 or 12 4 10 10 5 20 Table 4.10: Enactment Engine deployment scenarios CHOReOS FP7-257178 61 Figure 4.24: Choreography synthetic topology Table 4.11: Choreography enactment time Scenario Deployment Successful Successful time (s) Compositions Services 1 467.9 ± 34.8 10.0 (100%) ± 0 100.0 (100%) ± 0 2 1477.1 ± 130.0 9.3 (93%) ± 0.3 999.3 (99.9%) ± 0.4 3 1455.2 ± 159.1 98.9 (98.9%) ± 0.8 998.5 (99.8%) ± 1.3 4 585.2 ± 38.1 10.0 (100%) ± 0.1 100.0 (100%) ± 0.1 Figure 4.24: Choreography synthetic topology Table 4.11: Choreography enactment time Scenario Deployment Successful Successful time (s) Compositions Services 1 467.9 ± 34.8 10.0 (100%) ± 0 100.0 (100%) ± 0 2 1477.1 ± 130.0 9.3 (93%) ± 0.3 999.3 (99.9%) ± 0.4 3 1455.2 ± 159.1 98.9 (98.9%) ± 0.8 998.5 (99.8%) ± 1.3 4 585.2 ± 38.1 10.0 (100%) ± 0.1 100.0 (100%) ± 0.1 Figure 4.24: Choreography synthetic topology Table 4.11: Choreography enactment time Table 4.11: Choreography enactment time Scenario Deployment Successful Successful time (s) Compositions Services 1 467.9 ± 34.8 10.0 (100%) ± 0 100.0 (100%) ± 0 2 1477.1 ± 130.0 9.3 (93%) ± 0.3 999.3 (99.9%) ± 0.4 3 1455.2 ± 159.1 98.9 (98.9%) ± 0.8 998.5 (99.8%) ± 1.3 4 585.2 ± 38.1 10.0 (100%) ± 0.1 100.0 (100%) ± 0.1 ditional service. The idle nodes reserve size was ﬁve and the node creation timeout was 300 seconds. We used Amazon EC2 as the cloud computing service and the virtual machines were EC2 small in- stances, each one with 1.7 GiB of RAM, one vCPU with processing power equivalent to 1.0–1.2 GHz, and Ubuntu GNU/Linux 12.04. Results & Findings The results on Table 4.11 show that the Enactment Engine scales well in terms of the number of services being deployed. Although the number of services was multiplied by 10, the deployed time increased approximately only 3 times in scenarios 2 and 3. This time increment was caused mainly because the higher the number of services, the higher the likelihood of a fault that triggers the Enactment Engine fault-recovery mechanism, causing the re-execution of one or more tasks. The results also show that when the number of services per node was doubled (scenario 4), the deployment time increased nearly 25%. Part of this overhead was caused by the increase on the CHOReOS FP7-257178 62 Table 4.12: Enactment Engine scalability analysis Size of each Number of Deployment Successful composition available nodes time (s) Services 200 10 623 ± 92 199.8 (99.9%) ± 0.4 600 30 1170 ± 260 599.2 (99.9%) ± 0.8 1000 50 1467 ± 140 998.4 (99.8%) ± 1.1 1400 70 1910 ± 374 1390.6 (99.3%)± 11.5 Table 4.12: Enactment Engine scalability analysis number of Chef scripts that must be executed (sequentially) on the nodes to reconﬁgure During our experiments, we observed that the amount of failures was low: all the services were successfully deployed in more than 75% of the executions. By one failure, we mean that one service was not properly deployed due to problems discussed in section 2.4. Considering all the 30 executions, we got no failures in scenario 1, whereas we had only one failure in scenario 4. In scenario 2, the worst execution had 3 of 1,000 services not successfully deployed. In scenario 3, we got an execution with 20 failures, but it was an exceptional event, since the second worst situation had only 3 failures. Regarding the reserve of idle nodes, as explained in section 2.4, we observed that 80% of the exe- cutions did not use it. When the node reserve was used, there was a maximum of six requests, but in most of the time there was only one. We also observed that the deployment time was not signiﬁcantly affected when the (relatively frequent) failures on the cloud environment occurred, because new nodes were immediately retrieved from the reserve. The results on Table 4.12 also show a good scalability in terms of deployed services. 5 Conclusion The implementation of the CHOReOS Middleware has achieved a good level of maturity for an open source project and it is ready to be used in real-world, complex, distributed applications. Table 5.1 presents achievements, deﬁciencies, and needed enhancements of middleware components regarding Future Internet challenges. We hope the middleware components will continue to be developed after the end of the CHOReOS project and that further reﬁnements, improvements, as well as new functionalities will be added. The use of the middleware by the sub-teams working on the CHOReOS use-cases provided valu- able feedback for the middleware development teams that were able to improve the implementation. The synthetic experiments and simulations performed help to give an idea of the current state of the implementation and provide hints on where the next optimization and enhancements should be made. In the near future, the challenge will be to attract a community of developers and users to further continue the development of the system, which, we believe, can be very valuable for the next generation of computer systems for the Future Internet The use of the middleware by the sub-teams working on the CHOReOS use-cases provided valu- able feedback for the middleware development teams that were able to improve the implementation. The synthetic experiments and simulations performed help to give an idea of the current state of the implementation and provide hints on where the next optimization and enhancements should be made. In the near future, the challenge will be to attract a community of developers and users to further continue the development of the system, which, we believe, can be very valuable for the next generation of computer systems for the Future Internet. In the table below, we summarize the major beneﬁts (Pros), limitations (Cons), and possibilities for future enhancements for each middleware component. Middleware Component Pros Cons Future Enhancements Things C&E Semantics-based automated service composition: Com- plex physical properties are derived from simpler ones. Returns all possible compositions, which might be too many. Optimizations to return the best set of services to be composed for a given re- quest. Service Substitution Enables service substitution addressing the FI adaptation issue FI adaptation further involves issues like recomposition, com- pensation, and re- negotiation. Reducing the overhead of the substitution mechanism, along with combining the pro- posed approach with scal- able recomposition, compen- sation, and re-negotiation. Results & Findings Increasing the number of deployed services in 5 times, the deployment time nearly increases 2.4 times, whereas increasing the number of deployed services in 7 times, the deployment time nearly increases 3.1 times. In absolute numbers, each increase in 200 deployed services was responsible for increasing the de- ployment time from 300 to 550 seconds. CONCLUSION: The Enactment Engine scales well for different cases of choreography deployment, behaving well both with a large number of small choreographies and with a small number of large choreographies. In all cases, most of the time is spent by the underlying Cloud infrastructure to provide the nodes to host the services. The failures of the Amazon EC2 infrastructure are frequent and a special mechanism was inserted into the Enactment Engine to cope with them in a transparent way to the user. The concurrent deployment of a large number of large choreographies (e.g., simultaneously deploying 100 choreographies of 100 services each) is not advisable because it would take too long to complete (possibly over an hour). Thus, choreography-based systems and applications should be designed with the idea that large choreographies are deployed not very often, but then used for a long time, e.g., a choreography may be executing for months. We also consider that even the most higher deployed time (about 30 minutes for 1400 services) may be considered low if considering the long period a choreography should run and even considering the frequency of updates in choreographies, since both are much larger then 30 minutes. CHOReOS FP7-257178 63 CHOReOS FP7-257178 64 5 Conclusion XSB Interconnection of services employing heterogeneous in- teraction paradigms; deploy- able on top of different com- munication substrates; exten- sibility to support new service middleware or new interac- tion paradigms. In current proto- type, throughput and latency values are satisfactory for relatively low num- bers of concurrent service clients. Optimizations for improving throughput and latency val- ues in the case of many con- current service clients; empir- ical evaluation of the frame- work facilities by different de- velopers; extend with support for data streaming protocols. Optimizations for improving throughput and latency val- ues in the case of many con- current service clients; empir- ical evaluation of the frame- work facilities by different de- velopers; extend with support for data streaming protocols. CHOReOS FP7-257178 65 Middleware Component Pros Cons Future Enhancements EasyESB Efﬁcient integration of multi- protocol technologies; com- pliance with cloud infrastruc- tures and ability for a dis- tributed deployment; efﬁcient support for the coordination delegates for enacting the choreographies; ability of pro- ﬁling EasyESB into a moni- toring infrastructure for con- trolling services widely dis- tributed. EasyESB is a re- search prototype and thus needs maintenance and debugging. Optimizations in resource us- age; extending the cloud ap- pliance to other solutions and improving the nodes auto- matic deployment. LSB Universal access to hetero- geneous things-based ser- vices. Dependence on proxy for 3G access may lead to issues at ultra large scale. Investigate proxy-less solu- tions to access of thing-based services. Service Dis- covery Enables efﬁcient service lookup over service abstrac- tions, addressing the FI scale issue. Service registration becomes more com- plex due to the ex- traction of service abstractions. Selecting a small number of “important” service abstrac- tions and making the extrac- tion of abstractions semantic- aware and more interactive, without compromising perfor- mance. Things Dis- covery Scalable discovery thanks to the use of probabilistic ap- proaches. Fine-grained loca- tion data of services stored in registry, thus possibly lead- ing to privacy issues. Use only coarse-grained lo- cation data during registra- tion, and ﬁne-grained loca- tion data of services only dur- ing the lookup and access phases. Grid as a Service Better performance for CPU- and data-intensive tasks. Requires a working grid installation. Automate Grid installation writing Chef cookbooks for the Enactment Engine Middleware component. 5 Conclusion Enactment Engine Helps choreography devel- opers in tackling scalability by enabling a fully auto- mated deployment process; copes with technological heterogeneity by exten- sion mechanisms; provides some mechanisms to en- able cross-organizational choreographies; copes with adaptability by deploying monitoring infrastructure and providing means to ser- vice replication and service migration. Does not tackle se- curity issues; im- poses some restric- tions and obligations on services develop- ers (although most of them may be also handled by EE ex- tension). Improving support to cross- organizational choreogra- phies by federating different EE instances; better in- tegrating the monitoring infrastructure with services scale up/down mechanisms; support to more technologies and cloud infrastructures. 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Genera- tion and analysis of a large-scale urban vehicular mobility dataset. IEEE Transactions on Mobile Computing, 99(PrePrints):1, 2013. CHOReOS FP7-257178 68 CHOReOS FP7-257178 69 CHOReOS FP7-257178 70 A Enactment Engine Listings The listings cited on Section 4.1.5, Cloud Enactment Engine Use-Case-Based Evaluation, are the fol- lowing. A Enactment Engine Listings 1 package org.ow2.choreos; 2 3 import java.util.Collections; 4 5 import org.ow2.choreos.chors.datamodel.ChoreographySpec; 6 import org.ow2.choreos.nodes.datamodel.ResourceImpact; 7 import org.ow2.choreos.services.datamodel.DeployableServiceSpec; 8 import org.ow2.choreos.services.datamodel.PackageType; 9 import org.ow2.choreos.services.datamodel.ServiceDependency; 10 import org.ow2.choreos.services.datamodel.ServiceType; 11 12 public class ThalesSpecs { 13 14 public static ﬁnal String AIRPORT = ”airport”; 15 public static ﬁnal String AIRPORT BUS COMPANY = ”airportbuscompany”; 16 public static ﬁnal String AIRPORT DISPLAY ACTUATORS AGGREGATOR = ”airportdisplayactuatorsaggregator”; 17 public static ﬁnal String AIRPORT INFRARED SENSORS AGGREGATOR = ”airportinfraredsensorsaggregator”; 18 public static ﬁnal String AIRPORT NOISE SENSORS AGGREGATOR = ”aiportnoisesensorsaggregator”; 19 public static ﬁnal String AIRPORT PRESSURE SENSORS AGGREGATOR = ”aiportpressuresensorsaggregator”; 20 public static ﬁnal String AIRPORT SIGN ACTUATORS AGGREGATOR = ”airportsignactuatorsaggregator”; 21 public static ﬁnal String AIRPORT SPEAKER ACTUATORS AGGREGATOR = ”airportspeakeractuatorsaggregator”; 22 public static ﬁnal String BOOKABLE AMENITY = ”bookableamenity”; 23 public static ﬁnal String LUGGAGE HANDLING COMPANY = ”luggagehandlingcompany”; 24 public static ﬁnal String MID DISPLAY ACTUATORS AGGREGATOR = ”middisplayactuatorsaggregator”; 25 public static ﬁnal String MID LOCATION SENSORS AGGREGATOR = ”midlocationsensorsaggregator”; 26 public static ﬁnal String MID MICROPHONE SENSORS AGGREGATOR = ”midmicrophonesensorsaggregator”; 27 public static ﬁnal String SECURITY COMPANY = ”securitycompany”; 28 public static ﬁnal String STAND AND GATE MANAGEMENT = ”standandgatemanagement”; 29 30 public static ﬁnal String AIRPORT JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airport−service.jar”; 31 public static ﬁnal String AIRPORT BUS COMPANY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportbuscompany−service.jar”; 32 public static ﬁnal String AIRPORT DISPLAY ACTUATORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportdisplayactuatorsaggregator− service.jar”; 33 public static ﬁnal String AIRPORT INFRARED SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportinfraredsensorsaggregator− service.jar”; 34 public static ﬁnal String AIRPORT NOISE SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportnoisesensorsaggregator−service.jar”; 35 public static ﬁnal String AIRPORT PRESSURE SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportpressuresensorsaggregator− service.jar”; 36 public static ﬁnal String AIRPORT SIGN ACTUATORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportsignactuatorsaggregator−service.jar” 37 public static ﬁnal String AIRPORT SPEAKER ACTUATORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportspeakeractuatorsaggregator− service.jar”; 38 public static ﬁnal String BOOKABLE AMENITY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/bookableamenity−service.jar”; 39 public static ﬁnal String LUGGAGE HANDLING COMPANY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/luggagehandlingcompany−service.jar”; 40 public static ﬁnal String MID DISPLAY ACTUATORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/middisplayactuatorsaggregator−service.jar”; 41 public static ﬁnal String MID LOCATION SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/midlocationsensorsaggregator−service.jar”; 42 public static ﬁnal String MID MICROPHONE SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/midmicrophonesensorsaggregator−servic .jar”; 43 public static ﬁnal String SECURITY COMPANY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/securitycompany−service.jar”; 44 public static ﬁnal String STAND AND GATE MANAGEMENT JAR = ”http://sd−49168.dedibox.fr/DeployableServices/standandgatemanagement−service.jar”; 45 46 public static ﬁnal int AIRPORT PORT = 8004; 47 public static ﬁnal int AIRPORT BUS COMPANY PORT = 8023; 48 public static ﬁnal int AIRPORT DISPLAY ACTUATORS AGGREGATOR PORT = 8006; 49 public static ﬁnal int AIRPORT INFRARED SENSORS AGGREGATOR PORT = 8007; 50 public static ﬁnal int AIRPORT NOISE SENSORS AGGREGATOR PORT = 8008; 51 public static ﬁnal int AIRPORT PRESSURE SENSORS AGGREGATOR PORT = 8024; 52 public static ﬁnal int AIRPORT SIGN ACTUATORS AGGREGATOR PORT = 8010; 53 public static ﬁnal int AIRPORT SPEAKER ACTUATORS AGGREGATOR PORT = 8011; 54 public static ﬁnal int BOOKABLE AMENITY PORT = 8013; 55 public static ﬁnal int LUGGAGE HANDLING COMPANY PORT = 8017; 56 public static ﬁnal int MID DISPLAY ACTUATORS AGGREGATOR PORT = 8018; 57 public static ﬁnal int MID LOCATION SENSORS AGGREGATOR PORT = 8019; 58 public static ﬁnal int MID MICROPHONE SENSORS AGGREGATOR PORT = 8020; 59 public static ﬁnal int SECURITY COMPANY PORT = 8021; 60 public static ﬁnal int STAND AND GATE MANAGEMENT PORT = 8022; 61 62 private ﬁnal ResourceImpact resourceImpact = new ResourceImpact(); 63 64 private ﬁnal String serviceVersion = ”0.1”; 65 66 private ChoreographySpec chorSpec; 67 68 private DeployableServiceSpec airportSpec; 69 private DeployableServiceSpec airportBusCompanySpec; 70 private DeployableServiceSpec airportDisplayActuatorsAggregatorSpec; 71 private DeployableServiceSpec airportInfraredSensorsAggregatorSpec; 72 private DeployableServiceSpec airportNoiseSensorsAggregatorSpec; CHOReOS FP7-257178 1 package org.ow2.choreos; 2 3 import java.util.Collections; 4 5 import org.ow2.choreos.chors.datamodel.ChoreographySpec; 6 import org.ow2.choreos.nodes.datamodel.ResourceImpact; 7 import org.ow2.choreos.services.datamodel.DeployableServiceSpe 8 import org.ow2.choreos.services.datamodel.PackageType; 9 import org.ow2.choreos.services.datamodel.ServiceDependency; 10 import org.ow2.choreos.services.datamodel.ServiceType; 11 12 public class ThalesSpecs { 13 14 public static ﬁnal String AIRPORT = ”airport”; 15 public static ﬁnal String AIRPORT BUS COMPANY = ”airportb 16 public static ﬁnal String AIRPORT DISPLAY ACTUATORS AG 17 public static ﬁnal String AIRPORT INFRARED SENSORS AG 18 public static ﬁnal String AIRPORT NOISE SENSORS AGGRE 19 public static ﬁnal String AIRPORT PRESSURE SENSORS AG 20 public static ﬁnal String AIRPORT SIGN ACTUATORS AGGRE 21 public static ﬁnal String AIRPORT SPEAKER ACTUATORS AG 22 public static ﬁnal String BOOKABLE AMENITY = ”bookableam 23 public static ﬁnal String LUGGAGE HANDLING COMPANY = 24 public static ﬁnal String MID DISPLAY ACTUATORS AGGREG 25 public static ﬁnal String MID LOCATION SENSORS AGGREG 26 public static ﬁnal String MID MICROPHONE SENSORS AGG 27 public static ﬁnal String SECURITY COMPANY = ”securitycom 28 public static ﬁnal String STAND AND GATE MANAGEMENT = 29 30 public static ﬁnal String AIRPORT JAR = ”http://sd−49168.ded 31 public static ﬁnal String AIRPORT BUS COMPANY JAR = ”htt 32 public static ﬁnal String AIRPORT DISPLAY ACTUATORS AG service.jar”; 33 public static ﬁnal String AIRPORT INFRARED SENSORS AG service.jar”; 34 public static ﬁnal String AIRPORT NOISE SENSORS AGGRE 35 public static ﬁnal String AIRPORT PRESSURE SENSORS AG service.jar”; 36 public static ﬁnal String AIRPORT SIGN ACTUATORS AGGRE 37 public static ﬁnal String AIRPORT SPEAKER ACTUATORS AG service.jar”; 38 public static ﬁnal String BOOKABLE AMENITY JAR = ”http://s 39 public static ﬁnal String LUGGAGE HANDLING COMPANY JA 40 public static ﬁnal String MID DISPLAY ACTUATORS AGGREG 41 public static ﬁnal String MID LOCATION SENSORS AGGREG 42 public static ﬁnal String MID MICROPHONE SENSORS AGG .jar”; 43 public static ﬁnal String SECURITY COMPANY JAR = ”http://s 44 public static ﬁnal String STAND AND GATE MANAGEMENT J 45 46 public static ﬁnal int AIRPORT PORT = 8004; 47 public static ﬁnal int AIRPORT BUS COMPANY PORT = 8023 48 public static ﬁnal int AIRPORT DISPLAY ACTUATORS AGGR 49 public static ﬁnal int AIRPORT INFRARED SENSORS AGGR 50 public static ﬁnal int AIRPORT NOISE SENSORS AGGREGA 51 public static ﬁnal int AIRPORT PRESSURE SENSORS AGGR 52 public static ﬁnal int AIRPORT SIGN ACTUATORS AGGREGA 53 public static ﬁnal int AIRPORT SPEAKER ACTUATORS AGG 54 public static ﬁnal int BOOKABLE AMENITY PORT = 8013; 55 public static ﬁnal int LUGGAGE HANDLING COMPANY PORT 56 public static ﬁnal int MID DISPLAY ACTUATORS AGGREGAT 57 public static ﬁnal int MID LOCATION SENSORS AGGREGAT 58 public static ﬁnal int MID MICROPHONE SENSORS AGGRE 59 public static ﬁnal int SECURITY COMPANY PORT = 8021; 60 public static ﬁnal int STAND AND GATE MANAGEMENT POR 61 62 private ﬁnal ResourceImpact resourceImpact = new ResourceI 63 64 private ﬁnal String serviceVersion = ”0.1”; 65 66 private ChoreographySpec chorSpec; 67 68 private DeployableServiceSpec airportSpec; 69 private DeployableServiceSpec airportBusCompanySpec; 70 private DeployableServiceSpec airportDisplayActuatorsAggrega 71 private DeployableServiceSpec airportInfraredSensorsAggregat 72 private DeployableServiceSpec airportNoiseSensorsAggregator CHOReOS FP7-257178 package org.ow2.choreos; g ublic static ﬁnal String AIRPORT SIGN ACTUATORS AGGREGATOR = ”air p g public static ﬁnal String AIRPORT SPEAKER ACTUATORS AGGREGATO p g public static ﬁnal String BOOKABLE AMENITY = ”bookableamenity”; g y lic static ﬁnal String LUGGAGE HANDLING COMPANY = ”luggagehandlingco p g gg g g p y 24 public static ﬁnal String MID DISPLAY ACTUATORS AGGREGATOR = ”middisplayactuatorsaggregator”; p g gg g g p y public static ﬁnal String MID DISPLAY ACTUATORS AGGREGATOR = ”middisplayactuatorsaggregator”; p g public static ﬁnal String MID LOCATION SENSORS AGGREGATOR = ”m p g gg g ; public static ﬁnal String MID MICROPHONE SENSORS AGGREGATOR = ”midmicrophonesensorsaggregator”; p g gg g 26 public static ﬁnal String MID MICROPHONE SENSORS AGGREGATOR = ”midmicrophonesensorsaggregator”; p g public static ﬁnal String MID MICROPHONE SENSORS AGGREGATOR p g public static ﬁnal String SECURITY COMPANY = ”securitycompany”; public static ﬁnal String AIRPORT JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airport−service.jar”; S O S CO S 30 public static ﬁnal String AIRPORT JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airport−service.jar”; 31 public static ﬁnal String AIRPORT BUS COMPANY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airpo public static ﬁnal String AIRPORT BUS COMPANY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportbuscompany−service.jar”; public static ﬁnal String AIRPORT DISPLAY ACTUATORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportdisplayactua service.jar”; pub c stat c a St g O US CO J ttp //sd 9 68 ded bo / ep oyab eSe ces/a po tbusco pa y se ce ja public static ﬁnal String AIRPORT DISPLAY ACTUATORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportdi service.jar”; j ; tatic ﬁnal String AIRPORT INFRARED SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportinfraredsensorsaggregator− ervice.jar”; public static ﬁnal String AIRPORT INFRARED SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportin service.jar”; service.jar ; public static ﬁnal String AIRPORT NOISE SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportnoisesensorsaggregator−service.jar”; public static ﬁnal String AIRPORT PRESSURE SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportpressuresensorsaggregator− service jar”; pub c stat c a St g O O S S SO S GG G O J ttp //sd 9 68 ded bo / ep oyab eSe ces/a po t o sese so sagg egato se ce ja ; public static ﬁnal String AIRPORT PRESSURE SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportpressuresensorsaggregator− service.jar”; j ; tatic ﬁnal String AIRPORT SIGN ACTUATORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportsignactuatorsaggregator−servic tatic ﬁnal String AIRPORT SPEAKER ACTUATORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/airportspeakeractuatorsaggregato i j ” j ; public static ﬁnal String BOOKABLE AMENITY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/bookableamenity−service.jar”; public static ﬁnal String LUGGAGE HANDLING COMPANY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/luggagehandlingcompa j ; tatic ﬁnal String BOOKABLE AMENITY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/bookableamenity−service.jar”; tatic ﬁnal String LUGGAGE HANDLING COMPANY JAR ”http://sd 49168 dedibox fr/DeployableServices/luggagehandlingcom p g p p y y j ; public static ﬁnal String LUGGAGE HANDLING COMPANY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/luggagehandlingcompany−service.jar”; public static ﬁnal String MID DISPLAY ACTUATORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/middisplayactuatorsaggregator−service.jar”; public static ﬁnal String MID LOCATION SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/midlocationsensorsaggregator−service.jar”; public static ﬁnal String MID MICROPHONE SENSORS AGGREGATOR JAR = ”http://sd−49168.dedibox.fr/DeployableServices/midmicrophonesensorsaggregator−service j ” j ; blic static ﬁnal String SECURITY COMPANY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/securitycompany−service.jar”; blic static ﬁnal String STAND AND GATE MANAGEMENT JAR = ”http://sd−49168.dedibox.fr/DeployableServices/standandgatemanagemen j ; public static ﬁnal String SECURITY COMPANY JAR = ”http://sd−49168.dedibox.fr/DeployableServices/securitycompany−service.jar”; public static ﬁnal String STAND AND GATE MANAGEMENT JAR = ”http://sd−49168.dedibox.fr/DeployableServices/standandgatemanag 46 public static ﬁnal int AIRPORT PORT = 8004; p public static ﬁnal int AIRPORT BUS COMPANY PORT = 8023; p ; public static ﬁnal int AIRPORT DISPLAY ACTUATORS AGGREGATOR PORT = 8006; bli t ti ﬁ l i t AIRPORT INFRARED SENSORS AGGREGATOR PORT 8007 p public static ﬁnal int AIRPORT DISPLAY ACTUATORS AGGREGATOR POR ublic static ﬁnal int AIRPORT INFRARED SENSORS AGGREGATOR PORT = p ; public static ﬁnal int AIRPORT SIGN ACTUATORS AGGREGATOR PORT = 8010; p public static ﬁnal int AIRPORT SPEAKER ACTUATORS AGGREGATOR PO p public static ﬁnal int BOOKABLE AMENITY PORT = 8013; p ; public static ﬁnal int LUGGAGE HANDLING COMPANY PORT = 8017; ; static ﬁnal int MID DISPLAY ACTUATORS AGGREGATOR PORT = 8018; ; ic static ﬁnal int MID LOCATION SENSORS AGGREGATOR PORT = 8019; ic static ﬁnal int MID MICROPHONE SENSORS AGGREGATOR PORT = 802 ublic static ﬁnal int SECURITY COMPANY PORT = 8021; p public static ﬁnal int STAND AND GATE MANAGEMENT PORT = 8022; private ﬁnal ResourceImpact resourceImpact = new ResourceImpact(); CHOReOS FP7-257178 71 73 private DeployableServiceSpec airportPressureSensorsAggregatorSpec; 74 private DeployableServiceSpec airportSignActuatorsAggregatorSpec; 75 private DeployableServiceSpec airportSpeakerActuatorsAggregatorSpec; 76 private DeployableServiceSpec bookableAmenitySpec; 77 private DeployableServiceSpec luggageHandlingCompanySpec; 78 private DeployableServiceSpec midDisplayActuatorsAggregatorSpec; 79 private DeployableServiceSpec midLocationSensorsAggregatorSpec; 80 private DeployableServiceSpec midMicrophoneSensorsAggregatorSpec; 81 private DeployableServiceSpec securityCompanySpec; 82 private DeployableServiceSpec standAndGateManagementSpec; 83 84 public ThalesSpecs() { 85 initAirportSpecs(); 86 initAirportBusCompanySpecs(); 87 initAirportDisplayActuatorsAggregatorSpecs(); 88 initAirportInfraredSensorsAggregatorSpecs(); 89 initAirportNoiseSensorsAggregatorSpecs(); 90 initAirportPressureSensorsAggregatorSpecs(); 91 initAirportSignActuatorsAggregatorSpecs(); 92 initAirportSpeakerActuatorsAggregatorSpecs(); 93 initBookableAmenitySpecs(); 94 initLuggageHandlingCompanySpecs(); 95 initMIDDisplayActuatorsAggregatorSpecs(); 96 initMIDLocationSensorsAggregatorSpecs(); 97 initMIDMicrophoneSensorsAggregatorSpecs(); 98 initsecurityCompanySpecs(); 99 initStandAndGateManagementSpecs(); 100 createChorSpec(); 101 } 102 103 private void createChorSpec() { 104 this.chorSpec = new ChoreographySpec(this.airportSpec, this.airportBusCompanySpec, 105 this.airportDisplayActuatorsAggregatorSpec, this.airportInfraredSensorsAggregatorSpec, 106 this.airportNoiseSensorsAggregatorSpec, this.airportPressureSensorsAggregatorSpec, 107 this.airportSignActuatorsAggregatorSpec, this.airportSpeakerActuatorsAggregatorSpec, 108 this.bookableAmenitySpec, this.luggageHandlingCompanySpec, this.midDisplayActuatorsAggregatorSpec, 109 this.midLocationSensorsAggregatorSpec, this.midMicrophoneSensorsAggregatorSpec, 110 this.securityCompanySpec, this.standAndGateManagementSpec); 111 } 112 113 private void initStandAndGateManagementSpecs() { 114 standAndGateManagementSpec = new DeployableServiceSpec(STAND AND GATE MANAGEMENT, ServiceType.SOAP, 115 PackageType.COMMAND LINE, resourceImpact, serviceVersion, STAND AND GATE MANAGEMENT JAR, 116 STAND AND GATE MANAGEMENT PORT, STAND AND GATE MANAGEMENT, 1); 117 standAndGateManagementSpec.setRoles(Collections.singletonList(STAND AND GATE MANAGEMENT)); 118 standAndGateManagementSpec.addDependency(new ServiceDependency(AIRPORT, AIRPORT)); 119 } 120 121 private void initsecurityCompanySpecs() { 122 securityCompanySpec = new DeployableServiceSpec(SECURITY COMPANY, ServiceType.SOAP, PackageType.COMMAND LINE, 123 resourceImpact, serviceVersion, SECURITY COMPANY JAR, SECURITY COMPANY PORT, SECURITY COMPANY, 1); 124 securityCompanySpec.setRoles(Collections.singletonList(SECURITY COMPANY)); 125 securityCompanySpec.addDependency(new ServiceDependency(AIRPORT, AIRPORT)); 126 } 127 128 private void initMIDMicrophoneSensorsAggregatorSpecs() { 129 midMicrophoneSensorsAggregatorSpec = new DeployableServiceSpec(MID MICROPHONE SENSORS AGGREGATOR, 130 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 131 MID MICROPHONE SENSORS AGGREGATOR JAR, MID MICROPHONE SENSORS AGGREGATOR PORT, 132 MID MICROPHONE SENSORS AGGREGATOR, 1); 133 midMicrophoneSensorsAggregatorSpec.setRoles(Collections.singletonList(MID MICROPHONE SENSORS AGGREGATOR)); 134 } 135 136 private void initMIDLocationSensorsAggregatorSpecs() { 137 midLocationSensorsAggregatorSpec = new DeployableServiceSpec(MID LOCATION SENSORS AGGREGATOR, ServiceType.SOAP, 138 PackageType.COMMAND LINE, resourceImpact, serviceVersion, MID LOCATION SENSORS AGGREGATOR JAR, 139 MID LOCATION SENSORS AGGREGATOR PORT, MID LOCATION SENSORS AGGREGATOR, 1); 140 midLocationSensorsAggregatorSpec.setRoles(Collections.singletonList(MID LOCATION SENSORS AGGREGATOR)); 141 } 142 143 private void initMIDDisplayActuatorsAggregatorSpecs() { 144 midDisplayActuatorsAggregatorSpec = new DeployableServiceSpec(MID DISPLAY ACTUATORS AGGREGATOR, 145 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 146 MID DISPLAY ACTUATORS AGGREGATOR JAR, MID DISPLAY ACTUATORS AGGREGATOR PORT, 147 MID DISPLAY ACTUATORS AGGREGATOR, 1); 148 midDisplayActuatorsAggregatorSpec.setRoles(Collections.singletonList(MID DISPLAY ACTUATORS AGGREGATOR)); 149 } 150 151 private void initLuggageHandlingCompanySpecs() { 152 luggageHandlingCompanySpec = new DeployableServiceSpec(LUGGAGE HANDLING COMPANY, ServiceType.SOAP, 153 PackageType.COMMAND LINE, resourceImpact, serviceVersion, LUGGAGE HANDLING COMPANY JAR, 154 LUGGAGE HANDLING COMPANY PORT, LUGGAGE HANDLING COMPANY, 1); 155 luggageHandlingCompanySpec.setRoles(Collections.singletonList(LUGGAGE HANDLING COMPANY)); 156 luggageHandlingCompanySpec.addDependency(new ServiceDependency(AIRPORT, AIRPORT)); 157 } 158 159 private void initBookableAmenitySpecs() { 160 bookableAmenitySpec = new DeployableServiceSpec(BOOKABLE AMENITY, ServiceType.SOAP, PackageType.COMMAND LINE, 161 resourceImpact, serviceVersion, BOOKABLE AMENITY JAR, BOOKABLE AMENITY PORT, BOOKABLE AMENITY, 1); 162 bookableAmenitySpec.setRoles(Collections.singletonList(BOOKABLE AMENITY)); 163 } 164 165 private void initAirportSpeakerActuatorsAggregatorSpecs() { 166 airportSpeakerActuatorsAggregatorSpec = new DeployableServiceSpec(AIRPORT SPEAKER ACTUATORS AGGREGATOR, 167 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 168 AIRPORT SPEAKER ACTUATORS AGGREGATOR JAR, AIRPORT SPEAKER ACTUATORS AGGREGATOR PORT, 169 AIRPORT SPEAKER ACTUATORS AGGREGATOR, 1); 170 airportSpeakerActuatorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT SPEAKER ACTUATORS AGGREGATOR)); 171 } 172 77 private DeployableServiceSpec luggageHandlingCompanySpec; 78 private DeployableServiceSpec midDisplayActuatorsAggregatorSpec; 79 private DeployableServiceSpec midLocationSensorsAggregatorSpec; 80 private DeployableServiceSpec midMicrophoneSensorsAggregatorSpec; 81 private DeployableServiceSpec securityCompanySpec; 82 private DeployableServiceSpec standAndGateManagementSpec; 83 84 public ThalesSpecs() { 85 initAirportSpecs(); 86 initAirportBusCompanySpecs(); 87 initAirportDisplayActuatorsAggregatorSpecs(); 88 initAirportInfraredSensorsAggregatorSpecs(); 89 initAirportNoiseSensorsAggregatorSpecs(); 90 initAirportPressureSensorsAggregatorSpecs(); 91 initAirportSignActuatorsAggregatorSpecs(); 92 initAirportSpeakerActuatorsAggregatorSpecs(); 93 initBookableAmenitySpecs(); 94 initLuggageHandlingCompanySpecs(); 95 initMIDDisplayActuatorsAggregatorSpecs(); 96 initMIDLocationSensorsAggregatorSpecs(); 97 initMIDMicrophoneSensorsAggregatorSpecs(); 98 initsecurityCompanySpecs(); 99 initStandAndGateManagementSpecs(); 100 createChorSpec(); 101 } 102 103 private void createChorSpec() { 104 this.chorSpec = new ChoreographySpec(this.airportSpec, this.airportBusCompanySpec, 105 this.airportDisplayActuatorsAggregatorSpec, this.airportInfraredSensorsAggregatorSpec, 106 this.airportNoiseSensorsAggregatorSpec, this.airportPressureSensorsAggregatorSpec, 107 this.airportSignActuatorsAggregatorSpec, this.airportSpeakerActuatorsAggregatorSpec, 108 this.bookableAmenitySpec, this.luggageHandlingCompanySpec, this.midDisplayActuatorsAggregatorSpec, 109 this.midLocationSensorsAggregatorSpec, this.midMicrophoneSensorsAggregatorSpec, 110 this.securityCompanySpec, this.standAndGateManagementSpec); 111 } 112 113 private void initStandAndGateManagementSpecs() { 114 standAndGateManagementSpec = new DeployableServiceSpec(STAND AND GATE MANAGEMENT, ServiceType.SOAP, 115 PackageType.COMMAND LINE, resourceImpact, serviceVersion, STAND AND GATE MANAGEMENT JAR, 116 STAND AND GATE MANAGEMENT PORT, STAND AND GATE MANAGEMENT, 1); 117 standAndGateManagementSpec.setRoles(Collections.singletonList(STAND AND GATE MANAGEMENT)); 118 standAndGateManagementSpec.addDependency(new ServiceDependency(AIRPORT, AIRPORT)); 119 } 120 121 private void initsecurityCompanySpecs() { 122 securityCompanySpec = new DeployableServiceSpec(SECURITY COMPANY, ServiceType.SOAP, PackageType.COMMAND LINE, 123 resourceImpact, serviceVersion, SECURITY COMPANY JAR, SECURITY COMPANY PORT, SECURITY COMPANY, 1); 124 securityCompanySpec.setRoles(Collections.singletonList(SECURITY COMPANY)); 125 securityCompanySpec.addDependency(new ServiceDependency(AIRPORT, AIRPORT)); 126 } 127 128 private void initMIDMicrophoneSensorsAggregatorSpecs() { 129 midMicrophoneSensorsAggregatorSpec = new DeployableServiceSpec(MID MICROPHONE SENSORS AGGREGATOR, 130 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 131 MID MICROPHONE SENSORS AGGREGATOR JAR, MID MICROPHONE SENSORS AGGREGATOR PORT, 132 MID MICROPHONE SENSORS AGGREGATOR, 1); 133 midMicrophoneSensorsAggregatorSpec.setRoles(Collections.singletonList(MID MICROPHONE SENSORS AGGREGATOR)); 134 } 135 136 private void initMIDLocationSensorsAggregatorSpecs() { 137 midLocationSensorsAggregatorSpec = new DeployableServiceSpec(MID LOCATION SENSORS AGGREGATOR, ServiceType.SOAP, 138 PackageType.COMMAND LINE, resourceImpact, serviceVersion, MID LOCATION SENSORS AGGREGATOR JAR, 139 MID LOCATION SENSORS AGGREGATOR PORT, MID LOCATION SENSORS AGGREGATOR, 1); 140 midLocationSensorsAggregatorSpec.setRoles(Collections.singletonList(MID LOCATION SENSORS AGGREGATOR)); 141 } 142 143 private void initMIDDisplayActuatorsAggregatorSpecs() { 144 midDisplayActuatorsAggregatorSpec = new DeployableServiceSpec(MID DISPLAY ACTUATORS AGGREGATOR, 145 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 146 MID DISPLAY ACTUATORS AGGREGATOR JAR, MID DISPLAY ACTUATORS AGGREGATOR PORT, 147 MID DISPLAY ACTUATORS AGGREGATOR, 1); 148 midDisplayActuatorsAggregatorSpec.setRoles(Collections.singletonList(MID DISPLAY ACTUATORS AGGREGATOR)); 149 } 150 151 private void initLuggageHandlingCompanySpecs() { 152 luggageHandlingCompanySpec = new DeployableServiceSpec(LUGGAGE HANDLING COMPANY, ServiceType.SOAP, 153 PackageType.COMMAND LINE, resourceImpact, serviceVersion, LUGGAGE HANDLING COMPANY JAR, 154 LUGGAGE HANDLING COMPANY PORT, LUGGAGE HANDLING COMPANY, 1); 155 luggageHandlingCompanySpec.setRoles(Collections.singletonList(LUGGAGE HANDLING COMPANY)); 156 luggageHandlingCompanySpec.addDependency(new ServiceDependency(AIRPORT, AIRPORT)); 157 } 158 159 private void initBookableAmenitySpecs() { 160 bookableAmenitySpec = new DeployableServiceSpec(BOOKABLE AMENITY, ServiceType.SOAP, PackageType.COMMAND LINE, 161 resourceImpact, serviceVersion, BOOKABLE AMENITY JAR, BOOKABLE AMENITY PORT, BOOKABLE AMENITY, 1); 162 bookableAmenitySpec.setRoles(Collections.singletonList(BOOKABLE AMENITY)); 163 } 164 165 private void initAirportSpeakerActuatorsAggregatorSpecs() { 166 airportSpeakerActuatorsAggregatorSpec = new DeployableServiceSpec(AIRPORT SPEAKER ACTUATORS AGGREGATOR, 167 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 168 AIRPORT SPEAKER ACTUATORS AGGREGATOR JAR, AIRPORT SPEAKER ACTUATORS AGGREGATOR PORT, 169 AIRPORT SPEAKER ACTUATORS AGGREGATOR, 1); 170 airportSpeakerActuatorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT SPEAKER ACTUATORS AGGREGATOR)); 171 } 172 CHOReOS 136 private void initMIDLocationSensorsAggregatorSpecs() { 137 midLocationSensorsAggregatorSpec = new DeployableServiceSpec(MID LOCATION SENSORS AGGREGATOR, ServiceType.SOAP, 138 PackageType.COMMAND LINE, resourceImpact, serviceVersion, MID LOCATION SENSORS AGGREGATOR JAR, 139 MID LOCATION SENSORS AGGREGATOR PORT, MID LOCATION SENSORS AGGREGATOR, 1); 140 midLocationSensorsAggregatorSpec.setRoles(Collections.singletonList(MID LOCATION SENSORS AGGREGATOR)); 141 } CHOReOS FP7-257178 72 173 private void initAirportSignActuatorsAggregatorSpecs() { 174 airportSignActuatorsAggregatorSpec = new DeployableServiceSpec(AIRPORT SIGN ACTUATORS AGGREGATOR, 175 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 176 AIRPORT SIGN ACTUATORS AGGREGATOR JAR, AIRPORT SIGN ACTUATORS AGGREGATOR PORT, 177 AIRPORT SIGN ACTUATORS AGGREGATOR, 1); 178 airportSignActuatorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT SIGN ACTUATORS AGGREGATOR)); 179 } 180 181 private void initAirportPressureSensorsAggregatorSpecs() { 182 airportPressureSensorsAggregatorSpec = new DeployableServiceSpec(AIRPORT PRESSURE SENSORS AGGREGATOR, 183 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 184 AIRPORT PRESSURE SENSORS AGGREGATOR JAR, AIRPORT PRESSURE SENSORS AGGREGATOR PORT, 185 AIRPORT PRESSURE SENSORS AGGREGATOR, 1); 186 airportPressureSensorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT PRESSURE SENSORS AGGREGATOR)); 187 } 188 189 private void initAirportNoiseSensorsAggregatorSpecs() { 190 airportNoiseSensorsAggregatorSpec = new DeployableServiceSpec(AIRPORT NOISE SENSORS AGGREGATOR, 191 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 192 AIRPORT NOISE SENSORS AGGREGATOR JAR, AIRPORT NOISE SENSORS AGGREGATOR PORT, 193 AIRPORT NOISE SENSORS AGGREGATOR, 1); 194 airportNoiseSensorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT NOISE SENSORS AGGREGATOR)); 195 } 196 197 private void initAirportInfraredSensorsAggregatorSpecs() { 198 airportInfraredSensorsAggregatorSpec = new DeployableServiceSpec(AIRPORT INFRARED SENSORS AGGREGATOR, 199 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 200 AIRPORT INFRARED SENSORS AGGREGATOR JAR, AIRPORT INFRARED SENSORS AGGREGATOR PORT, 201 AIRPORT INFRARED SENSORS AGGREGATOR, 1); 202 airportInfraredSensorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT INFRARED SENSORS AGGREGATOR)); 203 } 204 205 private void initAirportDisplayActuatorsAggregatorSpecs() { 206 airportDisplayActuatorsAggregatorSpec = new DeployableServiceSpec(AIRPORT DISPLAY ACTUATORS AGGREGATOR, 207 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 208 AIRPORT DISPLAY ACTUATORS AGGREGATOR JAR, AIRPORT DISPLAY ACTUATORS AGGREGATOR PORT, 209 AIRPORT DISPLAY ACTUATORS AGGREGATOR, 1); 210 airportDisplayActuatorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT DISPLAY ACTUATORS AGGREGATOR)); 211 } 212 213 private void initAirportBusCompanySpecs() { 214 airportBusCompanySpec = new DeployableServiceSpec(AIRPORT BUS COMPANY, ServiceType.SOAP, 215 PackageType.COMMAND LINE, resourceImpact, serviceVersion, AIRPORT BUS COMPANY JAR, 216 AIRPORT BUS COMPANY PORT, AIRPORT BUS COMPANY, 1); 217 airportBusCompanySpec.setRoles(Collections.singletonList(AIRPORT BUS COMPANY)); 218 airportBusCompanySpec.addDependency(new ServiceDependency(AIRPORT, AIRPORT)); 219 } 220 221 private void initAirportSpecs() { 222 airportSpec = new DeployableServiceSpec(AIRPORT, ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, 223 serviceVersion, AIRPORT JAR, AIRPORT PORT, AIRPORT, 1); 224 airportSpec.setRoles(Collections.singletonList(AIRPORT)); 225 airportSpec.addDependency(new ServiceDependency(AIRPORT BUS COMPANY, AIRPORT BUS COMPANY)); 226 airportSpec.addDependency(new ServiceDependency(AIRPORT DISPLAY ACTUATORS AGGREGATOR, 227 AIRPORT DISPLAY ACTUATORS AGGREGATOR)); 228 airportSpec.addDependency(new ServiceDependency(AIRPORT INFRARED SENSORS AGGREGATOR, 229 AIRPORT INFRARED SENSORS AGGREGATOR)); 230 airportSpec.addDependency(new ServiceDependency(AIRPORT NOISE SENSORS AGGREGATOR, 231 AIRPORT NOISE SENSORS AGGREGATOR)); 232 airportSpec.addDependency(new ServiceDependency(AIRPORT PRESSURE SENSORS AGGREGATOR, 233 AIRPORT PRESSURE SENSORS AGGREGATOR)); 234 airportSpec.addDependency(new ServiceDependency(AIRPORT SIGN ACTUATORS AGGREGATOR, 235 AIRPORT SIGN ACTUATORS AGGREGATOR)); 236 airportSpec.addDependency(new ServiceDependency(AIRPORT SPEAKER ACTUATORS AGGREGATOR, 237 AIRPORT SPEAKER ACTUATORS AGGREGATOR)); 238 airportSpec.addDependency(new ServiceDependency(BOOKABLE AMENITY, BOOKABLE AMENITY)); 239 airportSpec.addDependency(new ServiceDependency(LUGGAGE HANDLING COMPANY, LUGGAGE HANDLING COMPANY)); 240 airportSpec.addDependency(new ServiceDependency(MID DISPLAY ACTUATORS AGGREGATOR, 241 MID DISPLAY ACTUATORS AGGREGATOR)); 242 airportSpec.addDependency(new ServiceDependency(MID LOCATION SENSORS AGGREGATOR, 243 MID LOCATION SENSORS AGGREGATOR)); 244 airportSpec.addDependency(new ServiceDependency(MID MICROPHONE SENSORS AGGREGATOR, 245 MID MICROPHONE SENSORS AGGREGATOR)); 246 airportSpec.addDependency(new ServiceDependency(SECURITY COMPANY, SECURITY COMPANY)); 247 airportSpec.addDependency(new ServiceDependency(STAND AND GATE MANAGEMENT, STAND AND GATE MANAGEMENT)); 248 } 249 250 public ChoreographySpec getChorSpec() { 251 return chorSpec; 252 } 253 254 } 173 private void initAirportSignActuatorsAggregatorSpecs() { 174 airportSignActuatorsAggregatorSpec = new DeployableServiceSpec(AIRPORT SIGN ACTUATORS AGGREGATOR, 175 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 176 AIRPORT SIGN ACTUATORS AGGREGATOR JAR, AIRPORT SIGN ACTUATORS AGGREGATOR PORT, 177 AIRPORT SIGN ACTUATORS AGGREGATOR, 1); 178 airportSignActuatorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT SIGN ACTUATORS AGGREGATOR)); 179 } 180 181 private void initAirportPressureSensorsAggregatorSpecs() { 182 airportPressureSensorsAggregatorSpec = new DeployableServiceSpec(AIRPORT PRESSURE SENSORS AGGREGATOR, 183 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 184 AIRPORT PRESSURE SENSORS AGGREGATOR JAR, AIRPORT PRESSURE SENSORS AGGREGATOR PORT, 185 AIRPORT PRESSURE SENSORS AGGREGATOR, 1); 186 airportPressureSensorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT PRESSURE SENSORS AGGREGATOR)); 187 } 188 189 private void initAirportNoiseSensorsAggregatorSpecs() { 190 airportNoiseSensorsAggregatorSpec = new DeployableServiceSpec(AIRPORT NOISE SENSORS AGGREGATOR, 191 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 192 AIRPORT NOISE SENSORS AGGREGATOR JAR, AIRPORT NOISE SENSORS AGGREGATOR PORT, 193 AIRPORT NOISE SENSORS AGGREGATOR, 1); 194 airportNoiseSensorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT NOISE SENSORS AGGREGATOR)); 195 } 196 197 private void initAirportInfraredSensorsAggregatorSpecs() { 198 airportInfraredSensorsAggregatorSpec = new DeployableServiceSpec(AIRPORT INFRARED SENSORS AGGREGATOR, 199 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 200 AIRPORT INFRARED SENSORS AGGREGATOR JAR, AIRPORT INFRARED SENSORS AGGREGATOR PORT, 201 AIRPORT INFRARED SENSORS AGGREGATOR, 1); 202 airportInfraredSensorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT INFRARED SENSORS AGGREGATOR)); 203 } 204 205 private void initAirportDisplayActuatorsAggregatorSpecs() { 206 airportDisplayActuatorsAggregatorSpec = new DeployableServiceSpec(AIRPORT DISPLAY ACTUATORS AGGREGATOR, 207 ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, serviceVersion, 208 AIRPORT DISPLAY ACTUATORS AGGREGATOR JAR, AIRPORT DISPLAY ACTUATORS AGGREGATOR PORT, 209 AIRPORT DISPLAY ACTUATORS AGGREGATOR, 1); 210 airportDisplayActuatorsAggregatorSpec.setRoles(Collections.singletonList(AIRPORT DISPLAY ACTUATORS AGGREGATOR)); 211 } 212 213 private void initAirportBusCompanySpecs() { 214 airportBusCompanySpec = new DeployableServiceSpec(AIRPORT BUS COMPANY, ServiceType.SOAP, 215 PackageType.COMMAND LINE, resourceImpact, serviceVersion, AIRPORT BUS COMPANY JAR, 216 AIRPORT BUS COMPANY PORT, AIRPORT BUS COMPANY, 1); 217 airportBusCompanySpec.setRoles(Collections.singletonList(AIRPORT BUS COMPANY)); 218 airportBusCompanySpec.addDependency(new ServiceDependency(AIRPORT, AIRPORT)); 219 } 220 221 private void initAirportSpecs() { 222 airportSpec = new DeployableServiceSpec(AIRPORT, ServiceType.SOAP, PackageType.COMMAND LINE, resourceImpact, 223 serviceVersion, AIRPORT JAR, AIRPORT PORT, AIRPORT, 1); 224 airportSpec.setRoles(Collections.singletonList(AIRPORT)); 225 airportSpec.addDependency(new ServiceDependency(AIRPORT BUS COMPANY, AIRPORT BUS COMPANY)); 226 airportSpec.addDependency(new ServiceDependency(AIRPORT DISPLAY ACTUATORS AGGREGATOR, 227 AIRPORT DISPLAY ACTUATORS AGGREGATOR)); 228 airportSpec.addDependency(new ServiceDependency(AIRPORT INFRARED SENSORS AGGREGATOR, 229 AIRPORT INFRARED SENSORS AGGREGATOR)); 230 airportSpec.addDependency(new ServiceDependency(AIRPORT NOISE SENSORS AGGREGATOR, 231 AIRPORT NOISE SENSORS AGGREGATOR)); 232 airportSpec.addDependency(new ServiceDependency(AIRPORT PRESSURE SENSORS AGGREGATOR, 233 AIRPORT PRESSURE SENSORS AGGREGATOR)); 234 airportSpec.addDependency(new ServiceDependency(AIRPORT SIGN ACTUATORS AGGREGATOR, 235 AIRPORT SIGN ACTUATORS AGGREGATOR)); 236 airportSpec.addDependency(new ServiceDependency(AIRPORT SPEAKER ACTUATORS AGGREGATOR, 237 AIRPORT SPEAKER ACTUATORS AGGREGATOR)); 238 airportSpec.addDependency(new ServiceDependency(BOOKABLE AMENITY, BOOKABLE AMENITY)); 239 airportSpec.addDependency(new ServiceDependency(LUGGAGE HANDLING COMPANY, LUGGAGE HANDLING COMPANY)) 240 airportSpec.addDependency(new ServiceDependency(MID DISPLAY ACTUATORS AGGREGATOR, 241 MID DISPLAY ACTUATORS AGGREGATOR)); 242 airportSpec.addDependency(new ServiceDependency(MID LOCATION SENSORS AGGREGATOR, 243 MID LOCATION SENSORS AGGREGATOR)); 244 airportSpec.addDependency(new ServiceDependency(MID MICROPHONE SENSORS AGGREGATOR, 245 MID MICROPHONE SENSORS AGGREGATOR)); 246 airportSpec.addDependency(new ServiceDependency(SECURITY COMPANY, SECURITY COMPANY)); 247 airportSpec.addDependency(new ServiceDependency(STAND AND GATE MANAGEMENT, STAND AND GATE MANAGEMENT 248 } 249 250 public ChoreographySpec getChorSpec() { 251 return chorSpec; 252 } 253 Listing A.1: WP6 choreography speciﬁcation used as Enactment Engine input Listing A.1: WP6 choreography speciﬁcation used as Enactment Engine input CHOReOS FP7-257178 73 1 package org.ow2.choreos; 2 3 import org.ow2.choreos.chors.ChoreographyDeployer; 4 import org.ow2.choreos.chors.ChoreographyNotFoundException; 5 import org.ow2.choreos.chors.EnactmentException; 6 import org.ow2.choreos.chors.client.ChorDeployerClient; 7 import org.ow2.choreos.chors.datamodel.Choreography; 8 import org.ow2.choreos.chors.datamodel.ChoreographySpec; 9 import org.ow2.choreos.utils.Alarm; 10 import org.ow2.choreos.utils.CommandLineException; 11 12 public class ThalesEnact { 13 14 public static void main(String[] args) throws EnactmentException, ChoreographyNotFoundException, CommandLineException { 15 16 ﬁnal String CHOR DEPLOYER URI = ”http://localhost:9102/choreographydeployer”; 17 ChoreographyDeployer chorDeployer = new ChorDeployerClient(CHOR DEPLOYER URI); 18 ThalesSpecs thalesSpecs = new ThalesSpecs(); 19 ChoreographySpec chorSpec = thalesSpecs.getChorSpec(); 20 21 String chorId = chorDeployer.createChoreography(chorSpec); 22 Choreography chor = chorDeployer.enactChoreography(chorId); 23 24 System.out.println(chor); // just to check EE output 25 } 26 } Listing A.2: Program to invoke Enactment Engine and launch WP6 choreography deployment 1 package org.ow2.choreos; 2 3 import org.ow2.choreos.chors.ChoreographyDeployer; 4 import org.ow2.choreos.chors.ChoreographyNotFoundException; 5 import org.ow2.choreos.chors.EnactmentException; 6 import org.ow2.choreos.chors.client.ChorDeployerClient; 7 import org.ow2.choreos.chors.datamodel.Choreography; 8 import org.ow2.choreos.chors.datamodel.ChoreographySpec; 9 import org.ow2.choreos.utils.Alarm; 10 import org.ow2.choreos.utils.CommandLineException; 11 12 public class ThalesEnact { 13 14 public static void main(String[] args) throws EnactmentException, ChoreographyNotFoundException, CommandLineException { 15 16 ﬁnal String CHOR DEPLOYER URI = ”http://localhost:9102/choreographydeployer”; 17 ChoreographyDeployer chorDeployer = new ChorDeployerClient(CHOR DEPLOYER URI); 18 ThalesSpecs thalesSpecs = new ThalesSpecs(); 19 ChoreographySpec chorSpec = thalesSpecs.getChorSpec(); 20 21 String chorId = chorDeployer.createChoreography(chorSpec); 22 Choreography chor = chorDeployer.enactChoreography(chorId); 23 24 System.out.println(chor); // just to check EE output 25 } 26 } Listing A.2: Program to invoke Enactment Engine and launch WP6 choreography deployment 1 package org.ow2.choreos; 2 Listing A.2: Program to invoke Enactment Engine and launch WP6 choreography deployment Listing A.2: Program to invoke Enactment Engine and launch WP6 choreography deployment CHOReOS FP7-257178 74
https://openalex.org/W4247974659	https://www.researchsquare.com/article/rs-16812/v4.pdf?c=1606325744000	English	null	Comparative analysis, applications, and interpretation of electronic health record-based stroke phenotyping methods	Research Square (Research Square)	2,020	cc-by	9,678	Comparative analysis, applications, and interpretation of electronic health record-based stroke phenotyping methods Phyllis Thangaraj Columbia University Medical Center https://orcid.org/0000-0003-3968-1563 Benjamin R Kummer Icahn School of Medicine at Mount Sinai Tal Lorberbaum Columbia University Medical Center Mitchell S.V. Elkind Columbia University Medical Center Nicholas P Tatonetti (  nick.tatonetti@columbia.edu ) Columbia University https://orcid.org/0000-0002-2700-2597 Research Keywords: phenotyping algorithms, acute ischemic stroke, machine learning, electronic health record studies Posted Date: November 25th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-16812/v4 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 December 7th, 2020. See the published version at https://doi.org/10.1186/s13040-020-00230-x. Comparative analysis, applications, and interpretation of electronic health record-based stroke phenotyping methods Phyllis Thangaraj Columbia University Medical Center https://orcid.org/0000-0003-3968-1563 Benjamin R Kummer Icahn School of Medicine at Mount Sinai Tal Lorberbaum Columbia University Medical Center Mitchell S.V. Elkind Columbia University Medical Center Nicholas P Tatonetti (  nick.tatonetti@columbia.edu ) Columbia University https://orcid.org/0000-0002-2700-2597 Research Keywords: phenotyping algorithms, acute ischemic stroke, machine learning, electronic health record studies Posted Date: November 25th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-16812/v4 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 December 7th, 2020. See the published version at https://doi.org/10.1186/s13040-020-00230-x. Research 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 December 7th, 2020. See the published version at https://doi.org/10.1186/s13040-020-00230-x. Page 1/23 Abstract Background: Accurate identification of acute ischemic stroke (AIS) patient cohorts is essential for a wide range of clinical investigations. Automated phenotyping methods that leverage electronic health records (EHRs) represent a fundamentally new approach cohort identification without current laborious and ungeneralizable generation of phenotyping algorithms. We systematically compared and evaluated the ability of machine learning algorithms and case-control combinations to phenotype acute ischemic stroke patients using data from an EHR. Materials and Methods: Using structured patient data from the EHR at a tertiary-care hospital system, we built and evaluated machine learning models to identify patients with AIS based on 75 different case- control and classifier combinations. We then estimated the prevalence of AIS patients across the EHR. Finally, we externally validated the ability of the models to detect AIS patients without AIS diagnosis codes using the UK Biobank. Results: Across all models, we found that the mean AUROC for detecting AIS was 0.963±0.0520 and average precision score 0.790±0.196 with minimal feature processing. Classifiers trained with cases with AIS diagnosis codes and controls with no cerebrovascular disease codes had the best average F1 score (0.832±0.0383). In the external validation, we found that the top probabilities from a model-predicted AIS cohort were significantly enriched for AIS patients without AIS diagnosis codes (60-150 fold over expected). Conclusions: Our findings support machine learning algorithms as a generalizable way to accurately identify AIS patients without using process-intensive manual feature curation. When a set of AIS patients is unavailable, diagnosis codes may be used to train classifier models. Background phenotyping) with commonly accessible EHR data and develop new approaches to etiologic identification and subtyping, the optimal combination of cases and controls to train such models remains unclear. Reproducibility and computability of phenotyping algorithms stem from the use of structured data, standardized terminologies, and rule-based logic.6 Phenotyping features from the EHR have been traditionally culled and curated by experts to manually construct algorithms, 7 but machine learning techniques present the potential advantage of automating this process of feature selection and refinement.8-11 Recent machine learning approaches have also combined publicly available knowledge sources with EHR data to facilitate feature curation.12,13 Additionally, while case and control phenotyping using EHR data has also relied on a small number of expert curated cohorts, recent studies have demonstrated that ML approaches can expand upon and identify such cohorts using automated feature selection and imperfect case definitions in a high-throughput manner.14-18 Studies have also shown that case and control selection with diagnosis codes can significantly affect model performance, the hierarchical organization of structured medical data can be utilized for feature reduction and model performance improvement, and calibration is essential for understanding the clinical utility of a phenotyping model.19-22 Stroke phenotyping algorithms have also used machine learning to enhance the classification performance of a diagnosis-code based AIS phenotyping algorithm.23-26 However, while ML models present an opportunity to automate identification of AIS patients (i.e. phenotyping) with commonly accessible EHR data and develop new approaches to etiologic identification and subtyping, the optimal combination of cases and controls to train such models remains unclear. Given the limitations of manual and diagnosis-code cohort identification, we sought to develop phenotypic classifiers for AIS using machine learning approaches, with the objective of specifically identifying AIS patients that were missing diagnosis codes. Additionally, considering the challenge of identifying true controls in the EHR for the purpose of model training, we also attempted to determine the optimal grouping of cases and controls by selecting and comparing model discriminatory performance with multiple case-control group combinations. We also sought to contrast model training based on cases defined by diagnostic code with that using manually-curated cohorts. Our phenotyping method utilizes machine learning classifiers with minimal data processing to increase the number of stroke patients recovered within the EHR and reduce the time and effort needed to find them for research studies. Background Stroke is a complex disease that is a leading cause of death and severe disability for millions of survivors worldwide.1 Accurate identification of stroke etiology, which is most commonly ischemic but encompasses several other causative mechanisms, is essential for risk stratification, optimal treatment, and support of clinical research. While electronic health records (EHR) are an emerging resource that can be used to study stroke patients, identification of stroke patient cohorts using the EHR requires the integration of multiple facets of data, including medical notes, labs, imaging reports, and medical expertise of neurologists. This process is often manually performed and time-consuming, and can reveal mis-classification errors.2 One simple approach to identify acute ischemic stroke (AIS) is the diagnosis- code based algorithm created by Tirschwell and Longstreth.3 However, identifying every AIS patient using these criteria can be difficult due to the inaccuracy and incompleteness of diagnosis recording through insurance billing. 3–5 Additionally, this approach prevents the identification of AIS patients until after hospital discharge, thereby limiting the clinical usability of identification algorithms in time-sensitive situations, such as in-hospital care management, research protocol enrollment, or acute treatment. Page 2/23 Page 2/23 Reproducibility and computability of phenotyping algorithms stem from the use of structured data, standardized terminologies, and rule-based logic.6 Phenotyping features from the EHR have been traditionally culled and curated by experts to manually construct algorithms, 7 but machine learning techniques present the potential advantage of automating this process of feature selection and refinement.8-11 Recent machine learning approaches have also combined publicly available knowledge sources with EHR data to facilitate feature curation.12,13 Additionally, while case and control phenotyping using EHR data has also relied on a small number of expert curated cohorts, recent studies have demonstrated that ML approaches can expand upon and identify such cohorts using automated feature selection and imperfect case definitions in a high-throughput manner.14-18 Studies have also shown that case and control selection with diagnosis codes can significantly affect model performance, the hierarchical organization of structured medical data can be utilized for feature reduction and model performance improvement, and calibration is essential for understanding the clinical utility of a phenotyping model.19-22 Stroke phenotyping algorithms have also used machine learning to enhance the classification performance of a diagnosis-code based AIS phenotyping algorithm.23-26 However, while ML models present an opportunity to automate identification of AIS patients (i.e. Study cohort Table 1 presents the data and the total number of patients available for each set of cases and controls used in the training and internal and external validation parts of this study. Out of the Columbia University Irving Medical Center (CUIMC) Clinical Data Warehouse (CDW), which has a total of 6.4 million patients, we extracted 4,844 stroke service patients, which we found to have a 4-16% false positive rate for stroke through manual review. Supplementary Table 2 presents demographic characteristics for the training sets, and Supplementary Table 3 and Supplementary Table 4 present demographic and feature category coverage for the testing sets. Page 3/23 Page 3/23 Variable Identification N Samples Total Patients CUIMC CDW Person ID 6,377,222 Diagnosis Codes ICD9-CM, ICD10-CM, SNOMED 140,300,457 Procedure Codes ICD9-CM, ICD10-CM, CPT, SNOMED 64,383,775 Prescription Orders RxNorm 40,759,814 Training Categories: Cases (S) Cases: Stroke Service Patients Seen by NYP Stroke Service 4,484 (T) Cases: AIS Tirschwell Criteria ICD9-CM: 434.x1, 433.x1, ICD10-CM: I63.xxx 79,306 (C) Cases: CCS Cerebrovascular Disease ICD9-CM: 346.6x,430, 431, 432.x, 433.xx 181,698 Training Categories: Controls (N) Controls: AIS Mimetic Diseases ICD9-CM: 191.x, 225.x, 340, 250.0, 431 8,438 (I) Controls: Without AIS Tirschwell Criteria No (T) Codes 5,243,646 (C) Controls: Without CCS Cerebrovascular Disease No (C) Codes 5,149,975 (CI) Controls: With CCS Cerebrovascular disease, w/o AIS Tirschwell Criteria (C) codes, No (T) codes 102,435 (R) Random set of patients With >=1 ICD9-CM or ICD10-CM diagnosis code 5,396,172 Table 1: Select Structured Data and Sample Case/Controls for models available in Table 1: Select Structured Data and Sample Case/Controls for models available in Columbia University Irving Medical Center Common Data Warehouse. NYP= New York Table 1: Select Structured Data and Sample Case/Controls for mod Table 1: Select Structured Data and Sample Case/Controls for models available in Columbia University Irving Medical Center Common Data Warehouse. NYP= New York Presbyterian, AIS= Acute Ischemic Stroke, CCS = Clinical Classifications Software Columbia University Irving Medical Center Common Data Warehouse. NYP= New York Presbyterian, AIS= Acute Ischemic Stroke, CCS = Clinical Classifications Software Feature importance We found the most commonly chosen features associated with stroke diagnosis were procedures used in evaluation of AIS, including extra- and intra-cranial arterial scans, computerized tomography (CT) scans and magnetic resonance imaging (MRI) of the brain, and MR angiography (Figure 3A). We also found that all 75 models relied on incremental contributions from many different features (Figure 3B, Supplementary Figures 20-34). Algorithm performance We trained 75 models using all combinations of cases, controls, and model types after excluding 15 neural network models due to poor performance (architecture described in supplemental methods). Logistic regression classifiers with L1 penalty gave the best area under the receiving operator curve (AUROC) performance (0.913-0.997) and the best average precision score (0.662-0.969), followed by logistic regression classifiers with elastic net penalty (Figure 2, Supplementary Table 5). Across all classifier types, the models using the T-C case-control combination had the best average F1 score (0.832±0.0383), whereas logistic regression models with L1 penalty (LR) and elastic-net penalty had the best classifier average F1 score (0.705±0.146 and 0.710±0.134 respectively) (Figure 2B, Supplementary Table 8). Use of cases from the CUIMC stroke service gave the highest average precision Page 4/23 Page 4/23 (0.932±.0536), while cases identified through AIS diagnosis codes and controls without cerebrovascular disease or acute ischemic stroke (AIS)-related diagnosis codes (TC, TI) gave high precision as well (0.896±0.0488 and 0.918±0.0316, respectively). The sensitivity of the models ranged widely, between 0.18 and 0.96, while specificity narrowly ranged between 0.993-1.0 (Supplementary Table 9). (0.932±.0536), while cases identified through AIS diagnosis codes and controls without cerebrovascular disease or acute ischemic stroke (AIS)-related diagnosis codes (TC, TI) gave high precision as well (0.896±0.0488 and 0.918±0.0316, respectively). The sensitivity of the models ranged widely, between 0.18 and 0.96, while specificity narrowly ranged between 0.993-1.0 (Supplementary Table 9). We also evaluated the AUROC and maximum F1 Score using a hold-out test set of Tirschwell (T) criteria cases and a random selection of (I) controls. We trained on S, T, and C cases and C controls, and found AUROC of 0.932-0.937 for the TC and CC trained sets and 0.69-0.87 for the SC trained sets. We also see a maximum F1 score of 0.351-0.432 for the TC and CC trained sets and 0.298-0.321 for the SC trained sets (Supplementary Figure 35). Internal validation in institutional EHR We applied the 75 models to the entire CUIMC EHR with at least one diagnosis code, totaling between 5,324,725 and 5,315,923 patients depending on the case/control set. We found that the results varied widely across models, but most predicted a prevalence of between 0.2-2% of patients in the EHR were AIS patients. The models with controls with cerebrovascular disease codes but no AIS codes predicted the lowest prevalence of AIS patients, and found 50.3-100% of the proposed patients had AIS diagnosis codes. The models with the best performance and robustness, 1) stroke service cases and controls without cerebrovascular disease codes and 2) cases with AIS codes and controls without cerebrovascular disease codes with 1) Logistic Regression and L1 Penalty classifier and 2) Adaboost classifier, had sensitivities between 0.822-0.959, specificities 0.994-0.999, and estimated AIS prevalence in the EHR ranging between 1.3-2.0% (Supplementary Table 9, Table 2). Within these proposed AIS patients, 37.7- 41.4% had an AIS diagnosis code (Table 2). Page 5/23 Case/ Control Combo LR EHR Prev RF EHR Prev AB EHR Prev GB EHR Prev EN EHR Prev LR with AIS codes RF with AIS codes AB with AIS codes GB with AIS codes EN with AIS codes SN 0.7 0.7 1.0 1.3 0.7 41.3 32.2 35.6 29.0 26.4 SI 1.1 2.0 1.5 1.7 1.1 40.5 23.0 35.7 29.8 27.1 SC 1.3 1.7 1.5 1.8 1.3 37.7 25.4 37.9 30.8 28.5 SCI 0.2 0.1 0.2 0.3 0.2 83.1 82.6 76.9 72.2 63.5 SR 0.2 0.2 0.3 0.5 0.2 75.4 63.2 68.8 58.2 48.9 TN 0.9 0.8 0.9 1.0 0.9 44.7 28.5 47.2 35.6 22.5 TI 1.6 2.3 1.4 4.7 1.6 43.8 31.4 47.9 21.8 8.10 TC 1.7 2.7 2.0 1.6 1.7 41.4 28.2 39.0 43.1 32.6 TCI 0.1 0.0 0.1 0.1 0.1 94.6 96.1 85.9 95.3 79.0 TR 0.8 0.8 0.8 0.4 0.8 46.1 40.0 44.0 61.4 31.1 CN 1.3 1.3 1.3 1.0 1.3 34.0 17.1 33.5 31.5 21.4 CI 2.0 3.3 1.9 1.9 2.0 37.5 24.2 39.5 39.8 39.9 CC 2.3 3.3 2.2 2.1 2.3 35.6 25.3 37.2 37.1 29.9 CCI 0.0 0.0 0.1 0.0 0.0 97.5 100 50.3 92.8 74.2 CR 1.0 0.9 0.9 0.7 1.0 37.3 35.6 37.7 42.6 29.6 Table 2. Prevalence of acute ischemic stroke patients identified by each classifier across the EHR and proportion of those patients with T-L criteria. Prev=prevalence. See Supplementary Table 1 for case-control and model abbreviations' definitions. Internal validation in institutional EHR EHR and proportion of those patients with T-L criteria. Prev=prevalence. See Supplementary Table 1 for case-control and model abbreviations' definitions. Supplementary Table 1 for case-control and model abbreviations' definitions. Page 6/23 Page 6/23 UK Biobank Variables Identification Subject Data (T) Cases: AIS Tirschwell Criteria (C) Controls: Without Cerebrovascular Disease Self-reported AIS but no diagnosis codes Biobank. External validation We evaluated the performance of the TC models on identifying 2,624 patients without AIS ICD10 codes. The top 50, 100, 500, and 2,624 probabilities had a precision of over 29%, and up to 80% (Figure 4). Since within the test set only 0.5% of the patients had AIS, this translates to a 60-150-fold increase in AIS detection over random choice. Discussion Using a feature-agnostic, data-driven approach with minimal data transformation, we developed models that identify acute ischemic stroke (AIS) patients from commonly-accessible EHR data at the time of patient hospitalization without making use of AIS-related ICD9 and ICD10 codes as defined by Tirschwell and Longstreth. In demonstrating that AIS patients can be recovered from other EHR-available structured clinical features without AIS codes, this approach is in contrast to previous machine learning phenotyping algorithms, which have relied on manually curated features or use AIS-related diagnosis codes as the sole nonzero features in their models. 23,24,3 Cases and controls for training of phenotyping algorithms can be challenging to identify and define given the richness of available EHR data. From the sparsity of diagnosis codes in the EHR, it follows that patients lacking an AIS-related diagnosis code may not always be considered as a control in stroke cohorts. Similarly, it is difficult to determine whether patients with cerebrovascular diseases, which can serve as risk factors for AIS, or share genetic and pathophysiologic underpinnings with AIS should be considered controls. Additionally, due to the prevalence of AIS mimics, cohort definitions based on diagnosis code criteria may be unreliable. In light of the problems in defining patient cohorts from EHR data, we found marked differences in classifying performance across 15 different case-control training sets. While training with cases from the CUIMC stroke service cases identified stroke patients most accurately and with the highest precision and recall, we also found that training with cases identified from AIS codes with controls from either 1) no cerebrovascular disease or 2) no AIS codes afforded high Page 7/23 Page 7/23 precision (Supplementary Table 5). These findings suggest that a manually curated cohort may not be necessary to train the phenotyping models, and the AIS codes may be enough to define a training set. Using these models, we also increased our AIS patient cohort by 60% across the EHR, suggesting that the AIS codes themselves are not sufficient to identify all AIS patients. We found that stroke evaluation procedures, such as a CT scan or MRI, were important features in many of the models, which corroborates with a previous study. 23 Since none of these models use AIS diagnosis codes as features, this suggests that procedures may serve as proxies for them when identifying AIS cohorts. In some cases, the AIS code will only be added during outpatient follow up. Discussion For example, while in the stroke service set, 13.5% of cases did not have AIS codes in the inpatient setting but did in the outpatient setting, and 90% of these patients had had a CT scan of the head. We also found evidence that procedures provided a significant contribution to classification in the models in supplementary analysis (Supplementary Methods, Results, and Supplementary Figure 4). We found that as measured by AUROC and AP, discriminatory performance of the random forest, logistic regression with L1 and elastic net penalties, and gradient boosting models was robust, even when up to 95% of the training set was removed. These findings showed that a training set size as small as 70-350 samples can maintain high performance, depending on the model. Our results from traditional model performance and robustness evaluations show that our best machine learning phenotyping algorithm used Logistic Regression with L1 penalty or AdaBoost classifiers trained with controls without any cerebrovascular disease-related codes and a stroke service case population. However, we found that a similar model performed comparably well using cases identified by AIS-related diagnosis codes, suggesting that these models do not require manual case curation for high performance. In addition, our validation study in the UK Biobank detected AIS patients without ICD10-CM codes up to 150-fold better than random selection. In light of our findings, we recommend using machine learning models trained on all available structured EHR data, not just AIS diagnosis codes, to identify AIS patients. Previous studies required time- consuming manual curation of features or trained on only AIS codes, which would have missed AIS patients identified through a CT scan or MRI but without AIS diagnosis codes.23,24 Our thorough In light of our findings, we recommend using machine learning models trained on all available structured EHR data, not just AIS diagnosis codes, to identify AIS patients. Previous studies required time- consuming manual curation of features or trained on only AIS codes, which would have missed AIS patients identified through a CT scan or MRI but without AIS diagnosis codes.23,24 Our thorough investigation of feature importance shows that each feature contributes to the improved performance of the models. We also recommend restricting controls further to patients without cerebrovascular disease diagnosis codes, rather than just without AIS diagnosis codes to improve discriminatory ability. Discussion In addition, we show improved AUROC and specificity, and comparable sensitivity, precision, recall, and F1- score using SC and TC case-control sets, to previous studies.23,24 Finally, as shown in Table 2, we show the vast potential for identifying AIS cases in the EHR that do not have an AIS diagnosis code. In light of our findings, we recommend using machine learning models trained on all available structured EHR data, not just AIS diagnosis codes, to identify AIS patients. Previous studies required time- consuming manual curation of features or trained on only AIS codes, which would have missed AIS patients identified through a CT scan or MRI but without AIS diagnosis codes.23,24 Our thorough investigation of feature importance shows that each feature contributes to the improved performance of the models. We also recommend restricting controls further to patients without cerebrovascular disease diagnosis codes, rather than just without AIS diagnosis codes to improve discriminatory ability. In addition, we show improved AUROC and specificity, and comparable sensitivity, precision, recall, and F1- score using SC and TC case-control sets, to previous studies.23,24 Finally, as shown in Table 2, we show the vast potential for identifying AIS cases in the EHR that do not have an AIS diagnosis code. g g g investigation of feature importance shows that each feature contributes to the improved performance of the models. We also recommend restricting controls further to patients without cerebrovascular disease diagnosis codes, rather than just without AIS diagnosis codes to improve discriminatory ability. In addition, we show improved AUROC and specificity, and comparable sensitivity, precision, recall, and F1- score using SC and TC case-control sets, to previous studies.23,24 Finally, as shown in Table 2, we show the vast potential for identifying AIS cases in the EHR that do not have an AIS diagnosis code. This study has several limitations. First, we relied on noisy labels and proxies for training our models, as evidenced by our manual review false positive rate. Without a gold standard set of cases, model performance is difficult to definitively evaluate. We relied on pre-defined codes, the Tirschwell criteria, and Page 8/23 Page 8/23 patients evaluated for stroke as our cases. We included a random set of patients as our holdout control test set for representation of all patients in the EHR. This is a limitation, however, because patients with Tirschwell criteria could be labeled as random controls. Discussion We addressed this by removing any Tirschwell criteria patients from the hold out controls. In general, the use of random controls could lead to overlapping of cases and controls, especially in common disease, but one can use known diagnostic codes for the disease to separate cases and controls. Our method importantly does not include any codes used in the case and control definitions in our machine learned features in order to identify other features involved in defining stroke patients. We do this so that our models are not reidentifying Tirschwell criteria, and instead are identifying novel features complementary to the criteria. This removal of overlapping cases and controls can also influence our calibration results described in the supplementary materials by changing the proportion of expected stroke cases at each probability score; however, this only amounted to a removal of 0.05% of overlapping patients. We also do see a marked decreased in F1 performance and a slight decrease in AUROC when testing on hold out Tirschwell criteria cases instead of Stroke Service cases in the Columbia EHR. This may be due to better documentation of structured EHR data, particularly procedures and medications, in Stroke Service patients as seen in Supplementary Table 4. However, in the UK Biobank, which used Tirschwell criteria cases as a holdout test set, we see high precision in identifying AIS patients over random. This would suggest reduction in sensitivity of our model. Second, we used only structured features contained within standard terminologies across the patients' entire timeline, and did not use clinical notes. In addition, the biases inherent in phenotyping with billing codes are a significant limitation. Often the data is missing not at random, and data completeness relies on patient interaction with the healthcare system, which can lead to ascertainment bias towards diagnoses and tests that doctors already suspect or patients who actively seek care and make generalizing outcomes from these patients difficult. 27-31 Diagnosis also often are chosen for reimbursement purposes rather than actual diagnosis, and diagnosis code use changes over time, leading to inaccuracies in phenotyping. 27,28 Given previous studies, however, it has been established that stroke can be identified by diagnosis codes with high sensitivity, specificity, and positive predictive value. 3,46 While clinical notes may contain much highly relevant information, they may also give rise to less reproducible and generalizable feature sets. Discussion Additionally, each feature contributed incrementally to high performance of the models and required minimal processing to acquire. Third, due to limitations of time and computational complexity, we did not exhaustively explore all possible combinations of cases and controls, including other potential AIS mimetic diseases. Despite these limitations, precision in the internal validation using the held-out set was high, and when applied to an external validation cohort, the developed models improved detection of AIS patients between 60 and 150-fold over random patient identification. Fourth, we did not study clinical implementation of the models. However, the discriminatory ability of the classifiers in the external validation suggest that although these models have not been implemented clinically, they may potentially be useful for improving the power of existing clinical and h t d h t patients evaluated for stroke as our cases. We included a random set of patients as our holdout control test set for representation of all patients in the EHR. This is a limitation, however, because patients with Tirschwell criteria could be labeled as random controls. We addressed this by removing any Tirschwell criteria patients from the hold out controls. In general, the use of random controls could lead to overlapping of cases and controls, especially in common disease, but one can use known diagnostic codes for the disease to separate cases and controls. Our method importantly does not include any codes used in the case and control definitions in our machine learned features in order to identify other features involved in defining stroke patients. We do this so that our models are not reidentifying Tirschwell criteria, and instead are identifying novel features complementary to the criteria. This removal of overlapping cases and controls can also influence our calibration results described in the supplementary materials by changing the proportion of expected stroke cases at each probability score; however, this only amounted to a removal of 0.05% of overlapping patients. We also do see a marked decreased in F1 performance and a slight decrease in AUROC when testing on hold out Tirschwell criteria cases instead of Stroke Service cases in the Columbia EHR. This may be due to better documentation of structured EHR data, particularly procedures and medications, in Stroke Service patients as seen in Supplementary Table 4. Discussion However, in the UK Biobank, which used Tirschwell criteria cases as a holdout test set, we see high precision in identifying AIS patients over random. This would suggest reduction in sensitivity of our model. Second, we used only structured features contained within standard terminologies across the patients' entire timeline, and did not use clinical notes. In addition, the biases inherent in phenotyping with billing codes are a significant limitation. Often the data is missing not at random, and data completeness relies on patient interaction with the healthcare system, which can lead to ascertainment bias towards diagnoses and tests that doctors already suspect or patients who actively seek care and make generalizing outcomes from these patients difficult. 27-31 Diagnosis also often are chosen for reimbursement purposes rather than actual diagnosis, and diagnosis code use changes over time, leading to inaccuracies in phenotyping. 27,28 Given previous studies, however, it has been established that stroke can be identified by diagnosis codes with high sensitivity, specificity, and positive predictive value. 3,46 While clinical notes may contain much highly relevant information, they may also give rise to less reproducible and generalizable feature sets. Additionally, each feature contributed incrementally to high performance of the models and required minimal processing to acquire. Third, due to limitations of time and computational complexity, we did not exhaustively explore all possible combinations of cases and controls, including other potential AIS mimetic diseases. Despite these limitations, precision in the internal validation using the held-out set was high, and when applied to an external validation cohort, the developed models improved detection of AIS patients between 60 and 150-fold over random patient identification. Fourth, we did not study clinical implementation of the models. However, the discriminatory ability of the classifiers in the external validation suggest that although these models have not been implemented clinically, they may potentially be useful for improving the power of existing clinical and research study cohorts. Our study benefits from several strengths. First, to address the current deficiencies in developing phenotyping algorithms, we developed an approach that demonstrates comparable discriminatory ability Page 9/23 Page 9/23 of identifying patients with AIS to past methods but has the added benefit of using EHR data that is generally available during inpatient hospitalization. Second, our model features were composed of structured data that encompass a larger feature variety than purely ICD-code based algorithms. Discussion Third, because our model incorporated structured data from standard terminologies, they therefore may be generalizable to other health systems outside CUIMC, whereas recent studies have relied on manually curated feature sets.23 Fourth, we examined several different combinations of cases, controls and classifiers for the purposes of training phenotyping models. Finally, our phenotype classifiers assign probability of having had an AIS, which moves beyond binary classification of patients to develop a mor granular description of patient’s disease state. Conclusions In addition to research tasks such as cohort identification, future models could focus on timely interventions such as care planning prior to discharge and risk stratification. We showed that structured data may be sufficiently accurate for classification, allowing for widespread usability of the algorithm. We also demonstrated the potential for using machine learning classifiers for cohort identification, which achieve high performance with many features acquired through minimal processing. In addition, patient cohorts derived using AIS diagnosis codes may obviate the need for manually-curated cohorts of patients with AIS, and procedure codes may be useful in identifying patients with AIS that may not have been coded with AIS-related diagnosis codes. We, and others, hypothesize that expanding cohort size by assigning a probability of disease may improve the power of heritability and genome-wide association studies.32–37 Utilizing the structured framework present in many current EHRs, along with machine learning models may provide a generalizable approach for expanding research study cohort size. Study design In this study, we developed several machine learning phenotyping models for AIS using combinations of different case and control groups derived from our institution’s EHR data. Use of Columbia patient data was approved by Columbia’s institutional review board and UK Biobank data approved with UK Biobank Research Ethics Committee (REC) approval number 16/NW/0274. We also applied key methods to optimize number of features for generalizability, as well as calibration to ensure a clinically meaningful model output, and model robustness to missing data. To estimate the prevalence of potential AIS patients without AIS-related International Classification of Diseases-Clinical Modification (ICD-CM) codes, we then applied the developed models to all patients in our institutional EHR. Finally, we externally validated our best-performing model in an independent cohort from the UK Biobank to evaluate its ability to detect AIS patients without the requisite ICD codes. Figure 1 shows the overall workflow of training and testing the models, the models' evaluation, and its testing in an independent test set. Patient population We defined 3 case groups. We first included all patients from the CUIMC stroke service that were recorded as having AIS (cohort S). We then defined all patients in the CDW that met the Tirschwell-Longstreth (T-L) diagnosis code criteria for AIS (cohort T), which comprise ICD9-CM codes 434.x1, 433.x1, 436 (where x is any number) and the code is in the primary diagnostic position. 3 Our dataset did not specify the diagnostic position of codes. We also included ICD10-CM code equivalents, I63.xxx or I67.89, with the ICD10-CM codes being determined from ICD9-CM from Centers for Medicaid and Medicare Services (CMS) General Equivalence Mappings,] with a “10000” flag.39 Because patients with cerebrovascular disease are also likely to have suffered AIS, but may not have an attached AIS-related diagnosis code, we also created a group of cases according to cerebrovascular disease-related ICD codes defined by the ICD- 9-Clinical Modification (CM) Clinical Classifications Software tool (CCS), as well as their ICD10-CM equivalents (cohort C).40 We then defined 4 control groups (Figure 1, Table 1). First, we defined a control group of patients without AIS-related diagnosis codes (I). Due to the fact that cerebrovascular disease is a major risk factor for stroke,41,42 and to test a more stringent control definition than that of group (I), we also defined an additional group without any of the CCS cerebrovascular disease codes defined in cohort (C). Then, we defined a control set using CCS cerebrovascular disease diagnosis codes other than AIS (CI). Because multiple clinical entities can present as AIS, we also defined a group of controls according to diagnosis codes for AIS mimetic diseases (N), including hemiplegic migraine (ICD9-CM 346.3), brain tumor (191.xx, 225.0), multiple sclerosis (340), cerebral hemorrhage (431), and hypoglycemia with coma (251.0). Finally, we identified a control group culled from a random sample of patients (R). Data sources Page 10/23 We used data from patients in the Columbia University Irving Medical Center Clinical Data Warehouse (CUIMC CDW), which contains longitudinal health records of 6.4 million patients from CUIMC's EHR, spanning 1985-2018. The data are organized into tables and standardized vocabularies and terminologies in the format of the Observational Health Data Sciences and Informatics (OHDSI) Observational Medical Outcomes Partnership Common Data Model (OMOP CDM).38 The data include structured medical data such as conditions, procedures, medication orders, lab measurement values, visit type, demographics, and observations. This includes patients from the CUIMC stroke service (Figure 1, Table 1) that were part of a larger group of patients with acute cerebrovascular diseases and were prospectively identified upon admission to New York Presbyterian Hospital and recorded as part of daily research activities by a CUIMC stroke physician between 2011 and 2018. Two researchers (PT and BK) each manually reviewed 50 patients' charts for a total of 100 patients from this cohort to determine baseline false positive rates. Model features From the CDW, we gathered race, ethnicity, age, sex, diagnostic and procedure insurance billing codes as well as medication prescriptions for all patients. We dichotomized each feature based on its presence or Page 11/23 Page 11/23 absence in the data. Because Systematized Nomenclature of Medicine (SNOMED) concept IDs perform similarly to ICD9-CM and ICD10-CM codes for phenotyping, 43 we mapped diagnoses and procedure features from ICD9-CM, ICD10-CM, and Current Procedural Terminology 4 (CPT4) codes to SNOMED concept IDs using the OHDSI OMOP mappings, and used RxNorm IDs for medication prescriptions. We identified patients with Hispanic ethnicity using an algorithm combining race and ethnicity codes.44 The most recent diagnosis in the medical record served as the age end point and we dichotomized age as greater than or equal, or less than 50 years. We excluded from our feature set any diagnosis codes that were used in any case or control definitions. Because approximately 5 million patients exist in the CUIMC CDW without a cerebrovascular disease diagnosis code, we addressed this large resultant imbalance in cases and controls by randomly sampling controls to create a balanced, or 1:1 case to control ratio. In addition, we set the maximum sample size to 16,000 patients in order to control the size of the feature set. See Supplementary Methods for model development. Internal validation using all EHR patients To identify the number of patients classified as having AIS in our institutional EHR, we applied each of the 75 models to the entire patient population in the CUIMC CDW with at least one diagnosis code. We chose a probability threshold based on the maximum F1 score determined for each model from the training set. We also determined the percentage of patients that had AIS ICD9-CM codes as defined by T-L criteria and associated ICD10-CM codes. Acknowledgements An earlier version of this manuscript was included in the doctoral dissertation of Phyllis M. Thangaraj. We thank Dr. Patrick Ryan, Dr. Fernanda Polubriaginof, Dr. Theresa Koleck, Dr. Prashanth Selvaraj, Dr. Rami Vanguri, Dr. Joseph Romano, Alexandre Yahi, and Dr. Kayla Quinnies for their feedback and guidance. Consent for publication Not applicable. Funding PMT is funded by F30HL14094601, and previously was funded by 5T32GM007367, 5R01GM107145 and 10T3TR002027. NPT is funded by R35GM131905 and was funded by 5R01GM107145. Authors’ contributions PMT and NPT designed the study and drafted the original manuscript; BRK and MSE provided list of stroke service patients; BRK and PMT performed chart evaluation of stroke service patients; PMT and NPT performed analyses and wrote code. TL wrote code; MSE and NPT provided supervision; and PMT, BRK, TL, MSE, and NPT provided critical feedback on the manuscript. External validation The UK Biobank is a prospective health study of over 500,000 participants, ages 40-69, containing comprehensive EHR and genetic data.45 Given that this dataset contains 4,922 patients with an AIS related ICD10 code, similar to our T case cohort criteria, and 163 patients with self-reported AIS, the UK Biobank can evaluate our machine learning models’ ability to recover potential AIS patients that lack AIS- related ICD10 codes. In a systemic review, the UK Biobank Stroke Outcomes group found positive predictive value between 22-87% and negative predictive value between 88-99% for self-reported strokes.46 One difference between the UK Biobank definition of the AIS related ICD10 codes and our definition is their addition of code I64, which translates as “Stroke, not specified as haemorrhage or infarction”. We chose the most accurate and robust case-control combination from our models (cases defined by the T-L AIS codes (T) and controls without codes for cerebrovascular disease (C) in a 1:1 case- control ratio as our training set) to train the phenotyping model using conditions specified by ICD10 codes, procedures specified by OCPS4 codes, medications specified by RxNorm codes, and demographics as features, excluding features that were used to create the training and testing cohorts. We trained on half of the patients with AIS related ICD10 codes, and then tested our models on the rest of the UK Biobank data which included self-reported AIS cases and the other half of the patients with AIS related ICD10 codes. We added these patients to improve the power of detecting cases, and we removed the AIS related ICD10 codes from our feature set to prevent recovery of patients due to these codes. We Page 12/23 resampled the control set 50 times and evaluated the performance of the algorithm through AUROC, AP, and precision at the top 50, 100, 500 and 2,624 patients (ordered by model probability). resampled the control set 50 times and evaluated the performance of the algorithm through AUROC, AP, and precision at the top 50, 100, 500 and 2,624 patients (ordered by model probability). Ethics approval and consent to participate Use of Columbia patient data was approved by Columbia’s institutional review board and UK Biobank data approved with UK Biobank Research Ethics Committee (REC) approval number 16/NW/0274. This research has been conducted using the UK Biobank Resource under Application Number 41039. Availability of data and materials Code for analysis and figure generation in this study is publicly available online at https://github.com/pthangaraj/Stroke-Phenotyping. Electronic health record data structured in OMOP OHDSI format is required for use. Competing interests None. 1. Benjamin EJ, Virani SS, Callaway CW, et al. Heart Disease and Stroke Statistics—2018 Update: A Report From the American Heart Association. Circulation. 2018;137:e67–e492. References Page 13/23 Page 13/23 1. Benjamin EJ, Virani SS, Callaway CW, et al. Heart Disease and Stroke Statistics—2018 Update: A Report From the American Heart Association. Circulation. 2018;137:e67–e492. 2. 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Deep Phenotyping on Electronic Health Records Facilitates Genetic Diagnosis by Clinical Exomes. American journal of human genetics. 2018;103:58–73. 37. Hripcsak G, Albers DJ. High-fidelity phenotyping: richness and freedom from bias. Journal of the American Medical Informatics Association : JAMIA. 2017 38. Reich, C., and Ryan, P.B., and Belenkaya, R., Natarajan,K., and Blacketer, C. OMOP Common Data Model v6.0 Specifications. https://github.com/OHDSI/CommonDataModel/wiki Accessed September 2019 9. 2018 ICD-10 CM and GEMs. U.S. Centers for Medicare & Medicaid Service 39. 2018 ICD-10 CM and GEMs. U.S. Centers for Medicare & Medicaid Services. https://www.cms.gov/medicare/coding/icd10/2018-icd-10-cm-and-gems.html. Accessed February 2018 https://www.cms.gov/medicare/coding/icd10/2018-icd-10-cm-and-gems.html. Accessed February 2018. 40. HCUP CCS-Services and Procedures. Healthcare Cost and Utilization Project (HCUP). March 2017. Agency for Healthcare Research and Quality. https://www.hcup-us.ahrq.gov/toolssoftware/ccs/ccs.jsp. Accessed March 2019. 40. HCUP CCS-Services and Procedures. Healthcare Cost and Utilization Project (HCUP). March 2017. Agency for Healthcare Research and Quality. https://www.hcup-us.ahrq.gov/toolssoftware/ccs/ccs.jsp. Accessed March 2019. 41. Boehme AK, Esenwa C, Elkind M. Stroke Risk Factors, Genetics, and Prevention. Circ Res. 2017;120:472–495. 42. Benjamin EJ, Blaha MJ, Chiuve SE, et al. Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association. Circulation. 2017;135(10):e146. Epub 2017 Jan 25 43. Hripcsak G, Levine ME, Shang N, et al. OUP accepted manuscript. J Am Med Inform Assn. 2018; 44. Polubriaginof F, Vanguri R, Quinnies K, et al. Disease Heritability Inferred from Familial Relationships Reported in Medical Records. Cell. 2018;173:1692-1704.e11. 45. Sudlow C, Gallacher J, Allen N, et al. UK Biobank: An Open Access Resource for Identifying the Causes of a Wide Range of Complex Diseases of Middle and Old Age. Plos Med. 2015;12:e1001779. 46. Woodfield, R., Group, U. B. S. O., Group, U. B. F. and O. W. & Sudlow, C. L. M. Accuracy of Patient Self- Report of Stroke: A Systematic Review from the UK Biobank Stroke Outcomes Group. PLOS ONE 10, e0137538 (2015). Figures Figures Page 17/23 Figure 1 Schematic of Model Training, Testing, Evaluation, and Application to UK Biobank. See methods for case/control abbreviations. Case: Control ratio was 1:1, subjects overlapping in the case and control definitions were removed from the control set, and subjects overlapping between the training and testing sets were removed from the testing set before any training or testing. Models included Random Forest (RF), Logistic Regression with L1 penalty (LR), Neural Network (NN), Gradient Boosting (GB), Logistic Regression with Elastic Net Penalty (EN) and Adaboost (AB). AUROC: Area Under the Receiver Operating Curve, AUPR: Area under the Precision-Recall Curve, Sens: Sensitivity, Spec: Specificity, PPV: Positive Predictive Value, NPV: Negative Predictive Value. Figure 1 Schematic of Model Training, Testing, Evaluation, and Application to UK Biobank. See methods for case/control abbreviations. Case: Control ratio was 1:1, subjects overlapping in the case and control definitions were removed from the control set, and subjects overlapping between the training and testing sets were removed from the testing set before any training or testing. Models included Random Forest (RF), Logistic Regression with L1 penalty (LR), Neural Network (NN), Gradient Boosting (GB), Logistic Regression with Elastic Net Penalty (EN) and Adaboost (AB). AUROC: Area Under the Receiver Operating Curve, AUPR: Area under the Precision-Recall Curve, Sens: Sensitivity, Spec: Specificity, PPV: Positive Predictive Value, NPV: Negative Predictive Value. Page 18/23 Figure 1 Schematic of Model Training, Testing, Evaluation, and Application to UK Biobank. See methods for case/control abbreviations. Case: Control ratio was 1:1, subjects overlapping in the case and control definitions were removed from the control set, and subjects overlapping between the training and testing sets were removed from the testing set before any training or testing. Models included Random Forest (RF), Logistic Regression with L1 penalty (LR), Neural Network (NN), Gradient Boosting (GB), Logistic Regression with Elastic Net Penalty (EN) and Adaboost (AB). AUROC: Area Under the Receiver Operating Curve, AUPR: Area under the Precision-Recall Curve, Sens: Sensitivity, Spec: Specificity, PPV: Positive Predictive Value, NPV: Negative Predictive Value. Figure 2 Performance of select models on Stroke Service holdout test set ((a): AUROC (circle: median, bars: 50% CI), (b): F1 (circle: median, bars: 50% CI)). Different combinations of cases and controls are shown on the y-axis. (LR) logistic regression with l1 penalty, (RF) random forest, (AB) AdaBoost, (GB) gradient boosting, (EN) logistic regression with elastic net penalty. Different combinations of cases and controls are shown on the y-axis. Cases (first letter) may be one of cerebrovascular (C), T-L (T), or Stroke Service (S). Controls (second and third letters) may be one of random (R), cerebrovascular disease but no AIS code (CI), no cerebrovascular disease (C), no AIS code (I), or a stroke mimetic disease (N), See Methods and Supplementary Table 1 for definitions of sets. Threshold to compute the F1 score on the testing set was chosen as the threshold that yielded the maximum F1 in cross-validation on the training set (Methods, Supplementary Table 10). Supplementary Table 10). Figure 2 Performance of select models on Stroke Service holdout test set ((a): AUROC (circle: median, bars: 50% CI), (b): F1 (circle: median, bars: 50% CI)). Different combinations of cases and controls are shown on the y-axis. (LR) logistic regression with l1 penalty, (RF) random forest, (AB) AdaBoost, (GB) gradient boosting (EN) logistic regression with elastic net penalty. Different combinations of cases and controls are shown on the y-axis. Cases (first letter) may be one of cerebrovascular (C), T-L (T), or Stroke Service (S). Controls (second and third letters) may be one of random (R), cerebrovascular disease but no AIS code (CI), no cerebrovascular disease (C), no AIS code (I), or a stroke mimetic disease (N), See Methods and Supplementary Table 1 for definitions of sets. Threshold to compute the F1 score on the testing set was chosen as the threshold that yielded the maximum F1 in cross-validation on the training set (Methods, Supplementary Table 10). Figure 1 Schematic of Model Training, Testing, Evaluation, and Application to UK Biobank. See methods for case/control abbreviations. Case: Control ratio was 1:1, subjects overlapping in the case and control definitions were removed from the control set, and subjects overlapping between the training and testing sets were removed from the testing set before any training or testing. Models included Random Forest (RF), Logistic Regression with L1 penalty (LR), Neural Network (NN), Gradient Boosting (GB), Logistic Regression with Elastic Net Penalty (EN) and Adaboost (AB). AUROC: Area Under the Receiver Operating Curve, AUPR: Area under the Precision-Recall Curve, Sens: Sensitivity, Spec: Specificity, PPV: Positive Predictive Value, NPV: Negative Predictive Value. Page 19/23 , g Figure 3 A: Common top 10 features in the models. After each of the 75 models were trained, we counted the number of times each feature was represented as one of the top ten by absolute coefficient weight, for methods like logistic regression, or by feature importance, for methods like random forest. Above are features from this analysis along with the proportion of models in which they were in the top ten (% Models), the average frequency in the cases (Ave. Freq. Cases) and the average frequency in the controls (Ave. Freq. Controls). B: Prevalence of features in cases vs controls in the TC AB model. Axes were on a logarithmic scale. Increasing size of blue dot correlates with higher feature importance or beta coefficient weight, depending on the classifier type. Gray dots are features with zero importance. Page 21/23 gure 3 : Common top 10 features in the models. After each of the 75 models were trained, we counted the umber of times each feature was represented as one of the top ten by absolute coefficient weight, for Figure 2 s on Stroke Service holdout test set ((a): AUROC (circle: median, bars: 50% Performance of select models on Stroke Service holdout test set ((a): AUROC (circle: median, bars: 50% CI), (b): F1 (circle: median, bars: 50% CI)). Different combinations of cases and controls are shown on the y-axis. (LR) logistic regression with l1 penalty, (RF) random forest, (AB) AdaBoost, (GB) gradient boosting, (EN) logistic regression with elastic net penalty. Different combinations of cases and controls are shown on the y-axis. Cases (first letter) may be one of cerebrovascular (C), T-L (T), or Stroke Service (S). Controls (second and third letters) may be one of random (R), cerebrovascular disease but no AIS code (CI), no cerebrovascular disease (C), no AIS code (I), or a stroke mimetic disease (N), See Methods and Supplementary Table 1 for definitions of sets. Threshold to compute the F1 score on the testing set was chosen as the threshold that yielded the maximum F1 in cross-validation on the training set (Methods, Supplementary Table 10). Page 20/23 igure 3 : Common top 10 features in the models. After each of the 75 models were trained, we counted the umber of times each feature was represented as one of the top ten by absolute coefficient weight, for methods like logistic regression, or by feature importance, for methods like random forest. Above are eatures from this analysis along with the proportion of models in which they were in the top ten (% Figure 3 A: Common top 10 features in the models. After each of the 75 models were trained, we counted the number of times each feature was represented as one of the top ten by absolute coefficient weight, for Page 21/23 Page 21/23 methods like logistic regression, or by feature importance, for methods like random forest. Above are features from this analysis along with the proportion of models in which they were in the top ten (% Models), the average frequency in the cases (Ave. Freq. Cases) and the average frequency in the controls (Ave. Freq. Controls). B: Prevalence of features in cases vs controls in the TC AB model. Axes were on a logarithmic scale. Increasing size of blue dot correlates with higher feature importance or beta coefficient weight, depending on the classifier type. Gray dots are features with zero importance. Figure 4 Precision-fold over random sampling of acute ischemic stroke cases without related ICD10 codes at top 50, 100, 500, and 2,624 patient probabilities assigned by machine learning algorithms. With 95% confidence intervals in error bars. See Supplementary Table 1 for model abbreviations' definitions. Figure 4 Precision-fold over random sampling of acute ischemic stroke cases without related ICD10 codes at top 50, 100, 500, and 2,624 patient probabilities assigned by machine learning algorithms. With 95% confidence intervals in error bars. See Supplementary Table 1 for model abbreviations' definitions. Figure 4 Precision-fold over random sampling of acute ischemic stroke cases without related ICD10 codes at top 50, 100, 500, and 2,624 patient probabilities assigned by machine learning algorithms. With 95% confidence intervals in error bars. See Supplementary Table 1 for model abbreviations' definitions. Page 22/23 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SUPPLEMENTARYMATERIALSBioDataMiningThangarajetalFinal.docx SUPPLEMENTARYMATERIALSBioDataMiningThangarajetalFinal.docx SUPPLEMENTARYMATERIALSBioDataMiningThangarajetalFinal.docx SUPPLEMENTARYMATERIALSBioDataMiningThangarajetalFinal.docx SUPPLEMENTARYMATERIALSBioDataMiningThangarajetalFinal.docx SUPPLEMENTARYMATERIALSBioDataMiningThangarajetalFinal.docx Page 23/23 Page 23/23
https://openalex.org/W4386451654	http://www.journal.umuslim.ac.id/index.php/rkt/article/download/2020/1508	Indonesian	null	Sampul Vol. 7 No. 1 Januari 2023	Jurnal Rekayasa Teknik dan Teknologi	2,023	cc-by-sa	757	Vol. 7 No. 1 Januari 2023 Vol. 7 No. 1 Januari 2023 Muhammad Yanis, ST., MT Fakultas Teknik - Universitas Al Muslim- Bireuen Aceh - Indonesia PENGANTAR DARI KETUA PENYUNTING (EDITOR IN CHIEF) Puji syukur kehadirat Allah Subhanawataala yang telah melimpahkan Rahmat dan KaruniaNya serta Salawat kepada Nabi Besar Muhammad SAW, kami kembali menghadirkan Jurnal Rekayasa Teknik dan Teknologi (REKATEK) Volume 7 Nomor 1 Tahun 2023 edisi bulan Januari. Jurnal Rekayasa Teknik dan Teknologi (REKATEK) adalah jurnal ilmiah yang diterbitkan oleh Fakultas Teknik Universitas Al Muslim Bireuen Aceh. Jurnal Rekayasa Teknik dan Teknologi (REKATEK) diterbitkan dua kali dalam setahun yakni periode Januari dan Juli. y p Artikel-artikel yang termuat dalam Jurnal Rekayasa Teknik dan Teknologi (REKATEK) ini adalah artikel-artikel yang sudah melalui proses penilaian atau review (Double Blind Peer Review) oleh mitra bestari dan/atau dewan penyunting. Penulis harus memperhatikan kualitas isi artikel sesuai petunjuk penulisan artikel dan komentar dari Mitra Bestari. Jumlah artikel yang terbit pada nomor ini sebanyak 8 (Delapan) judul artikel yang menyajikan karya penulis dengan beragam afiliasi. Penghargaan sebesar- besarnya disampaikan kepada penulis yang telah mengisi naskah di Jurnal Rekayasa Teknik dan Teknologi (REKATEK), demikian juga kepada para mitra bestari yang telah memberikan komitmennya dalam bidang keilmuan spesifik yang tidak ternilai sehingga dapat dihasilkan tulisan yang lebih berkualitas, terima kasih yang sebesar- besarnya kepada para anggota dewan penyunting dan seluruh pihak yang terlibat dalam penerbitan jurnal ini. Menyadari bahwa penyusunan jurnal membutuhkan semangat dan stamina yang tinggi, maka untuk keberhasilan kami tetap membutuhkan sumbang saran dan pemikiran demi kemajuan dan kesempurnaan Jurnal ini, dan kami berharap semoga jurnal ini dapat menjadi bahan rujukan dan memberikan kontribusi pengetahuan yang bermanfaat. Wassalam Wassalam Ketua Penyunting (Editor In Chief) Indek Ketua Penyunting (Editor In Chief) Indek Ketua Penyunting (Editor In Chief) Indek Ketua Penyunting (Editor In Chief) Ketua Penyunting (Editor In Chief) Indek Kumita. ST., MT Fakultas Teknik - Universitas Al Muslim- Bireuen Aceh - Indonesia Fakultas Teknik - Universitas Al Muslim- Bireuen Aceh - Indonesia Fakultas Teknik - Universitas Al Muslim- Bireuen Aceh - Indonesia MITRA BESTARI JURNAL REKAYASA TEKNIK DAN TEKNOLOGI (REKATEK) Dr. Musbar., ST., MT DAFTAR ISI DAFTAR ISI Judul dan Penulis: Januari 2023, 7 (1) Hal Pengendalian Biaya dan Waktu Pada Proyek Rekonstruksi Jalan Pante Gurah – Tanohanoe Kecamatan Muara Batu Dengan Metode Earned Value Ismail, Darkasyi DOI : https://doi.org/10.51179/rkt.v7i1.1827 1-4 Evaluasi Pelaksanaan K3 Pada Proyek Konstruksi di Kabupaten Bireuen Munardy, Deni Iqbal DOI : https://doi.org/10.51179/rkt.v7i1.1828 5-11 Pengaruh Penambahan Serat Kawat Bendrat Pada Beton Terhadap Kuat Tekan Dan Kuat Belah Khairul Miswar, R. Dedi Iman Kurnia, Robi Yusmananda DOI : https://doi.org/10.51179/rkt.v7i1.1829 12-17 Analisa Tingkat Kerusakan Jalan Dengan Perkerasan Lentur Menggunakan Metode Pavement Condition Index [Studi Kasus: Jalan Line Pipa Kecamatan Nibong Kabupaten Aceh Utara] Iskandar, Aris Munandar, Suhaimi DOI : https://doi.org/10.51179/rkt.v7i1.1830 18-25 Peningkatan Kualitas Lingkungan Melalui Lubang Resapan Biopori Sebagai Upaya Penanggulangan Banjir Dengan Menggunakan Sampah Rumah Tangga Fitri Muliani, Romaynoor Ismy, Zaeri Tahrizi DOI : https://doi.org/10.51179/rkt.v7i1.1831 26-31 Pengaruh Hambatan Samping Terhadap Kinerja Jalan [Studi Kasus: Jalan Kolonel Husein Yusuf dan Jalan Medan-Banda Aceh Kecamatan Kota Juang Kabupaten Bireuen] Kumita, Idayani, Muhamad Syahroni DOI : https://doi.org/10.51179/rkt.v7i1.1832 32-36 Evaluasi Simpang Empat Tak Bersinyal Berdasarkan Metode PKJI 2014 [Studi Kasus: Simpang Empat Geudong – Geudong Kec. Kota Juang Kab. Bireuen] Musfirah, Idayani, Mahdi DOI : https://doi.org/10.51179/rkt.v7i1.1833 37-41 Bireuen Sebagai Kota Juang Zuraihan, Idayani, Kumita DOI : https://doi.org/10.51179/rkt.v7i1.1834 42-45 Judul dan Penulis: Januari 2023, 7 (1) Hal Pengendalian Biaya dan Waktu Pada Proyek Rekonstruksi Jalan Pante Gurah – Tanohanoe Kecamatan Muara Batu Dengan Metode Earned Value Ismail, Darkasyi DOI : https://doi.org/10.51179/rkt.v7i1.1827 1-4 Evaluasi Pelaksanaan K3 Pada Proyek Konstruksi di Kabupaten Bireuen Munardy, Deni Iqbal DOI : https://doi.org/10.51179/rkt.v7i1.1828 5-11 Pengaruh Penambahan Serat Kawat Bendrat Pada Beton Terhadap Kuat Tekan Dan Kuat Belah Khairul Miswar, R. Dedi Iman Kurnia, Robi Yusmananda DOI : https://doi.org/10.51179/rkt.v7i1.1829 12-17 Analisa Tingkat Kerusakan Jalan Dengan Perkerasan Lentur Menggunakan Metode Pavement Condition Index [Studi Kasus: Jalan Line Pipa Kecamatan Nibong Kabupaten Aceh Utara] Iskandar, Aris Munandar, Suhaimi DOI : https://doi.org/10.51179/rkt.v7i1.1830 18-25 Peningkatan Kualitas Lingkungan Melalui Lubang Resapan Biopori Sebagai Upaya Penanggulangan Banjir Dengan Menggunakan Sampah Rumah Tangga Fitri Muliani, Romaynoor Ismy, Zaeri Tahrizi DOI : https://doi.org/10.51179/rkt.v7i1.1831 26-31 Pengaruh Hambatan Samping Terhadap Kinerja Jalan [Studi Kasus: Jalan Kolonel Husein Yusuf dan Jalan Medan-Banda Aceh Kecamatan Kota Juang Kabupaten Bireuen] Kumita, Idayani, Muhamad Syahroni DOI : https://doi.org/10.51179/rkt.v7i1.1832 32-36 Evaluasi Simpang Empat Tak Bersinyal Berdasarkan Metode PKJI 2014 [Studi Kasus: Simpang Empat Geudong – Geudong Kec. Kota Juang Kab. MITRA BESTARI JURNAL REKAYASA TEKNIK DAN TEKNOLOGI (REKATEK) Bireuen] Musfirah, Idayani, Mahdi DOI : https://doi.org/10.51179/rkt.v7i1.1833 37-41 Bireuen Sebagai Kota Juang Zuraihan, Idayani, Kumita DOI : https://doi.org/10.51179/rkt.v7i1.1834 42-45 5-11
https://openalex.org/W4382178483	https://zenodo.org/record/8083987/files/3809%20fulltext.pdf	English	null	Information Visualization Research Publications during 1990-2018: A Scientometric Analysis	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	4,682	University of Nebraska - Lincoln University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Libraries at University of Nebraska-Lincoln Libraries at University of Nebraska-Lincoln Singh, Kunwar; Ranjan, Abhishek; and Rai, Somesh, "Information Visualization Research Publications during 1990-2018: A Scientometric Analysis" (2019). Library Philosophy and Practice (e-journal). 3809. https://digitalcommons.unl.edu/libphilprac/3809 1 Assistant Professor, Dept. of Library & Information Science, Banaras Hindu University, Varanasi - 221005 E-mail: kunwar.singh@bhu.ac.in 2 Student, Dept. of Library & Information Science, Banaras Hindu University, Varanasi - 221005 E-mail: abhishekranjan561@gmail.com 3 Junior Research Fellow, Dept. of Library & Information Science, Banaras Hindu University, Varanasi-221005 E-mail: somesh.rai15@bhu.ac.in Information visualization research publications during 1990-2018: A scientometric analysis Kunwar Singh1, Abhishek Ranjan2 & Somesh Rai3 Abstract: To understand the history and research status of information visualization, information visualization research citation data has been collected from the Scopus expanded during the period from 1990 to 2018. Results indicated that the research of information visualization has increased during the studied 29-year period. The country with the highest research output was the United States with 1996 publications, while the institution with the highest research output was the CNRS Centre National de la Recherche Scientifique. The majority of research articles have been contributed from developed countries. It also revealed that developed countries have more research advantages in comparison to developing countries. The top three outputs journals were Nucleic Acids Research, BMC Bioinformatics and Bioinformatics. Keywords: Information visualization, Data visualization, Scientometric analysis, scientific research. Information Visualization Research Publications during Information Visualization Research Publications during 1990-2018: A Scientometric Analysis 1990-2018: A Scientometric Analysis Abhishek Ranjan Dept. of Library & Information Science, Banaras Hindu University, Varanasi - 221005, abhishekranjan561@gmail.com Somesh Rai Dept. of Library & Information Science, Banaras Hindu University, Varanasi - 221005, somesh.rai15@bhu.ac.in Follow this and additional works at: https://digitalcommons.unl.edu/libphilprac Part of the Library and Information Science Commons Singh, Kunwar; Ranjan, Abhishek; and Rai, Somesh, "Information Visualization Research Publications during 1990-2018: A Scientometric Analysis" (2019). Library Philosophy and Practice (e-journal). 3809. https://digitalcommons.unl.edu/libphilprac/3809 Introduction Information visualization (or infovis) seeks to augment human cognition by leveraging human visual capabilities to make sense of abstract information (Card et al, 1999). It is a representation of data in a visual context, which helps to understand the significance of data (Chen, 2017, p.7). Information Visualization has been a method for humans to communicate knowledge about events beyond the boundary of space and time. The cave drawings are best example of information visualization used by humans in the absence of proper communicating languages. These drawings are very detailed and it helps in understanding the events happening at that time. Same is applicable in current use of visualization techniques; by using intuitive, meaningful and detailed representation, we can make the audience understand highly abstracted information or knowledge with mild efforts. It is already recognized fact that visual information is more easily understandable than information presented in linear textual form. Further, with the advent of human technological advancement new ways expression of abstract knowledge, are being invented. Virtual reality, Augmented reality, Holographic technologies are some current examples, which have high potential for abstract information visualization. It is necessary to find 1 new ways to present the exponentially increasing information in the knowledge-world. As we live in a three dimensional world and it is very well perceivable by our human senses, it is an excellent idea to explore the possibilities of representing information in 3-D space, it has the capability to present even more highly abstracted knowledge with more detail. Besides these, it has also been found to be highly useful for various analytics purposes, assisting in more confident decision making (Padilla, Creem-Regehr, Hegarty, & Stefanucci, 2018). All these advantages have guided the researchers over the years to explore the domain of Information Visualization. A simple search in the Web of Science database for the term Information Visualization results in more than 21000 documents published since 1989. This work attempts to quantify and understand the past research growth and collaboration patterns by means of scientometric analysis. Scientometrics is a quantitative method which utilises various indicators to assess the growth and pattern of publication of scholarly literature, author collaborations and citations. Objective of the study The main objectives of the present study are as follows: The main objectives of the present study are as follows: ● To identify the form wise distribution of publications; ● To find out the year wise distribution of the publications; ● To find out annual and compound growth rate of publications; ● To find out the authorship pattern of the publications; ● To scrutinize the authors productivity; ● To determine the degree of collaboration among single and multiple authors ● To explore the country wise and institutions (affiliation) wise publications; ● To find out the subject wise distribution of publications; ● To find out the most favored source titles; ● To find out the funding institutions and the highly cited publications Methodology Scopus is one of the largest abstract and citation databases of peer-reviewed scholarly publications. It covers the world's research output in the fields of science, technology, medicine, social sciences, and arts and humanities (https://www.elsevier.com/en-in/solutions/scopus). We first retrieved all research publications on information visualization via the Scopus database. We searched using keywords “information” AND “visualization” on 12 Sept 2019. Our search yielded 6192 records made up of several document types, including articles, review, conference paper, note, editorial, short survey, letter, erratum and data paper. The data were exported into a text-based format via the Scopus website, then imported into MS-Excel and analyzed. The output 2 and citations for each year, country, institution and journal were summarized; further, we analyzed high-impact articles, high-impact authors and research trends. and citations for each year, country, institution and journal were summarized; further, we analyzed high-impact articles, high-impact authors and research trends. Document type There were 09 document types acknowledged in the 6192 records. The majority of the documents were articles, which accounted for 90.42% of the total records, demonstrating that these are the main approach for scientific communication on information visualization. Review and conference papers, taking up of 6.27% and 2.36%, respectively, were two other significant ways to publish academic achievements in this field of research. Table 1 Form wise distribution of documents Type Documents % Article 5599 90.42 Review 388 6.27 Conference Paper 146 2.36 Note 18 0.29 Editorial 11 0.18 Short Survey 11 0.18 Letter 9 0.15 Erratum 7 0.11 Data Paper 3 0.05 Total 6192 100.00 Table 1 Form wise distribution of documents Type Documents % Article 5599 90.42 Review 388 6.27 Conference Paper 146 2.36 Note 18 0.29 Editorial 11 0.18 Short Survey 11 0.18 Letter 9 0.15 Erratum 7 0.11 Data Paper 3 0.05 Total 6192 100.00 Yearly research outputs Yearly research outputs Yearly research outputs Yearly research outputs Table 2 shows research output each year from 1990 to 2018, according to the data we collected on 12 September 2019. It indicates a fluctuating increase from approximately 3-899 records during the last 29 years after 1990. In this period, the overall trend has been an increase of yearly output. Results revealed that the research on information visualization was consistently the focus of scholars during the past 29 years. Table 2 Yearly research outputs Year TP % TC ACPP Year TP % TC ACPP 1990 3 0.05 173 57.67 2005 168 2.71 19329 115.05 1991 10 0.16 678 67.80 2006 165 2.66 8939 54.18 1992 12 0.19 169 14.08 2007 217 3.50 9781 45.07 1993 7 0.11 120 17.14 2008 237 3.83 11564 48.79 1994 12 0.19 457 38.08 2009 262 4.23 21745 83.00 1995 23 0.37 5128 222.96 2010 253 4.09 16201 64.04 1996 23 0.37 582 25.30 2011 313 5.05 14853 47.45 1997 25 0.40 824 32.96 2012 344 5.56 15771 45.85 1998 18 0.29 1776 98.67 2013 400 6.46 16224 40.56 1999 29 0.47 3135 108.10 2014 513 8.28 15182 29.59 2000 50 0.81 8363 167.26 2015 609 9.84 13486 22.14 3 2001 35 0.57 2313 66.09 2016 597 9.64 9292 15.56 2002 63 1.02 4262 67.65 2017 698 11.27 6449 9.24 2003 85 1.37 11773 138.51 2018 899 14.52 2603 2.90 2004 122 1.97 12331 101.07 Total 6192 100.00 233503 37.71 TP=Total paper, %= percentage out of Total, TC=Total Citation, ACPP=Average citation per paper Annual and cumulative growth rate of the publications Table 3 provides the AGR of the number of documents for period 1990-2018. Nd= Number of documents in the year, Nc= Cumulative number of documents till the year. 𝐴𝐺𝑅= 𝑒𝑛𝑑 𝑣𝑎𝑙𝑢𝑒−𝑓𝑖𝑟𝑠𝑡 𝑣𝑎𝑙𝑢𝑒 𝑓𝑖𝑟𝑠𝑡 𝑣𝑎𝑙𝑢𝑒 × 100 Table 3 shows that the annual growth rate of the total publication calculated year wise. Fluctuation is seen in throughout the study period. Here, the AGR has been determined as per the formula given above. In our study, the AGR for publications has decreasing trends from 233.33 in 1991 to -41.67 in 1993. However, the AGR has increased to 71.43 in 1994 to 91.67 in 1995. Since then, there is fluctuation in year after year as presented in Table 3. Table 3 also provides the CAGR of the number of documents for the period 1990-2018. The equation used to calculate CAGR is given below: CAGR= (ending value/beginning value) (1/# of years)-1 The compound annual growth rates of the publications are gradually decreased from 0.63 in 1991 to 0.09 in 1993 as seen in table 3. However, the CAGR has increased to 0.11 in 1994 to 0.15 in 1995. This indicates that though the yearly output is fluctuating year after year as seen in table 3. Table 3 Annual and cumulative growth rate Year Nd AGR Nc CAGR Year Nd AGR Nc CAGR 1990 3 0 3 0 2005 168 37.70 685 0.10 1991 10 233.33 13 0.63 2006 165 -1.79 850 0.07 1992 12 20.00 25 0.24 2007 217 31.52 1067 0.08 1993 7 -41.67 32 0.09 2008 237 9.22 1304 0.07 1994 12 71.43 44 0.11 2009 262 10.55 1566 0.06 1995 23 91.67 67 0.15 2010 253 -3.44 1819 0.05 1996 23 0.00 90 0.10 2011 313 23.72 2132 0.05 1997 25 8.70 115 0.09 2012 344 9.90 2476 0.05 1998 18 -28.00 133 0.05 2013 400 16.28 2876 0.05 1999 29 61.11 162 0.07 2014 513 28.25 3389 0.06 2000 50 72.41 212 0.09 2015 609 18.71 3998 0.06 2001 35 -30.00 247 0.05 2016 597 -1.97 4595 0.05 2002 63 80.00 310 0.08 2017 698 16.92 5293 0.05 2003 85 34.92 395 0.08 2018 899 28.80 6192 0.05 2004 122 43.53 517 0.09 Total 6192 841.8 - 2.77 Nd= Number of documents in the year, Nc= Cumulative number of documents till the year. 4 Authorship pattern p p Table 4 represents that the maximum number of research articles were published by multiple authors 5832 (94.19%) and 360(5.81%) were published by single authors. We observed from the study that the majority of papers were published by multi-authors. Table 4 represents that the maximum number of research articles were published by multiple authors 5832 (94.19%) and 360(5.81%) were published by single authors. We observed from the study that the majority of papers were published by multi-authors. Table 4 Authorship pattern Year Authors Year Authors Single Multiple Single Multiple 1990 0 3 2005 10 158 1991 1 9 2006 12 153 1992 3 9 2007 18 199 1993 0 7 2008 17 220 1994 1 11 2009 16 246 1995 5 18 2010 15 238 1996 1 22 2011 20 293 1997 1 24 2012 20 324 1998 2 16 2013 18 382 1999 2 27 2014 24 489 2000 4 46 2015 36 573 2001 2 33 2016 36 561 2002 6 57 2017 28 670 2003 6 79 2018 46 853 2004 10 112 Total 360 5832 Most prolific authors Most prolific authors Table 5 shows top ten prolific authors with their contributions. It is observed that Herrmjakob, H has contributed 14 papers scored first rank followed by Karp, P. D. 12 papers scored second rank and Chaussabel, D. 10 papers. On the other hand, Birney, E., Evelo, C. T., Kohlbacher, O., Markl, M., Pico, A. R., have 9 papers each whereas, Boughorbel, S. and Cherry, J. M. contributed 8 papers each. Table 5 Ten top most prolific authors Sl. No. Author Documents 1 Hermjakob, H. 14 2 Karp, P. D. 12 3 Chaussabel, D. 10 4 Birney, E. 9 5 Evelo, C.T. 9 6 Kohlbacher, O. 9 7 Markl, M. 9 8 Pico, A. R. 9 9 Boughorbel, S. 8 10 Cherry, J. M. 8 5 Degree of collaboration The DC is defined as the ratio of the number of collaborative research papers to the total number of research papers in the discipline during a certain period of time. The formula suggested by Subramaniam (1983) is used. It is expressed as: DC = 𝑁𝑚 𝑁𝑚+ 𝑁𝑠 Where, DC is Degree of Collaboration in a discipline, Where, DC is Degree of Collaboration in a discipline, Nm - Is the number of multi-authored research papers in the discipline published during the year, Ns - Is the number of single-authored papers in the discipline published during the same year. Using this formula, the DC is determined for each year. Table 6 shows year wise variation of DC. It can be observed that degree of collaboration has been above 90 percent. Table 6 Degree of collaboration Year Ns Nm Ns+Nm DC Year Ns Nm Ns+Nm DC 1990 0 3 3 1.00 2005 10 158 168 0.94 1991 1 9 10 0.90 2006 12 153 165 0.93 1992 3 9 12 0.75 2007 18 199 217 0.92 1993 0 7 7 1.00 2008 17 220 237 0.93 1994 1 11 12 0.92 2009 16 246 262 0.94 1995 5 18 23 0.78 2010 15 238 253 0.94 1996 1 22 23 0.96 2011 20 293 313 0.94 1997 1 24 25 0.96 2012 20 324 344 0.94 1998 2 16 18 0.89 2013 18 382 400 0.96 1999 2 27 29 0.93 2014 24 489 513 0.95 2000 4 46 50 0.92 2015 36 573 609 0.94 2001 2 33 35 0.94 2016 36 561 597 0.94 2002 6 57 63 0.90 2017 28 670 698 0.96 2003 6 79 85 0.93 2018 46 853 899 0.95 2004 10 112 122 0.92 Total 360 5832 6192 0.94 Nm= Number of multiple authors, Ns= Number of single authors, DC= Degree of Collaboration. Countries-wise distribution of research output Table 7 shows the analysis of the country-wise distribution of research output can help us to identify the capacity of a country and discover the capacity differences among different countries. 115 countries have been contributed in this domain, but it shows that more than 50 percent documents came from developed countries. As Table 7 shows, that among the top ten countries were United States, Germany, United Kingdom, China, Japan, Canada, Netherland, France, Spain and Australia; their published papers occupied 93.15% of the total output. These countries, except China, all are developed countries. 6 Table 7 Top ten most productive countries Sl. No. Country Documents 1 United States 1996 2 Germany 742 3 United Kingdom 694 4 China 616 5 Japan 460 6 Canada 283 7 Netherlands 274 8 France 262 9 Spain 234 10 Australia 207 Table 7 Top ten most productive countries Institution-wise distribution of research output Institution-wise distribution of research output Institution-wise distribution of research output can help us understand the research capacity and activities of institutions around the world. It also can help us to identify leading institutions in information visualization research. From Table 8, it can be seen that the highest institutional research output was from the CNRS Centre National de la Recherche Scientifique, from which we found 90 records. Results showed that the USA and UK allocated a large number of resources to information visualization research, especially to government and government- supported scientific organizations. The reason may be that the field of information visualization is one of the fundamental scientific research and primarily non-profit, therefore requiring funds and support from the government should be given. Table 8 Top twenty most prolific Institutions Sl. No. Institution (Affiliation) Documents Country 1 CNRS Centre National de la Recherche Scientifique 90 France 2 European Bioinformatics Institute 89 UK 3 Chinese Academy of Sciences 87 China 4 Harvard Medical School 71 USA 5 University of California, San Diego 70 USA 6 National Institutes of Health, Bethesda 68 USA 7 Inserm 55 France 8 Ministry of Education China 54 China 9 University of Toronto 54 Canada 10 Imperial College London 53 UK 11 European Molecular Biology Laboratory 49 Germany 12 University of Washington, Seattle 49 USA 13 University of Tokyo 46 Japan 14 University of Oxford 46 UK 15 University of Cambridge 46 UK 16 Stanford University 44 USA 17 University of California, Davis 44 USA 18 Massachusetts Institute of Technology 44 USA 19 University of California, San Francisco 44 USA 20 University of Michigan, Ann Arbor 44 USA Table 8 Top twenty most prolific Institutions 7 Subject-wise distribution of research output Tables 9 examine the subject wise distribution of publications which were produced during the stipulated period. This study identifies the authors’ interest and involvement of subjects in terms of producing the publication in their respective specialization. It shows that most of the subjects are overlapped with each other. The findings of the study reveal that the highest number 2604 (42.05%) of scientific scholarly publications were published in the subject of Biochemistry, Genetics and Molecular Biology study due to the rapid growth of development in the area that the majority of authors are very much interested to do their research work and followed by 31.31% of papers were from computer science and 24.85% of papers were from medicine. Table 9 Subject wise distribution of documents Sl. No. Subject area Documents % 1 Biochemistry, Genetics and Molecular Biology 2604 42.05 2 Computer Science 1939 31.31 3 Medicine 1539 24.85 4 Mathematics 1055 17.04 5 Engineering 836 13.50 6 Agricultural and Biological Sciences 662 10.69 7 Social Sciences 478 7.72 8 Earth and Planetary Sciences 379 6.12 9 Physics and Astronomy 355 5.73 10 Environmental Science 308 4.97 11 Chemistry 253 4.09 12 Neuroscience 237 3.83 13 Materials Science 228 3.68 14 Immunology and Microbiology 190 3.07 15 Multidisciplinary 167 2.70 16 Chemical Engineering 128 2.07 17 Health Professions 124 2.00 18 Pharmacology, Toxicology and Pharmaceutics 119 1.92 19 Business, Management and Accounting 83 1.34 20 Energy 77 1.24 Table 9 Subject wise distribution of documents 8 Journal-wise distribution of research output There were more than 170 journals that published 6192 articles in the area of information visualization. Table 10 shows the top 20 records journals, which had 2214 articles, thus comprising 35.76% of all 6192 articles. As Table 5 shows, the journal with the most outputs was Nucleic Acids Research, with 446 records, accounting for 7.2% of the total number of records. The following four journals were Nucleic Acids Research, BMC Bioinformatics, Bioinformatics and Plos One, which published 446, 383, 378 and 272 articles, occupying 7.2%, 6.2%, 6.1% and 4.4% of the total records, respectively. From the scope of top 20 records journals, information visualization research mostly concentrated on medical science, computer science and engineering. The researchers from these disciplines paid much attention to the field of information visualization. Table 10 Top twenty most prolific Journals Table 10 Top twenty most prolific Journals Sl. No. Subject-wise distribution of research output Source documents 1 Nucleic Acids Research 446 2 BMC Bioinformatics 383 3 Bioinformatics 378 4 Plos One 272 5 IEEE Access 80 6 BMC Genomics 70 7 Scientific Reports 64 8 Journal Of Biomedical Informatics 58 9 ISPRS International Journal Of Geo Information 55 10 IEICE Transactions On Information And Systems 53 11 Sensors Switzerland 49 12 F1000research 39 13 Journal of the American Medical Informatics Association 39 14 Database 36 Table 10 Top twenty most prolific Journals Sl. No. Source documents 1 Nucleic Acids Research 446 2 BMC Bioinformatics 383 3 Bioinformatics 378 4 Plos One 272 5 IEEE Access 80 6 BMC Genomics 70 7 Scientific Reports 64 8 Journal Of Biomedical Informatics 58 9 ISPRS International Journal Of Geo Information 55 10 IEICE Transactions On Information And Systems 53 11 Sensors Switzerland 49 12 F1000research 39 13 Journal of the American Medical Informatics Association 39 14 Database 36 9 15 Proceedings of The National Academy of Sciences of The United States of America 36 16 Journal of Medical Internet Research 35 17 International Journal of Health Geographics 32 18 Remote Sensing 31 19 Frontiers In Neuroinformatics 30 20 Plos Computational Biology 28 Funding Institutes of research output Table 11 shows the rank of top 20 research funding agencies / institutions. The National Institutes of Health, National Natural Science Foundation of China and National Science Foundation are the top contributing research project funding agencies which result 692(26.26 %) research publication during the year 1990-2018. Total 2635(42.55%) research papers were published through the funded research projects. Table 11 Top twenty funding Institutes Sl. No. Funding Institute Documents 1 National Institutes of Health 302 2 National Natural Science Foundation of China 222 3 National Science Foundation 168 4 Deutsche Forschungsgemeinschaft 69 5 Engineering and Physical Sciences Research Council 64 6 European Commission 62 7 Wellcome Trust 54 8 Biotechnology and Biological Sciences Research Council 52 9 Japan Society for the Promotion of Science 51 10 U.S. Department of Energy 48 11 European Research Council 44 12 Natural Sciences and Engineering Research Council of Canada 39 13 Bundesministerium für Bildung und Forschung 38 14 National Research Foundation of Korea 38 15 National Human Genome Research Institute 37 16 National Cancer Institute 35 17 Canadian Institutes of Health Research 32 18 Foundation for the National Institutes of Health 31 19 U.S. National Library of Medicine 30 20 Ministry of Education, Culture, Sports, Science and Technology 29 Top ten highly cited articles The highly cited articles got 5998 citations, about 2.57% of all citations. However, it was noticed that none of the prolific authors is a highly cited author. Further analysis of the highly cited papers indicates that these papers came from developed countries, mainly from the USA, Spain, Germany, England and so on. Most of the highly cited papers are in the area of genetics, medicine and computer science, which are cited more frequently as compared to other areas. 10 Table 12 Top ten highly cited articles Authors Cited by Title Year Country Conesa A, Götz S, García- Gómez JM, Terol J, Talón M, Robles M. 5998 Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics research 2005 Spain Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R 4811 DnaSP, DNA polymorphism analyses by the coalescent and other methods 2003 Spain Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, A., Buchner, A., Lai, T., Steppi, S., Jacob, G., Förster,, W., Brettske, I., Gerber, S., Ginhart, A.W., Gross, O., Grumann, S., Hermann, S., Jost, R.,König, A., Liss, T., Lüßman, R., May, M., Nonhoff, B., Reichel, B., Strehlow, R., Stamatakis, A., Stuckmann, N., Vilbig, A., Lenke, M., Ludwig, T., Bode, A., Schleifer, K.-H. 4545 ARB: A software environment for sequence data 2004 Germany Anselin, L. 4102 Local Indicators of Spatial Association— LISA 1995 USA Westrip, S.P. 3754 PublCIF: Software for editing, validating and formatting crystallographic information files 2010 England Krzywinski, M., Schein, J., Birol, I., Connors, J., Gascoyne, R., Horsman, D., Jones, S.J., Marra, M.A. 3497 Circos: An information aesthetic for comparative genomics 2009 Canada Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.-C., Sasaki, Y.F. 3382 Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing 2000 USA Cole, J.R., Wang, Q., Cardenas, E., Fish, J., Chai, B., Farris, R.J., Kulam-Syed-Mohideen, A.S., McGarrell, D.M., Marsh, T., Garrity, G.M., Tiedje, J.M. 3122 The Ribosomal Database Project: Improved alignments and new tools for rRNA analysis 2009 USA Thorvaldsdóttir H, Robinson J T, Mesirov J P. 2624 Integrative Genomics Viewer (IGV): High- performance genomics data visualization and exploration 2013 USA Punta, M., Coggill, P.C., Eberhardt, R.Y., Mistry, J., Tate, J., Boursnell, C., Pang, N., Forslund, K., Ceric, G., Clements, J., Heger, A., Holm, L., Sonnhammer, E.L.L., Eddy, S.R., Bateman, A., Finn, R.D. Top ten highly cited articles 2458 The Pfam protein families database 2012 UK 11 Findings Findings The major findings of this study are as follows: The major findings of this study are as follows: ● The highest number 14.52% of papers were published in 2018 and the lowest number 0.25% of research articles published in the year 1990. ● The result shows that majority of the documents were articles (90.42%) of the total records, it indicates that articles are the main approach for scientific communication on information visualization. ● It indicates a fluctuating increase from approximately 3-899 records during the last 29 years after 1990. ● It was observed from the study that the majority of papers were published by multi-authors 5832(94.19%). ● It was observed that Herrmjakob, H has contributed 14 papers scored first rank in most prolific authors’ group. ● It was observed that degree of collaboration has been above 90 percent. ● It revealed that the top ten most productive countries, except China, all are developed countries. ● The findings of the study reveal that the highest number 2604 (42.05%) of scientific scholarly publications were published in the subject of Biochemistry, Genetics and Molecular Biology study due to the rapid growth of development in the area. ● The finding of the study shows that total 2635(42.55%) research papers were published through the project funded research. ● It was also revealed that most of the highly cited papers are in the area of genetics, medicine and computer science, which are cited more frequently as compared to other areas. ● The maximum number of citations were 21745 (9.31%) in the year 2009 whereas, the minimum number of citations were 120 (0.05%) in the year 1993. ● The highest number 2604 (42.05%) of scientific scholarly publications were published in the subject of Biochemistry, Genetics and Molecular Biology, followed by 31.31% of papers were from Computer Science, 8.46% of papers were from Medicine. Conclusion Scoups: https://www.elsevier.com/en-in/solutions/scopus accessed on 12.10.2019 Conclusion As we all know that information visualization plays an important role in demonstrating raw data into a visual and meaningful way so that one can better understand it. Overall, the research trend of information visualization has increased from 2004 to 2018, with its research outputs accomplished approximately from 122 to 899 records. These trends implied that information visualization research as an important research area for addressing information and visualization issues obtained a reasonably stable intellectual attention. Among the top 20 highly productive countries, United States with (1996 publications) has scored top rank. The majority of the research papers were published from Biochemistry, Genetics and Molecular Biology subjects. Maximum research papers were published in the discipline-specific journals on information visualization, such as Nucleic Acids Research, BMC Bioinformatics and Bioinformatics. 12 Reference Reference 1. Card, S. K., Mackinlay, J. D., and Shneiderman, B. (1999). Readings in Information Visualization: Using Vision to Think. San Francisco, California: Morgan-Kaufmann. 2. Padilla, L. M., Creem-Regehr, S. H., Hegarty, M., & Stefanucci, J. K. (2018). Decision making with visualizations: a cognitive framework across disciplines. Cognitive research: principles and implications, 3(1), 29. https://dx.doi.org/10.1186%2Fs41235- 018-0120-9 3. Subramanyan, K. (1983). Bibliometric studies of research in collaboration: A review. Journal of information Science, 6(1), 33-38. 4. Sears, A., & Jacko, J. A. (2000). Understanding the Relation Between Network Quality of Service and the Usability of Distributed Multimedia Documents. 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https://openalex.org/W4251105985	https://www.researchsquare.com/article/rs-22357/latest.pdf	English	null	Reactional Ultrasonic Systems And Microwave Irradiation For Pretreatment Of Agro-Industrial Waste To Increase Enzymatic Activity	Research Square (Research Square)	2,020	cc-by	12,610	Simone Kubeneck Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Karina Paula Preczeski Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Caroline Dalastra Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Fabiane Fernanda Czapela Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Caroline Dalastra Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Thamarys Scapini Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Thamarys Scapini Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Thamarys Scapini Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Charline Bonatto Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Charline Bonatto Charline Bonatto Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Fábio Spitza Stefanski Fábio Spitza Stefanski Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Aline Frumi Camargo Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Jessica Zanivan Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Altemir José Mossi Altemir José Mossi Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul - UFFS, Rodovia ERS 135 - km 72, nº 200, Erechim, RS, Brazil. Zip code 99700-970 Gislaine Fongaro Page 1/36 Page 1/36 Department of Microbiology, Immunology and Parasitology, Universidade Federal de Florianópolis - UFSC, Engenheiro Agronômico Andrei Cristian Ferreira St., Trindade, Florianópolis, SC, Brazil. Zip code 88040-900 Helen Treichel (  helentreichel@gmail.com ) ( @g ) Universidade Federal da Fronteira Sul https://orcid.org/0000-0002-3810-3000 Abstract Pretreatment of keratinous residues using an ultrasonic reaction system provides greater enzymatic production in less time. This is a promising technology for measuring enzyme activity and microwave processes. In the present work, an ultrasonic probe reaction system was used to evaluate the potential of swine hair pretreatment. The pretreated material was submerged with non-pretreated residues for 9 days to obtain the enzyme. Enzyme activity was measured in the extracts obtained using the ultrasonic probe, ultrasonic bath, and microwave. We also used the enzymatic concentration technique with NaCl and acetone. Homemade enzymatic extracts were evaluated for their ability to degrade swine hair and chicken feathers by comparing them with the activities commercial enzymes. Macrobeads gave greater energy dissipation in less time, providing greater enzyme activity (50.8 U/mL over 3 days). In terms of waste degradation, non-pretreated swine hair was more promising. The ultrasonic probe reaction system had the potential to evaluate increased enzyme activity (38.4% relative activity) and the enzyme concentration increased activity by 53.5%. The homemade enzymatic extract showed promise for degradation of keratinous residues. Research Keywords: Reaction system, Keratin residues, Biotechnological processes. Posted Date: September 4th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-22357/v4 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: Reaction system, Keratin residues, Biotechnological processes. Posted Date: September 4th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-22357/v4 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 September 26th, 2020. See the published version at https://doi.org/10.1186/s40643-020-00338-2. Page 2/36 2.1 Keratin substrates Swine hair and chicken feathers were obtained from a food agroindustry in Rio Grande do Sul, Brazil. Residues were stored at –4 °C until use. Prior to use, the residues were washed with water and detergent, and immersed in 70% alcohol for 1 h, followed by drying at 70 ± 2 °C for 16 h (adapted from Călin et al. 2017; Preczeski et al. 2020). Chicken feathers were used in the degradation tests only. 1. Introduction Keratinous waste is generated in large quantities; managing this waste is made difficult by its resilient structure (Onifade et al, 1998; Łaba et al. 2015). These residues consist of fibrous proteins such as keratins that are resistant to physical, chemical and enzymatic actions (Korniłłowicz-Kowalska and Bohacz 2011). When found in their solid state, the structure of the keratin increases its stiffness because of cysteine bonds (Barone et al. 2006), as well as by α and β-keratin chains and disulfide bonds (Onifade et al. 1998). Alpha-keratin can be found in materials such as hair, fur, nails, hooves, and animal horns, while β-keratin is present in scales, bird feathers, and beaks (Korniłłowicz-Kowalska and Bohacz 2011; Mazotto et al. 2011; Ire and Onyenama 2017). One way to promote the degradation of these residues is via biotechnological processes, particularly degradation through the action of specific enzymes such as keratinases (Gupta and Ramnani 2006; Brandelli 2008; Brandelli, Daroit and Riffel 2010; Gegeckas et al. 2018). Keratinases (EC 3.4.21/24/99.11) are a class of proteases that hydrolyze keratin, an advantage over other proteases (Brandelli, Daroit and Riffel 2010; Gegeckas et al. 2018). These enzymes have applications such as biotechnology (Okoroma et al. 2012; Mazotto et al. 2013; Paul et al. 2014; Brandelli, Sala and Kalil 2015) and waste degradation (Fang et al. 2013a; Yusuf et al. 2016; Su et al. 2017; Abdel-Fattah et al. 2018; Thankaswamy et al. 2018). They can be obtained commercially or from submerged fermentation (FS) or solid-state fermentation (FES) through microorganisms (Mazotto et al. 2013) such as fungi (Kushwaha 1983; Santos et al. 1996), that are considered good producers of keratinases; they can also be isolated from keratinous waste disposal sites (Kaul and Sumbali 1997; Riffel and Brandelli 2006). When obtained in this way, they are designated “homemade” and can be used in crude or concentrated form, a technique that combines the interaction of the enzyme with salts and solvents so as to separate out interferents (Preczeski et al. 2018). Page 3/36 Page 3/36 Because of the biotechnological importance of keratinases, several studies have been performed to improve the activity and to evaluate the conformational change of enzymes in general, when exposed to different reaction systems, including ultrasonic probe and bath and microwave (Ma et al. 2011; Jin et al. 2015; Mazinani, DeLong and Yan 2015). Simple and efficient heating technologies increase enzyme activity and conformational change (Ma et al. 1. Introduction 2011); residue pretreatment techniques using an ultrasonic probe (Azmi, Idris and Yusof 2018), makes the structures more accessible to microbial and enzymatic attack (Yusof and Ashokkumar 2015). The present study aimed to evaluate the potential for pretreatment of agro-industrial residues using an ultrasonic probe and to investigate the behavior of keratinases exposed to the following reaction systems: ultrasonic probe, ultrasonic bath, and the microwave and enzymatic concentration technique. In addition to comparing the activity of homemade enzymes with commercial enzymes and finally, we applied enzymatic extracts for the degradation of keratinous residues such as swine hair and chicken feathers. Equation 2 Where: FM is final dry mass, and IM is initial dry mass. 2.2 Swine hair pretreatment by ultrasonic probe Swine hair (1 g) was treated using an ultrasonic probe, comparing the micropoint and macropoint. The variables used were power (100%), pulse (3) and exposure time (15 min) using distilled water (100 mL) as a liquid medium in a 250 mL glass beaker (Adapted Azmi, Idris and Yusof 2018). The tests were conducted in triplicate. These were performed with the ECO-SONICS Ultrasonic Probe/Cell Disrupter Sonicator, 20 KHz ultrasonic frequency and 550 W ultrasonic power, equipped with a 4-mm titanium micropoint and 13-mm diameter titanium macropoint. Its operation is the result of the conversion of electrical energy into mechanical energy (BIOVERA 2019). The acoustic energy of the ultrasonic probe was calculated using the calorimetric determination method, according to Equation 1 (Feng, Barbosa-Cánovas and Weiss 2011). This energy is subsequently converted into heat. The experiment was carried out in a 250 mL beaker containing 100 mL of distilled water, proceeding to the pretreatment in the ultrasonic probe with the micropoint and macropoint according to the conditions specified previously. The temperature was measured with the aid of a digital thermometer. Page 4/36 Page 4/36 Equation 1 Where: is the acoustic energy per unit of time (Power), is the mass of the sample, is the specific heat of the sample and is the temperature variation over a given time. The sonicated material was filtered on filter paper and the solid fraction was subjected to a microbial load reduction process using 70% alcohol and heating at 70 °C ± 2 °C for 16 h before starting fermentation (Adapted from Călin et al. 2017). 2.3 Submerged fermentation Submerged fermentation was performed with swine hair. To this end, 10 g L-1 of pretreated and not pretreated ultrasonic probe residue was poured into 50 mM Tris HCl buffer (pH 8.5) with the addition of 106 spores mL-1 of Fusarium oxysporum. The fermentative medium was incubated at 150 rpm and 28 °C for 9 days (Preczeski et al. 2020). The fungus used was isolated from soil-borne chicken feather residues and identified using the Next Generation Sequencing (NGS) method (Preczeski et al. 2020). At the end of the fermentation, the fermented medium was filtered and the retained solid fraction was dried to quantify the degradation percentage of the agro-industrial residue, according to Equation 2. The residues were dried in an oven at 70 ± 2 °C for 16 h then they remained in a desiccator for 1 day. Subsequently, initial and final dry mass measurements (residues before and after the FS) was performed (Preczeski et al. 2020). The filtrate was used to measure keratinolytic activity and exposure in various reaction systems. Keratinolytic activity was quantified at 3, 6, and 9 days, and the percentage of residue degradation was quantified after 9 days (Preczeski et al. 2020). Equation 2 2.5.1.1 Ultrasonic probe A 20 mL aliquot of the crude enzyme extract was exposed to the ECO-SONICS Cell Disruptor Probe (20 KHz and 550 W) equipped with a 4-mm diameter titanium micropoint. The behavior of keratinases was evaluated under different conditions of exposure time (2 to 10 minutes), power (40% to 80%) and pulse (1 to 3) following experimental design CCD 2³ (ECO-SONICS). 2.5.1 Exposure of enzyme extracts to reaction systems Enzymatic extracts produced using swine hair as substrate pretreated with ultrasonic probe with macropoint and not pretreated were exposed to ultrasonic probe, ultrasonic bath and microwave using the experimental design methodology, aiming to evaluate the behavior of the enzymes. Each reaction system was evaluated using central composite design (CCD) based on previously defined independent variables, according to reaction interest and system possibilities. The response variable was relative activity (%), resulting from the variation between keratinolytic activities of the extract before and after exposure to reaction systems (Equation 2). 2.4 Keratinase assay Keratinolytic activity was measured using 0.013 g azokeratin as substrate (SIGMA-ALDRICH K8500 α-K), 3.2 mL 50 mM Tris HCl buffer (pH 8.5) and 0.8 mL enzyme extract. The reaction took place in an ultrathermostatic bath at 50 °C for 1 h, after which 0.8 mL of 10% trichloroacetic acid (TCA) was added to stop the reaction, and was read at 595 nm in a spectrophotometer (Adapted from Bressollier et al. 1999). Page 5/36 One unit of keratinase activity was defined as the amount of enzyme required to produce a 0.01 absorbance increase under the described assay conditions (Bressollier et al. 1999). Enzyme activity was determined before and after exposure to reaction systems. Relative activity (%) was also calculated for activity increment purposes, according to Equation 3. 2.5 Enzyme activity increase 2.5.1 Exposure of enzyme extracts to reaction systems Equation 3 Where: Final activity is enzyme extract activity after exposure to reaction systems, and Initial activity is activity of crude enzyme extract before exposure to reaction systems. 2.5 Enzyme activity increase 2.5.1.2 Ultrasonic bath In this reaction system, 2 mL aliquots of the crude enzyme extract were exposed to the ultrasonic bath, varying the temperature (°C), the power (%) and the exposure time (min) of the enzyme extract according to experimental design. The equipment used was the UNIQUE Ultrasonic Washer, model USC-1800 A, Page 6/36 Page 6/36 frequency US 40 KHz and maximum power of 137 W (0.42 W cm-²). The tests were performed with ultrasonic bath temperature ranging from 30 to 80 ºC, ultrasonic power ranging from 0 to 100% and with exposure time of 10 to 40 minutes, following experimental design (Adapted from Golunski et al. 2017; Adapted from Mulinari et al. 2017). frequency US 40 KHz and maximum power of 137 W (0.42 W cm-²). The tests were performed with ultrasonic bath temperature ranging from 30 to 80 ºC, ultrasonic power ranging from 0 to 100% and with exposure time of 10 to 40 minutes, following experimental design (Adapted from Golunski et al. 2017; Adapted from Mulinari et al. 2017). frequency US 40 KHz and maximum power of 137 W (0.42 W cm-²). The tests were performed with ultrasonic bath temperature ranging from 30 to 80 ºC, ultrasonic power ranging from 0 to 100% and with exposure time of 10 to 40 minutes, following experimental design (Adapted from Golunski et al. 2017; Adapted from Mulinari et al. 2017). 2.5.1.3 Microwave Aliquots of 3 mL of crude enzyme extract were exposed to microwave, varying conditions according to experimental design. Exposure of the enzyme to microwaves was evaluated in the time range of 5 to 15 minutes, using temperatures between 30 and 80 °C, according to the methodology described by Mazinani, Delong and Yan, (2015) and adapted from Golunski et al. (2017). The equipment used was the ANTON PAAR Monowave 100 Microwave Reactor which has unpulsed 500 W output power (ANTON PAAR). 2.5.2 Enzymatic concentration To test the most economically viable enzymatic activity increment methods, the enzyme concentration technique was performed. Enzyme extracts obtained from the fermentation process were concentrated using 0.5 mol L-1 NaCl and 50% (v v-1) acetone. The tests were performed in an ice bath at 4 °C. After centrifugation at 9500 x g, 4 °C and 20 min, the supernatant phase was discarded and the precipitate was resuspended with 50 mM Tris HCl buffer (pH 8.5) (Preczeski et al. 2018). The concentrated enzymatic extract (homemade) was exposed to reaction systems under the conditions of greatest activity increase in experimental designs and applied to the degradation of swine hair and chicken feathers, aiming to evaluate the degradation potential of keratinous residues. 2.1 Keratin substrates 2.6 Commercial enzymes and reaction systems The activity of the homemade enzyme extract was compared with that of the commercial enzyme K4519 SIGMA with initial enzymatic activity of 2812.5 U mL-1. K4519 SIGMA keratinase was diluted in 5 mM phosphate buffer (pH 7.0) (SIGMA-ALDRICH). The enzyme was exposed to the optimal enzymatic increment conditions obtained in the experimental designs for the ultrasonic probe, ultrasonic bath and microwave and was subsequently used for the degradation 2.1 Keratin substrates Page 7/36 Page 7/36 Swine hair and chicken feathers were obtained from a food agroindustry in Rio Grande do Sul, Brazil. Residues were stored at –4 °C until use. Prior to use, the residues were washed with water and detergent, and immersed in 70% alcohol for 1 h, followed by drying at 70 ± 2 °C for 16 h (adapted from Călin et al. 2017; Preczeski et al. 2020). Chicken feathers were used in the degradation tests only. Equation 1 Where: is the acoustic energy per unit of time (Power), is the mass of the sample, is the specific heat of the sample and is the temperature variation over a given time. The sonicated material was filtered on filter paper and the solid fraction was subjected to a microbial load reduction process using 70% alcohol and heating at 70 °C ± 2 °C for 16 h before starting fermentation (Adapted from Călin et al. 2017). 2.2 Swine hair pretreatment by ultrasonic probe Swine hair (1 g) was treated using an ultrasonic probe, comparing the micropoint and macropoint. The variables used were power (100%), pulse (3) and exposure time (15 min) using distilled water (100 mL) as a liquid medium in a 250 mL glass beaker (Adapted Azmi, Idris and Yusof 2018). The tests were conducted in triplicate. These were performed with the ECO-SONICS Ultrasonic Probe/Cell Disrupter Sonicator, 20 KHz ultrasonic frequency and 550 W ultrasonic power, equipped with a 4-mm titanium micropoint and 13-mm diameter titanium macropoint. Its operation is the result of the conversion of electrical energy into mechanical energy (BIOVERA 2019). The acoustic energy of the ultrasonic probe was calculated using the calorimetric determination method, according to Equation 1 (Feng, Barbosa-Cánovas and Weiss 2011). This energy is subsequently converted into heat. The experiment was carried out in a 250 mL beaker containing 100 mL of distilled water, proceeding to the pretreatment in the ultrasonic probe with the micropoint and macropoint according to the conditions specified previously. The temperature was measured with the aid of a digital thermometer. 2.4 Keratinase assay Keratinolytic activity was measured using 0.013 g azokeratin as substrate (SIGMA-ALDRICH K8500 α-K), 3.2 mL 50 mM Tris HCl buffer (pH 8.5) and 0.8 mL enzyme extract. The reaction took place in an ultrathermostatic bath at 50 °C for 1 h, after which 0.8 mL of 10% trichloroacetic acid (TCA) was added to stop the reaction, and was read at 595 nm in a spectrophotometer (Adapted from Bressollier et al. 1999). One unit of keratinase activity was defined as the amount of enzyme required to produce a 0.01 absorbance increase under the described assay conditions (Bressollier et al. 1999). Enzyme activity was determined before and after exposure to reaction systems. Relative activity (%) was also calculated for activity increment purposes, according to Equation 3. 2.3 Submerged fermentation Submerged fermentation was performed with swine hair. To this end, 10 g L-1 of pretreated and not pretreated ultrasonic probe residue was poured into 50 mM Tris HCl buffer (pH 8.5) with the addition of 106 spores mL-1 of Fusarium oxysporum. The fermentative medium was incubated at 150 rpm and 28 °C Page 8/36 Page 8/36 Page 8/36 for 9 days (Preczeski et al. 2020). The fungus used was isolated from soil-borne chicken feather residues and identified using the Next Generation Sequencing (NGS) method (Preczeski et al. 2020). for 9 days (Preczeski et al. 2020). The fungus used was isolated from soil-borne chicken feather residues and identified using the Next Generation Sequencing (NGS) method (Preczeski et al. 2020). At the end of the fermentation, the fermented medium was filtered and the retained solid fraction was dried to quantify the degradation percentage of the agro-industrial residue, according to Equation 2. The residues were dried in an oven at 70 ± 2 °C for 16 h then they remained in a desiccator for 1 day. Subsequently, initial and final dry mass measurements (residues before and after the FS) was performed (Preczeski et al. 2020). The filtrate was used to measure keratinolytic activity and exposure in various reaction systems. Keratinolytic activity was quantified at 3, 6, and 9 days, and the percentage of residue degradation was quantified after 9 days (Preczeski et al. 2020). Equation 2 Equation 2 Equation 2 Where: FM is final dry mass, and IM is initial dry mass. 2.5.1.2 Ultrasonic bath In this reaction system, 2 mL aliquots of the crude enzyme extract were exposed to the ultrasonic bath, varying the temperature (°C), the power (%) and the exposure time (min) of the enzyme extract according to experimental design. The equipment used was the UNIQUE Ultrasonic Washer, model USC-1800 A, frequency US 40 KHz and maximum power of 137 W (0.42 W cm-²). The tests were performed with ultrasonic bath temperature ranging from 30 to 80 ºC, ultrasonic power ranging from 0 to 100% and with exposure time of 10 to 40 minutes, following experimental design (Adapted from Golunski et al. 2017; Adapted from Mulinari et al. 2017). Equation 3 Equation 3 Where: Final activity is enzyme extract activity after exposure to reaction systems, and Initial activity is activity of crude enzyme extract before exposure to reaction systems. 2.5 Enzyme activity increase 2.5 Enzyme activity increase 2.5.1 Exposure of enzyme extracts to reaction systems 2.5.1 Exposure of enzyme extracts to reaction systems Page 9/36 Enzymatic extracts produced using swine hair as substrate pretreated with ultrasonic probe with macropoint and not pretreated were exposed to ultrasonic probe, ultrasonic bath and microwave using the experimental design methodology, aiming to evaluate the behavior of the enzymes. Each reaction system was evaluated using central composite design (CCD) based on previously defined independent variables, according to reaction interest and system possibilities. The response variable was relative activity (%), resulting from the variation between keratinolytic activities of the extract before and after exposure to reaction systems (Equation 2). 2.5.1.1 Ultrasonic probe A 20 mL aliquot of the crude enzyme extract was exposed to the ECO-SONICS Cell Disruptor Probe (20 KHz and 550 W) equipped with a 4-mm diameter titanium micropoint. The behavior of keratinases was evaluated under different conditions of exposure time (2 to 10 minutes), power (40% to 80%) and pulse (1 to 3) following experimental design CCD 2³ (ECO-SONICS). 2.6 Commercial enzymes and reaction systems The activity of the homemade enzyme extract was compared with that of the commercial enzyme K4519 SIGMA with initial enzymatic activity of 2812.5 U mL-1. K4519 SIGMA keratinase was diluted in 5 mM phosphate buffer (pH 7.0) (SIGMA-ALDRICH). The enzyme was exposed to the optimal enzymatic increment conditions obtained in the experimental designs for the ultrasonic probe, ultrasonic bath and microwave and was subsequently used for the degradation of keratinous residues. 2.7 Evaluation of keratin residue degradation Swine hair and chicken feather degradation was evaluated using crude and concentrated enzyme extracts, both with and without exposure to reaction systems. The degradation potential of agro-industrial residues was also evaluated using the commercial enzyme extract K4519 SIGMA before and after exposure to reaction systems. One whole feather or 0.1 g of swine hair was used for each 8 mL of enzyme extract. Assays were incubated at 28 °C for 28 days (Scott and Untereiner 2004), and the result was evaluated based on visible residue degradation. 2.5.1.3 Microwave Aliquots of 3 mL of crude enzyme extract were exposed to microwave, varying conditions according to experimental design. Exposure of the enzyme to microwaves was evaluated in the time range of 5 to 15 minutes, using temperatures between 30 and 80 °C, according to the methodology described by Mazinani, Delong and Yan, (2015) and adapted from Golunski et al. (2017). The equipment used was the ANTON PAAR Monowave 100 Microwave Reactor which has unpulsed 500 W output power (ANTON PAAR). Page 10/36 Page 10/36 2.5.2 Enzymatic concentration To test the most economically viable enzymatic activity increment methods, the enzyme concentration technique was performed. Enzyme extracts obtained from the fermentation process were concentrated using 0.5 mol L-1 NaCl and 50% (v v-1) acetone. The tests were performed in an ice bath at 4 °C. After centrifugation at 9500 x g, 4 °C and 20 min, the supernatant phase was discarded and the precipitate was resuspended with 50 mM Tris HCl buffer (pH 8.5) (Preczeski et al. 2018). The concentrated enzymatic extract (homemade) was exposed to reaction systems under the conditions of greatest activity increase in experimental designs and applied to the degradation of swine hair and chicken feathers, aiming to evaluate the degradation potential of keratinous residues. 2.9 Statistical analysis The experiments were statistically evaluated using CCD and the results of the enzymatic activity were analyzed using Protimiza Experimental Design software (Rodrigues and Iemma, 2014), using 95% confidence analysis of variance (p <0.05). Results were presented as mean ± standard deviation of triplicate experiments. Statistical differences between data sets were verified using ANOVA and Tukey Test (Fegade et al. 2013) of keratinous residues. 2.8 Characterization of homemade keratinase enzymes For the purpose of obtaining the optimal pH and temperature of the enzyme used, a characterization was carried out evaluating different pH conditions, ranging from 5 to 9 and temperature, from 37 °C to 70 °C. The buffers used were: 50 mM citrate buffer pH 5.0 and 5.7; 50 mM sodium phosphate buffer pH 7.1, pH Page 11/36 Page 11/36 8.4 and pH 9.0; 50 mM Tris HCl buffer pH 8.5 (SIGMA-ALDRICH). The characterization was carried out in an ultrathermostatic bath and the activity measurement followed that specified in item 2.4. 2.7 Evaluation of keratin residue degradation Swine hair and chicken feather degradation was evaluated using crude and concentrated enzyme extracts, both with and without exposure to reaction systems. The degradation potential of agro-industrial residues was also evaluated using the commercial enzyme extract K4519 SIGMA before and after exposure to reaction systems. One whole feather or 0.1 g of swine hair was used for each 8 mL of enzyme extract. Assays were incubated at 28 °C for 28 days (Scott and Untereiner 2004), and the result was evaluated based on visible residue degradation. 2.8 Characterization of homemade keratinase enzymes For the purpose of obtaining the optimal pH and temperature of the enzyme used, a characterization was carried out evaluating different pH conditions, ranging from 5 to 9 and temperature, from 37 °C to 70 °C. The buffers used were: 50 mM citrate buffer pH 5.0 and 5.7; 50 mM sodium phosphate buffer pH 7.1, pH 8.4 and pH 9.0; 50 mM Tris HCl buffer pH 8.5 (SIGMA-ALDRICH). The characterization was carried out in an ultrathermostatic bath and the activity measurement followed that specified in item 2.4. 2.9 Statistical analysis Page 12/36 The experiments were statistically evaluated using CCD and the results of the enzymatic activity were analyzed using Protimiza Experimental Design software (Rodrigues and Iemma, 2014), using 95% confidence analysis of variance (p <0.05). Results were presented as mean ± standard deviation of Page 12/36 triplicate experiments. Statistical differences between data sets were verified using ANOVA and Tukey Test (Fegade et al. 2013) 3.1 Swine hair pretreatment by ultrasonic probe The amount of energy required for the pretreatment of swine hair with the ultrasonic probe was determined by the method of calorimetric determination for the micropoint and for the macropoint and calculated using Equation 1. Table 1 shows the initial temperature values and end for each tip used. Table 1 Initial and final temperature measurement for the ultrasound probe's micropoint and macropoint to determine the amount of energy required for the pretreatment of swine hair. Micropoint Macropoint Assays Initial Temperature Final Temperature Assays Initial Temperature Final Temperature 16.1 ± 0.2 °C 26.5 ± 1.2 °C 19.5 ± 0.3 °C 53.3 ± 2.4 °C Afterwards, the acoustic energy of the ultrasonic probe was calculated, obtaining the values of and for the micropoint and macropoint respectively. It is seen that when the macropoint is used, the amount of energy required is higher, consequently, causing a greater increase in temperature. Most of the effects caused by the ultrasonic probe occur through the cavitation process, which is nothing more than the collapse of bubbles in a medium when they reach a certain temperature (Kuijpers, Kemmere and Keurentjes 2002). The fermentative process to obtain the enzymatic extract was performed using swine hair as the substrate that had been pretreated with or without the ultrasonic probe. Enzymatic activities and degradation percentages were evaluated (Table 2). Table 2 Keratinolytic activity after pretreatment in an ultrasonic probe and without pretreatment at 3, 6 and 9 days, as well as the percentage of degradation obtained at the end of the fermentation process. Table 2 Keratinolytic activity after pretreatment in an ultrasonic probe and without pretreatment at 3, 6 and 9 days, as well as the percentage of degradation obtained at the end of the fermentation process. Swine hair Enzymatic activity (U mL-1) Degradation (%) Pretreatment by ultrasonic probe Micropoint 3 days 6 days 9 days 38.8 ± 5.3 a 81.0 ± 5.2 a 55.7 ± 3.0 a 31.8 ± 1.1 a Macropoint 3 days 6 days 9 days 50.8 ± 5.3 b 39.5 ± 4.8 b 43.8 ± 1.1 b 45.5 ± 0.8 b Without pretreatment 3 days 6 days 9 days 25.0 ± 5.6 a 82.2 ± 9.4 a 75.2 ± 3.2 c 36.3 ± 4.0 a Without pretreatment Equal lowercase letters do not differ from each other by the Tukey test on the column. 3.1 Swine hair pretreatment by ultrasonic probe Equal lowercase letters do not differ from each other by the Tukey test on the column. Equal lowercase letters do not differ from each other by the Tukey test on the column. Page 13/36 Page 13/36 The keratinolytic activity obtained on the third day of the fermentation in the swine hair pretreated with the ultrasonic probe macropoint was 50.8 ± 5.3 U mL-1. From this result, it can be inferred that the greater energy dissipation caused by the macropoint resulted in more relevant data of enzymatic activity, because it has a larger diameter than that of the micropoint, which achieved enzymatic activity (38.8 ± 5.3 U mL-1) similar to that obtained with the crude residue (25.0 ± 5.6 U mL-1). We found that the micropoint did not have the same impact on structure in terms of enzymatic activity for the 3rd day, because the ultrasonic system can be influenced by several factors, including energy dissipation, ultrasonic intensity, temperature, exposure time, and diameter (Lippert et al. 2018), all of which may have resulted in lower erosion efficiency of the structure when using the micropoint than when using the macropoint. The highest keratinolytic activity was observed for swine hair without pretreatment and with micropoint pretreatment at 6 days of fermentation: 82.2 ± 9.4 U mL-1 and 81.0 ± 5.2 U mL-1, respectively. Regarding the degradation percentage, the highest degradation occurred in the pretreatment test with ultrasonic probe with macropoint (45.5% ± 0.8%), and it can be inferred that the abrasion and erosion in the structure of swine hair, which resulted in the highest enzymatic activity in three days for the same assay, also presented the highest dissolution of structural chains that make up the residue in question, resulting in the largest mass loss in this experiment. The ultrasonic probe increases reaction rates, especially hydrolysis processes (Zou et al. 2016). Ultrasonic systems facilitate structural degradation via propagation of ultrasonic waves that cause bubble cavitation, causing temperature rise, favoring the formation of rapidly reacting radicals (Wang 1981; Manasseh et al. 2010; Yusof and Ashokkumar 2015), improving the enzymatic attack on the substrate (Yang and Fang 2015). The cavitation process is the fundamental factor for the efficiency of ultrasound pretreatment, because the explosion of bubbles near the surface of the material causes abrasion and erosion, reducing the surface area of the structure and, consequently, improving the processes. 3.2.1 Exposure of enzyme extracts to reaction systems Exposure to reaction systems was performed to evaluate the behavior of keratinases enzymes in various reaction systems. The ultrasonic probe, ultrasonic bath and microwave were evaluated. Two enzyme extracts were exposed to the reaction systems: a) enzymatic extract from FS carried out with swine hair pretreated with ultrasonic probe with macropoint; b) enzymatic extract from FS carried out with swine hair without pretreatment. 3.1 Swine hair pretreatment by ultrasonic probe hydrolysis and enzyme production (McClements 1995; Barton Bullock and Weir 1996; Mason et al. 2011). Similar observations were reported by Jin et al. (2015), who verified changes in the kinetic characteristics between proteins and substrates in ultrasonic systems, observing the increase in hydrolysis rate, improving the affinity between enzyme and substrate. Pretreatment with an ultrasonic probe can be considered viable with the use of the macropoint when the objective of the process is enzyme production in shorter times. Visualizing the system in search of an efficient residue degradation and relevant enzymatic activities, the system with swine hair without pretreatment presented greater viability in relation to the higher enzymatic productions of the other experiments, considering that in six days there was an enzymatic production of approximately 38% greater than with the pretreated macropoint residue in three days, and only a 10% difference in residue degradation from the same assay. Comparing the data from the pretreated residue with micropoint, there was no significant difference between the data, suggesting that using the residue without pretreatment is more promising, in addition to ensuring lower energy expenditure. Page 14/36 3.2 Enzyme activity increase 3.2.1 Exposure of enzyme extracts to reaction systems 3.2.1.1 Ultrasonic Probe The enzymatic extracts were exposed to an ultrasonic probe using the micropoint. This was studied based on an experimental matrix (Table 3), which presents the results obtained for the independent variables of exposure time, power and pulse, and evaluated relative activity in response. For the enzymatic extract produced from swine hair pretreated using the ultrasonic probe, up to 23.3% were observed in test 7, corresponding to the shortest time evaluated (2 min) (Table 3). When subjected to effects analysis, there was a significant negative effect only of the time variable: the shorter the exposure time to the ultrasonic probe, the greater the keratinolytic activity. Power and pulse were not significant. For the enzymatic extracts obtained with swine hair that was not pretreated, more promising increments were observed. Table 3 shows that test 1 showed the largest increase in keratinolytic activity in relation to crude enzyme extract, i.e., 38.4% increase in enzymatic activity. The analysis of the effects of the variables on keratinolytic activity showed that only there was only a significant negative effect on the time variable: the longer the exposure time to the ultrasonic probe, the lower the keratinolytic activity achieved. As before, power and pulse were not significant in the process. In both cases studied, the statistical model was not validated due to Fcal <Ftab. Table 3 Experimental planning matrix (real and coded values) and relative activity results (%) for the conditions evaluated in the reaction system ultrasonic probe with enzymatic extracts produced from swine hair pretreated by ultrasonic probe and without pretreatment. 3.2.1.1 Ultrasonic Probe Page 15/36 Page 15/36 Assays Ultrasonic probe Relative activity (%) Time (min) Power (%) Pulse Pretreatment by ultrasonic probe Without pretreatment 1 -1 (2) -1 (40) -1 (1) 11.3 38.4 2 +1 (10) -1 (40) -1 (1) 0.0 1.4 3 -1 (2) +1 (80) -1 (1) 2.7 16.3 4 +1 (10) +1 (80) -1 (1) 0.0 9.1 5 -1 (2) -1 (40) +1 (3) 0.0 21.1 6 +1 (10) -1 (40) +1 (3) 0.0 0.0 7 -1 (2) +1 (80) +1 (3) 23.3 25.2 8 +1 (10) +1 (80) +1 (3) 0.0 10.3 9 0 (6) 0 (60) 0 (2) 0.0 0.0 10 0 (6) 0 (60) 0 (2) 0.0 3.0 11 0 (6) 0 (60) 0 (2) 0.0 0.0 12 0 (6) 0 (60) 0 (2) 0.0 0.0 Crude enzymatic extract 75.0 U mL-1 124.3 U mL-1 zymatic extract obtained after FS without exposure to reaction systems. * Enzymatic extract obtained after FS without exposure to reaction systems. * Enzymatic extract obtained after FS without exposure to reaction systems. The largest increases in activity occurred in the shortest exposure time evaluated (2 min), corroborating the statistical analysis. Ultrasonic systems are responsible for generating waves that propagate through a liquid, which collapse at high temperatures. This phenomenon is called cavitation. The shear force resulting from the explosion of these microbubbles causes rapid heat and mass transfer, promoting increased enzymatic activity (Jin et al. 2015). Thus, the increase in activity may be a result of the shorter exposure time to the ultrasonic probe that favors the decomposition of interfering molecules and the change in enzymatic specificity, making enzymes more easily accessible to the reaction and consequently increasing their activity, providing an ideal environment for the reaction between enzyme and substrate (McClements 1995; Jin et al. 2015). The enzyme also remains more regular and flexible because thermodynamic parameters such as Ea, ΔH, and ΔS are reduced with the use of ultrasound, causing an improvement in its activity and improving the operational stability of enzymes (Ma et al. 2011; Wang, Chen and Zhu 2013). In assays where the exposure time was longer (from 6 to 10 min), the activity was lower and, in some cases, lower than the activity obtained in the enzyme extract without exposure to the ultrasonic probe, possibly due to the enzymatic denaturation caused by the longer exposure time to the ultrasonic probe (Kapturowska, Stolarzewicz and Krzyczkowska 2012). 3.2.1.1 Ultrasonic Probe Denaturation of enzymes can occur due to excess pressure, temperature or even shear force generated during the cavitation phenomenon in the ultrasonic system (Grintsevich et al. 2001; Potapovich, Eremin and Metelitza 2003). Page 16/36 Page 16/36 It is important to note that the use of ultrasonic systems is efficient in reducing reaction time, promoting increased enzymatic activity in many cases. On the other hand, depending on the conditions studied, there may be an enzymatic denaturation and consequently a decrease in enzyme activity. It is important to note that the use of ultrasonic systems is efficient in reducing reaction time, promoting increased enzymatic activity in many cases. On the other hand, depending on the conditions studied, there may be an enzymatic denaturation and consequently a decrease in enzyme activity. 3.2.1.2 Ultrasonic bath The greatest increase in enzymatic activity obtained for the ultrasonic bath occurred in the enzymatic extract from swine hair pretreated with ultrasonic probe (30.2%), with a temperature of 80 °C during 40 minutes of exposure and 0% power, corresponding to test 6 (Table 4). Analysis of effects on keratinolytic activity showed that temperature and power were important. The temperature variable had significant positive effects; that is, the higher the temperature, the higher the keratinolytic activity. The power variable affected the enzyme activity negatively; the lower the power, the higher the activity. Time had no influence on enzymatic activity. Because Fcal >Ftab, the statistical model was validated. For enzymatic extract produced from swine hair without pretreatment, only test 3, corresponding to a temperature of 30 °C during 10 minutes and 100% power, provided an increase of keratinolytic activity in relation to the crude enzyme, which was 5.9% (Table 4). However, when the results were subjected to effects analysis, no variable was found to be significant. For this enzyme extract studied, the model was not statistically validated due to Fcal <Ftab. In the enzymatic extracts evaluated, different behaviors of the enzyme regarding temperature were observed. For the pretreated residues, the enzyme extract increased the activity at a temperature of 80 °C and for the untreated residues, the only activity increase was at the temperature of 30 °C, the mildest within the study range. This shows that the enzymes produced during submerged fermentation with pretreated and untreated residues are different in the mode of action compared to the ultrasonic bath reaction system. Table 4 Experimental planning matrix (real and coded values) and relative activity results (%) for the conditions evaluated in the reaction system ultrasonic bath with enzymatic extracts produced from swine hair pretreated and non-pretreated with an ultrasonic probe. 3.2.1.2 Ultrasonic bath Page 17/36 Assays Ultrasonic Bath Relative activity (%) Temperature (°C) Power (%) Time (min) Pretreatment by ultrasonic probe Without pretreatment 1 -1 (30) -1 (0) -1 (10) 0.0 0.0 2 +1 (80) -1 (0) -1 (10) 9.4 0.0 3 -1 (30) +1 (100) -1 (10) 0.0 5.9 4 +1 (80) +1 (100) -1 (10) 0.0 0.0 5 -1 (30) -1 (0) +1 (40) 0.0 0.0 6 +1 (80) -1 (0) +1 (40) 30.2 0.0 7 -1 (30) +1 (100) +1 (40) 0.0 0.0 8 +1 (80) +1 (100) +1 (40) 0.0 0.0 9 0 (55) 0 (50) 0 (25) 0.0 0.0 10 0 (55) 0 (50) 0 (25) 0.0 0.0 11 0 (55) 0 (50) 0 (25) 0.0 0.0 12 0 (55) 0 (50) 0 (25) 0.0 0.0 Crude enzymatic extract* 79.5 U mL-1 118.5 U mL-1 * Enzymatic extract obtained after FS without exposure to reaction systems. Relative activity (%) Ultrasonic Bath * Enzymatic extract obtained after FS without exposure to reaction systems. * Enzymatic extract obtained after FS without exposure to reaction systems. The ideal temperature range for keratinases is between 30 and 80 °C, which may vary according to the microorganism that is used for the production of the enzyme, according to Kothari, Rani and Goyal (2017). Another fact that determines the term stability of the enzyme is the time of exposure to that temperature. The optimum temperature can also vary depending on the source and origin of the isolated microorganism (Brandelli, Daroit and Riffel 2010). The behavior observed for ultrasonic bath may be related to the synergistic effect of ultrasonic waves with temperature. Several studies show increased activity of enzymes exposed to ultrasonic bath (Leaes et al. 2013; Mulinari et al. 2017). According to the literature, temperature considerably influences the increase or decrease of enzymatic activity. With a rise in temperature the activity may be increased, but depending on the structure of the enzyme, if there is a very significant rise in temperature, denaturation may occur (Resa et al. 2009). This behavior was described in the studies by Wang et al. (2011) and Ovsianko et al. (2005), where enzymatic denaturation occurred due to the effect of ultrasonic sonication causing an increase in temperature, also stimulating the effect of cavitation. Ultrasonic bath alters the behavior of exposed enzymes. 3.2.1.2 Ultrasonic bath Thus, the use of ultrasonic systems can promote increases in enzymatic activity, possibly due to conformational changes in protein structure, in addition to considerably reducing the reaction time (Leaes et al. 2013) and also the decrease in activity by denaturing (Resa et al. 2009). 3.2.1.3 Microwave 3.2.1.3 Microwave Page 18/36 For the enzymatic extract produced from swine hair pretreated using the ultrasonic probe, test 2 showed an increase of activity, 15.1% at 80 °C for 5 minutes (Table 5). Performing statistical analysis, we noted that the temperature variable was significantly positive: the higher the temperature, the higher the keratinolytic activity achieved. Time had no influence on enzymatic activity. The model was statistically validated by Fcal >Ftab. For the enzymatic extract obtained from swine hair without pretreatment, we found that no assay showed an increase of keratinolytic activity (Table 5). However, performing the statistical analysis, we noted that the time variable was significantly negative; that is, the longer the microwave exposure time, the lower the keratinolytic activity achieved. The temperature had no influence on enzymatic activity. Table 5 Experimental planning matrix (real and coded values) and relative activity results (%) for the conditions evaluated in the reaction system by microwave irradiation with enzymatic extracts produced from swine hair pretreated and non-pretreated with an ultrasonic probe. Assays Microwave Relative Activity (%) Temperature (°C) Time (min) Pretreatment by ultrasonic probe Without pretreatment 1 -1 (30) -1 (5) 0.0 0.0 2 +1 (80) -1 (5) 15.1 0.0 3 -1 (30) +1 (15) 0.0 0.0 4 +1 (80) +1 (15) 8.8 0.0 5 0 (55) 0 (10) 0.0 0.0 6 0 (55) 0 (10) 0.0 0.0 7 0 (55) 0 (10) 0.0 0.0 8 0 (55) 0 (10) 0.0 0.0 Crude enzymatic extract* 79.5 U mL-1 128.8 U mL-1 * Enzymatic extract obtained after FS without exposure to reaction systems. * Enzymatic extract obtained after FS without exposure to reaction systems. The increase in activity obtained for enzymatic extract produced from swine hair pretreated with ultrasonic probe was because the microwaves provided an increase in enzymatic activity and an increase in reaction efficiency due to instantaneous overheating. Causing tremendous agitation of molecules that induces an increase in energy collisions, also increasing reaction and conversion rates (Ma et al. 2011; Mazinani, DeLong and Yan 2015). The mechanism of microwave operation occurs by the interaction of the electromagnetic field with matter. 3.2.1.4 Larger increments of enzymatic activity Given the results obtained for the three reaction systems evaluated, the ultrasonic probe showed greater potential for increased enzymatic activity compared to the other reaction systems (Fig. 1). Fig. 1. Larger increments of enzymatic activity of enzymatic extracts obtained from swine hair pretreated using the ultrasonic probe and without pretreatment subjected to the reaction systems ultrasonic probe, ultrasonic bath and microwave. The largest increase was obtained using the ultrasonic probe (38.4%) with the enzymatic extract produced from swine hair without pretreatment. Ultrasonic systems also showed good results of increased enzymatic activity for the enzymatic extract produced from swine hair pretreated by ultrasonic probe, with the increment values being 23.3% for ultrasonic probe and 30.2% for ultrasonic bath. In addition, these values were reached under mild conditions of time, power and pulse, making the process more viable. 3.2.1.2 Ultrasonic bath This causes a movement of ions, which in turn cause heat generation by two mechanisms, dipole rotation and ion conduction. In chemical reactions, microwaves cause molecular friction due to the polarization of molecules. This process is responsible for increasing the friction of these molecules, consequently increasing the temperature and the reaction rate (Lopes et al. 2015). In this manner, Page 19/36 microwave irradiation can cause conformational changes in the exposed structure, leading to increased enzyme activity or even damage such as enzymatic denaturation (Leonelli and Mason 2010; Lopes et al. 2015; Mazinani, DeLong and Yan 2015). microwave irradiation can cause conformational changes in the exposed structure, leading to increased enzyme activity or even damage such as enzymatic denaturation (Leonelli and Mason 2010; Lopes et al. 2015; Mazinani, DeLong and Yan 2015). Some authors point out that the use of microwaves can cause changes in thermodynamic properties, providing increased enzymatic activity (Mazinani, DeLong and Yan 2015; Golunski et al. 2017). These findings suggest that conformational changes in the structure of enzymes occur when exposed to microwaves, possibly due to cleavages that occur during the process and the formation of hot spots by instant heating. On the other hand, high temperatures and exposure times cause enzymatic denaturation. Possibly this occurred in the assays with enzymatic extract produced from untreated swine hair, where mild temperatures of 30 °C were sufficient to cause decreased enzyme activity. Temperature is one of the factors that most influence the behavior of enzymes exposed to microwave irradiation. This causes collisions between molecules to increase, and this causes an increase in energy, causing faster reaction rates. However, at very high temperatures, the reaction rate is reduced due to the denaturation of enzymes, caused by heat breakage and also the breakdown of ionic and hydrogen bonds, stabilizing the protein structure (Yadav and Borkar 2009; Khan and Rathod 2018). 3.2.1.4 Larger increments of enzymatic activity 3.2.2 Enzymatic concentration The enzyme concentration technique was performed to verify the possibility of increasing enzymatic activity from an economically viable and simple operation method. Enzyme extracts produced from swine hair without pretreatment were used and the enzyme concentration technique was performed in the presence of NaCl and acetone. Page 20/36 Page 20/36 The homemade extract showed an enzymatic activity of 159.3 U mL-1 after the concentration technique, increasing the activity value by 53.5% when compared to the crude enzyme extract (103.8 U mL-1). The concentrated enzymatic extract (homemade) was exposed to reaction systems under the conditions of greatest activity increase in experimental designs, where it presented stability against the ultrasonic probe, ultrasonic bath and microwave, possibly due to the absence of interferents that were separated during enzymatic concentration. After exposure, the concentrated enzymatic extract (homemade) was applied in the degradation of swine hair and chicken feathers, aiming to evaluate the degradation potential of keratinous residues. 3.3 Commercial enzymes and reaction systems 3.3 Commercial enzymes and reaction systems 3.3 Commercial enzymes and reaction systems The commercial enzyme K4519 SIGMA was exposed to ultrasonic probe, ultrasonic bath and microwave under the conditions of higher relative activity of homemade enzyme, in order to compare with the obtained data. The enzyme K4519 SIGMA keratinase had initial activity of 2812.5 U mL-1. When subjected to an ultrasonic probe and bath, it showed an 11.1% increase in enzymatic activity. In microwave irradiation, there was a reduction in activity. There was high stability because it is a highly pure enzyme, with little variation when subjected to reaction systems. The activity increase observed for the enzyme K4519 SIGMA keratinase was smaller than that obtained for the homemade enzyme when subjected to ultrasonic probe (38.4%). This behavior is expected because it is a pure enzyme with high stability. It should be noted that the production of the homemade enzyme is low-cost because it uses a highly available residue, being an inhomogeneous residue with large variations, in addition to the fact of disposal problems secondary to difficult degradation. The produced enzyme gave good values of enzymatic activity and activity increases when subjected to reaction systems, becoming competitive against commercial enzymes. 3.4 Evaluation of keratin residue degradation Crude and concentrated enzymatic extracts (homemade) without and with exposure to reaction systems were evaluated for degradation potential of keratinous residues. We also compared enzymatic extract with commercial enzyme K4519 SIGMA. Enzyme extracts were incubated at 28 °C for 28 days and the result was presented based on visible residue degradation and enzymatic activity. We found that, for the crude enzyme extract and after exposure to reaction systems, the values for keratinolytic activity of the assays containing swine hair and chicken feathers showed small decreases in relation to the initial value for enzymatic activity over the 28 days, however, remaining stable over time, suggesting that the enzyme was active and showing very promising enzyme activity. For the enzyme extract in concentrated form and after exposure to reaction systems, significant increases in keratinolytic activity were observed over the 28 days of study (Table 6). Page 21/36 Page 21/36 Page 21/36 Page 21/36 Table 6 Measure of keratinolytic activity at initial time and after 28 days for homemade enzymatic extract crude and concentrated and enzymatic extracts after exposure to ultrasonic reaction systems and microwave irradiation containing swine hair and chicken feathers. CRUDE Enzyme Extracts (homemade) Swine hair Chicken feathers Assays 0° day 28° day 0° day 28° day Enzymatic Activity (U mL-1) Enzymatic Activity (U mL-1) Ultrasonic Probe 172.0 137.0 172.0 133.0 Ultrasonic Bath 153.0 124.0 153.0 115.5 Microwave 96.0 106.5 96.0 141.0 Crude Extract 171.0 151.5 171.0 121.5 CONCENTRATED Enzyme Extracts (homemade) Swine hair Chicken feathers Assays 0° day 28° day 0° day 28° day Enzymatic Activity (U mL-1) Enzymatic Activity (U mL-1) Ultrasonic Probe 159.5 231.0 159.5 192.5 Ultrasonic Bath 141.8 202.0 141.8 181.5 Microwave 149.0 229.5 149.0 191.5 Crude Extract 103.8 - 103.8 - Concentrated Extract 159.3 - 159.3 - Almost total degradation of chicken feathers was observed in the crude enzyme extract and enzyme extracts after exposure to reaction systems (Fig. 2), while for swine hair, there was no degradation in any of the enzyme extracts evaluated. Possibly the enzyme could not access the resistant structure of swine hair. One factor that may explain the ease with which chicken feathers were degraded is the less rigid structure compared to swine hair. Because they are part of the keratin family, swine hair exhibits a structure that is extremely resistant to physical, chemical and also enzymatic actions (Choińska-Pulit, Łaba and Rodziewicz 2019). 3.4 Evaluation of keratin residue degradation The degradation potential of chicken feathers containing the commercial enzyme extract K4519 SIGMA was also evaluated. We found that, during the 28 days of incubation, the commercial enzyme was not able to degrade the chicken feathers, thereby favoring the homemade enzyme extract (Fig. 2). Fig. 2. Total degradation of chicken feathers observed in crude enzymatic extracts and after exposure to reaction systems. A) Control; B) Crude; C) Concentrated; D) K4519 SIGMA commercial enzyme. Page 22/36 Page 22/36 The results obtained for the degradation test containing the homemade extracts in crude form and after exposure to the reaction systems are promising as evidenced by the degradation potential on keratinous residues. The commercial enzyme K4519 SIGMA is expensive, highly concentrated and stable; nevertheless, it did not degrade the chicken feathers used in the tests. Swine hair and chicken feathers are part of the group of hard keratins owing to their high concentrations of cysteine, rendering them very resistant (Korniłłowicz-Kowalska and Bohacz 2011). However, α-keratin present in the swine hair structure has large amounts of sulfur, which confers highly resistant mechanical properties (Holkar et al. 2018). This explains why chicken feathers are more easily degraded than swine hair. In addition, the keratin structure formed by disulfide bonds ensures stability and resistance to enzymatic degradation. Kunert (1989) found that the reduction of disulfide bonds considerably influenced the degradation of keratin structure, ensuring efficient keratin hydrolysis. This suggests that the homemade enzyme extract acted on disulfide bonds and reduced the cystine concentration present in the keratin structure, as well as converting the sulfur molecule into smaller structures (Jaouadi et al. 2013). However, the exact process of degradation involves a series of mechanisms of action that occur in 3 stages. Mechanical keratinolysis occurs by the action of mycelium-producing fungi, involving the penetration of the mycelium into the keratin residue. Then, the hydrolysis of the disulfide bonds occurs, responsible for ensure the resistance and stability of the keratin structure. Finally, proteolysis occurs involving the action of the enzyme itself (Kunert 1989; Onifade et al. 1998). For the concentrated enzymatic extract, no residue degradation was observed in any of the evaluated extracts. In the tests where there were high values of keratinolytic activity, the degradation percentages were low or almost nonexistent. 3.4 Evaluation of keratin residue degradation This agrees with the study by Riffel and Brandelli (2006), where high values for keratinolytic activity were verified; nevertheless, the purified enzyme was not able to degrade chicken feathers. The authors suggest that in these cases, enzymes such as disulfide reductases and proteases need to act so that feather degradation occurs (Fang et al. 2013b). The presence of inhibitors may explain why there was no residue degradation (Hamiche et al. 2019). Despite the fact that the enzyme is concentrated and the amount of impurities is reduced, it has no potential for degradation of residues (Riffel and Brandelli 2006). 3.5 Characterization of homemade keratinase enzymes The characterization of homemade keratinases was carried out by evaluating different pH and temperature conditions in an ultrathermostatic bath for 1 h. The highest enzyme activity was achieved at pH 8.5 (50 mM Tris HCl buffer) and 50 °C temperature. Page 23/36 Most of keratinases producing microorganisms operate in the neutral and alkaline pH range, and the degradation of keratin is favored in alkaline pH, with the modification of cysteine residues and making them more accessible for the action of the enzyme. The optimum temperature is in the range of 28 °C to 50 °C, reaching 70 °C depending on the microorganism. In addition, most keratinases have good stability over wide pH and temperature ranges (Anbu et al. 2005). 50 °C, reaching 70 °C depending on the microorganism. In addition, most keratinases have good stability over wide pH and temperature ranges (Anbu et al. 2005). Consent for publication All authors agreed with this publication. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable 4. Conclusion Biotechnological processes involving the biodegradation of keratinolytic materials by action of microorganisms and enzymes are promising alternatives to improve the degradability of these materials, thereby adding value to the material. Ultrasonic and microwave reaction systems are viable alternatives for evaluating the behavior of keratinases and the potential for pretreatment of keratinous residues. The pretreatment in an ultrasonic probe using the macropoint causes greater energy dissipation due to the larger diameter in relation to the micropoint, thus increasing the reaction rates and facilitating the degradation process of the exposed structures due to the propagation of ultrasonic waves, resulting in greater weight loss. Thus, the use of the ultrasonic probe as a pretreatment can be quite useful when looking for greater enzyme production in a shorter time. For longer processes, the use of waste without pretreatment becomes more promising, also decreasing energy expenditure, since the enzymatic activity achieved over time is greater. Regarding the evaluation of the behavior of keratinases in relation to reaction systems, different behaviors were observed from enzymatic extracts, showing that it is possible to produce enzymes with different characteristics and behaviors. The ultrasonic probe proved to be more promising, generating the greatest increase in enzymatic activity. The enzymatic concentration showed a significant increase in activity, showing that simple and low-cost techniques can be efficient. It was found that the crude enzymatic extract has the potential to degrade chicken feathers, while the other extracts used did not show potential for degradation, thus making it a very interesting result. Availability of data and materials The datasets generated for this study are available on request to the corresponding author. The datasets generated for this study are available on request to the cor Competing interests There are not competing interests Page 24/36 Authors' contributions FFC, SK, KPP, CD, TS, CB, FSS, AFC and JZ: experimental procedures, results discussion, and data treatment AJM, GF, HT: research coordinators Acknowledgments The authors are grateful to the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGS, to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES; to the Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq; and to the research group at Laboratório de Microbiologia e Bioprocessos - LAMIBI da UFFS campus Erechim. Funding CAPES, CNPq and FAPERGS CAPES, CNPq and FAPERGS References Abdel-Fattah A M, El-Gamal M S, Ismail S A, Emran M A, Hashem A M (2018) Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1. 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Ultrason Sonochem 27: 46-53. doi: 10.1016/j.ultsonch.2015.04.031 Kapturowska A U, Stolarzewicz I A, Krzyczkowska J (2012) Studies on the lipolytic activity of sonicated enzymes from Yarrowia lipolytica. Ultrason Sonochem 19: 186-191. doi: 10.1016/j.ultsonch.2011.06.015 Page 28/36 Page 28/36 Kaul S, Sumbali G (1997) Keratinolysis by poultry farm soil fungi. Mycopathologia 139: 137-140. doi: 10.1023/a:1006896030739 Khan N R, Rathod V K (2018) Microwave assisted enzymatic synthesis of speciality esters: A mini - review. Process Biochem 75: 89-98. doi: 10.1016/j.procbio.2018.08.019 Korniłłowicz-Kowalska T, Bohacz J (2011) Biodegradation of keratin waste: Theory and practical aspects. Waste Manage 31: 1689-1701. doi: 10.1016/j.wasman.2011.03.02 Kothari D, Rani A, Goyal A (2017) Keratinases. In: Pandey A, Negi S, Soccol C R (ed) Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products, 1° edn. Elsevier. doi: https://doi.org/10.1016/B978-0-444-63662-1.00019-1 Kuijpers M W A, Kemmere M F, Keurentjes J T F (2002) Calorimetric study of the energy efficiency for ultrasound-induced radical formation. Ovsianko S L, Chernyavsky E A, Minchenya V T, Adzerikho I E, Shkumatov V M (2005) Effect of ultrasound on activation of serine proteases precursors. Ultrason Sonochem 12: 219-223. doi: 10.1016/j.ultsonch.2003.10.012 References Ultrasonics 40:675-678. doi: 10.1016/S0041-624X(02)00197-X Kunert K (1989) Biochemical mechanism of keratin degradation by the actinomycete Streptomyces fradiae and the fungus Microsporum gypseum: A comparison. J Basic Microbiol 29: 597-604. doi: 10.1002/jobm.3620290909 Kushwaha R K S (1983) The In Vitro Degradation of Peacock Feathers by Some Fungi. Mykosen 26: 324- 326. doi: 10.1111/j.1439-0507.1983.tb03218.x Łaba W, Kopeć W, Chorążyk D, Kancelista A (2015) Biodegradation of pretreated pig bristles by Bacillus cereus B5esz. Int Biodeterior Biodegrad 100: 116-123. doi: 10.1016/j.ibiod.2015.02.024 Page 29/36 Leaes E X, Lima D, Miklasevicius L, Ramon A P, Dal Prá V, Bassaco M M, Terra L M, Mazutti M A (2013) Effect of ultrasound-assisted irradiation on the activities of α-amylase and amyloglucosidase. Biocatal Agric Biotechnol 2: 21-25. doi: 10.1016/j.bcab.2012.08.003 doi: 10.1016/j.bcab.2012.08.003 Leonelli C, Mason T J (2010) Microwave and ultrasonic processing: Now a realistic option for industry. Chem Eng Process 49: 885-900. doi: 10.1016/j.cep.2010.05.006 Leonelli C, Mason T J (2010) Microwave and ultrasonic processing: Now a realistic option for industry. Chem Eng Process 49: 885-900. doi: 10.1016/j.cep.2010.05.006 Lippert T, Bandelin J, Musch A, Drewes J E, Koch K (2018) Energy-positive sewage sludge pre-treatment with a novel ultrasonic flatbed reactor at low energy input. Bioresour Technol 264: 298-305. doi: 10.1016/j.biortech.2018.05.073. Lopes L C, Barreto M T M, Gonçalves K M, Alvarez H M, Heredia M F, Souza R O M A, Cordeiro Y, Dariva C, Fricks A T (2015) Stability and structural changes of horseradish peroxidase: Microwave versus conventional heating treatment. Enzyme Microb Technol 69: 10-18. doi: 10.1016/j.enzmictec.2014.11.002 Ma H, Huang L, Jia J, He R, Luo L, Zhu W (2011) Effect of energy-gathered ultrasound on Alcalase. Ultrason Sonochem 18: 419-424. doi: 10.1016/j.ultsonch.2010.07.014 Manasseh R, Tho P, Ooi A, Petrovic-Duran K, Zhu Y (2010) Cavitation microstreaming and material transport around microbubbles. Phys Procedia 3: 427-432. Manasseh R, Tho P, Ooi A, Petrovic-Duran K, Zhu Y (2010) Cavitation microstreaming and material transport around microbubbles. Phys Procedia 3: 427-432. doi: 10.1016/j.phpro.2010.01.056 Mason T J, Cobley A J, Graves J E, Morgan D (2011) New evidence for the inverse dependence of mechanical and chemical effects on the frequency of ultrasound. Ultrason Sonochem 18: 226-230. doi: 10.1016/j.ultsonch.2010.05.008 Mazinani S A, DeLong B, Yan H (2015) Microwave radiation accelerates trypsin-catalyzed peptide hydrolysis at constant bulk temperature. Tetrahedron Lett 56: 5804-5807. Mazotto A M, Melo A C N, Macrae A, Rosado A S, Peixoto R, Cedrola S M L, Couri S, Zingali R B, Villa A L V, Rabinovitch L, Chaves J Q, Vermelho A B (2011) Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp. World J Microbiol Biotechnol 27: 1355-1365. doi: 10.1007/s11274-010-0586-1 References Paul T, Das A, Mandal A, Halder S K, Jana A, Maity C, DasMohapatra P K, Pati B R, Mondal K C (2014) An efficient cloth cleaning property of a crude keratinase combined with detergent: Towards industrial viewpoint. J Clean Prod 66: 672-684. References doi: 10.1016/j.tetlet.2015.09.003 Page 30/36 Mazotto A M, Melo A C N, Macrae A, Rosado A S, Peixoto R, Cedrola S M L, Couri S, Zingali R B, Villa A L V, Rabinovitch L, Chaves J Q, Vermelho A B (2011) Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp. World J Microbiol Biotechnol 27: 1355-1365. doi: 10.1007/s11274-010-0586-1 Mazotto A M, Melo A C N, Macrae A, Rosado A S, Peixoto R, Cedrola S M L, Couri S, Zingali R B, Villa A L V, Rabinovitch L, Chaves J Q, Vermelho A B (2011) Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp. World J Microbiol Biotechnol 27: 1355-1365. doi: 10.1007/s11274-010-0586-1 Mazotto A M, Couri S, Damaso M C T, Vermelho A B (2013) Degradation of feather waste by Aspergillus niger keratinases: Comparison of submerged and solid-state fermentation. Int Biodeterior Biodegrad 85: 189-195. doi: 10.1016/j.ibiod.2013.07.003 McClements D J (1995) Advances in the application of ultrasound in food analysis and processing. Trends Food Sci Tech 6: 293-299. doi: 10.1016/S0924-2244(00)89139-6 Mulinari J, Venturin B, Sbardelotto M, Dall Agnol A, Scapini T, Camargo A F, Baldissarelli D P, Modkovski T A, Rossetto V, Dalla Rosa C, Reichert Jr. F W, Golunski S M, Vargas G D L P, Dalla Rosa C, Mossi A J, Treichel H (2017) Ultrasound-assisted hydrolysis of waste cooking oil catalyzed by homemade lipases. Ultrason Sonochem 35: 313-318. doi: 10.1016/j.ultsonch.2016.10.007 Okoroma E A, Garelick H, Abiola O O, Purchase D (2012) Identification and characterisation of a Bacillus licheniformis strain with profound keratinase activity for degradation of melanised feather. Int Biodeterior Biodegrad 74: 54-60. doi: 10.1016/j.ibiod.2012.07.013 Onifade A A, Al-Sane N A, Al-Musallam A A, Al-Zarban S (1998) A Review: Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresour Technol 66: 1-11. doi: 10.1016/S0960- 8524(98)00033-9 Ovsianko S L, Chernyavsky E A, Minchenya V T, Adzerikho I E, Shkumatov V M (2005) Effect of ultrasound on activation of serine proteases precursors. Ultrason Sonochem 12: 219-223. doi: 10.1016/j.ultsonch.2003.10.012 Page 31/36 Paul T, Das A, Mandal A, Halder S K, Jana A, Maity C, DasMohapatra P K, Pati B R, Mondal K C (2014) An efficient cloth cleaning property of a crude keratinase combined with detergent: Towards industrial viewpoint. J Clean Prod 66: 672-684. doi: 10.1016/j.jclepro.2013.10.054 Potapovich M V, Eremin A N, Metelitza D I (2003) Kinetics of Catalase Inactivation Induced by Ultrasonic Cavitation. Appl Biochem Micro 39: 140-146. doi: 10.1023/A:1022577611056 Preczeski K P, Kamanski A B, Scapini T, Camargo A F, Modkoski T A, Rossetto V, Venturin B, Mulinari J, Golunski S M, Mossi A J, Treichel H (2018) Efficient and low-cost alternative of lipase concentrating aiming at the application in the treatment of waste cooking oils. Bioprocess Biosyst Eng 41: 851-857. doi: 10.1007/s00449-018-1919-y Preczeski K P, Dalastra C, Czapela F F, Kubeneck S, Scapini T, Camargo A F, Zanivan J, Bonatto C, Stefanski F S, Venturin B, Fongaro G, Treichel H (2020) Fusarium oxysporum and Aspergillus sp. as keratinase producers using swine hair from agroindustrial residues. 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Available in: https://www.sigmaaldrich.com/catalog/product/sigma/k4519?lang=pt&region=BR>. Accessed 28 May 2018. SIGMA-ALDRICH. K4519 SIGMA Keratinase. Available in: Su C, Gong J, Zhang R, Tao L, Dou W, Zhang D, Li H, Lu Z, Xu Z, Shi J (2017) A novel alkaline surfactant- stable keratinase with superior feather-degrading potential based on library screening strategy. Int J Biol Macromol 95: 404-411. doi: 10.1016/j.ijbiomac.2016.11.045 Thankaswamy S R, Sundaramoorthy S, Palanivel S, Ramudu K N (2018) Improved microbial degradation of animal hair waste from leather industry using Brevibacterium luteolum (MTCC 5982). J Clean Prod 189: 701-708. doi: 10.1016/j.jclepro.2018.04.095 Wang J, Yanping C, Sun B, Wang C, Mo Y (2011) Effect of ultrasound on the activity of alliinase from fresh garlic. Ultrason Sonochem 18: 534-540. Wang J, Yanping C, Sun B, Wang C, Mo Y (2011) Effect of ultrasound on the activity of alliinase from fresh garlic. Ultrason Sonochem 18: 534-540. doi: 10.1016/j.jclepro.2013.10.054 doi: 10.1016/j.ultsonch.2010.09.008 doi: 10.1016/j.ultsonch.2010.09.008 Wang F, Chen Z, Zhu H (2013) An efficient enzymatic modification of lily polysaccharide in ionic liquid under ultrasonic irradiation. Biochem Eng J 79: 25-28. doi: 10.1016/j.bej.2013.06.020 Yadav G D, Borkar I V (2009) Kinetic and Mechanistic Investigation of Microwave-Assisted Lipase Catalyzed Synthesis of Citronellyl Acetate. Ind Eng Chem Res 48: 7915-7922. doi: 10.1021/ie800591c Page 33/36 Equations For equations please see the equations PDF in the supplementary file section. For equations please see the equations PDF in the supplementary file section. Page 33/36 Yang C, Fang T J (2015) Kinetics for enzymatic hydrolysis of rice hulls by the ultrasonic pretreatment with a bio-based basic ionic liquid. Biochem Eng J 100: 23-29. Yang C, Fang T J (2015) Kinetics for enzymatic hydrolysis of rice hulls by the ultrasonic pretreatment with a bio-based basic ionic liquid. Biochem Eng J 100: 23-29. doi: 10.1016/j.bej.2015.04.012 doi: 10.1016/j.bej.2015.04.012 Yusof N S M, Ashokkumar M (2015) Sonochemical Synthesis of Gold Nanoparticles by Using High Intensity Focused Ultrasound. ChemPhysChem 16: 775-781. doi: 10.1002/cphc.201402697 Yusuf I, Ahmad S A, Phang L Y, Syed M A, Shamaan N A, Khalil K A, Dahalan F A, Shukor M Y (2016) Keratinase production and biodegradation of polluted secondary chicken feather wastes by a newly isolated multi heavy metal tolerant bacterium-Alcaligenes sp. AQ05-001. J Environ Manage 183: 182-195. doi: 10.1016/j.jenvman.2016.08.059 Zou S, Wang X, Chen Y, Wan H, Feng Y (2016) Enhancement of biogas production in anaerobic co- digestion by ultrasonic pretreatment. Energ Convers Manage 112: 226-235. doi: 10.1016/j.enconman.2015.12.087 Highlights The macropoint causes greater energy dissipation in less time; The macropoint causes greater energy dissipation in less time; Swine hair without pretreatment is more efficient in terms of residue d Reaction systems are promising in terms of increasing enzyme activity; The enzyme concentration technique increases enzymatic activity; Homemade enzymes have great potential for degradation of keratinous residues Figures Page 34/36 Page 34/36 Page 34/36 Page 34/36 Figure 1 Figure 1 Figure 2 Total degradation of chicken feathers observed in crude enzymatic extracts and after exposure to reaction systems. A) Control; B) Crude; C) Concentrated; D) K4519 SIGMA commercial enzyme. Total degradation of chicken feathers observed in crude enzymatic extracts and after exposure to reaction systems. A) Control; B) Crude; C) Concentrated; D) K4519 SIGMA commercial enzyme. Figure 1 Larger increments of enzymatic activity of enzymatic extracts obtained from swine hair pretreated using the ultrasonic probe and without pretreatment subjected to the reaction systems ultrasonic probe, ultrasonic bath and microwave. Page 35/36 Page 35/36 Figure 2 Total degradation of chicken feathers observed in crude enzymatic extracts and after exposure to reaction systems A) Control; B) Crude; C) Concentrated; D) K4519 SIGMA commercial enzyme Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. GraphicalAbstract.docx Equations.pdf Page 36/36
https://openalex.org/W3175649750	https://www.medrxiv.org/content/medrxiv/early/2021/06/27/2021.06.20.21259178.full.pdf	English	null	Taiwan on track to end third COVID-19 community outbreak	medRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	9,793	. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Introduction The ﬁrst outbreak started a little before February 16th and ended April 11th, 2020 (54 days), see Figure 1a. The second outbreak started a little before January 12th and ended February 9th, 2021 (27 days). The third outbreak started a little before April 20th and is still ongoing (56 days), but shows clear signs of having peaked on May 28th. The record of 721 conﬁrmed locally or unknown acquired infections in a day was reported on May 22nd. Between the ﬁrst and second outbreak Taiwan saw a period with 273 days without any reported local infection, excluding an isolated case of unknown source reported August 2nd and a local case reported December 22nd. Between the second and third outbreak Taiwan had 69 days without any reported local infection. Abstract Since the start of the COVID-19 pandemic on December 31st, 2019, with the World Health Organization being notiﬁed of pneumonia of unknown cause in Wuhan (China), Taiwan has successfully ended two COVID-19 community outbreaks. For 19 days, the third community outbreak has now been successfully suppressed, putting Taiwan on path to end it too around Aug. 16th based on our forecast using an exponential model. Since May 28th the 7-day average of reported conﬁrmed infected, which peaked at 593, has been falling to 204 on June 16th and the 7-day average of reported suspected and excluded cases increased to above 25 000. Resulting in a decrease in the ratio of the 7-day average of local & unknown conﬁrmed to suspected cases–the identiﬁed control variable–to less than one third of its peak value. The later is a hallmark of working contact tracing, which together with testing and isolation of infected are the keys to ending the community outbreak. Taiwan on track to end third COVID-19 community outbreak Torbj¨orn E. M. Nordling and Yu-Heng Wu Department of Mechanical Engineering, National Cheng Kung University, No. 1 University Road, Tainan City 701, Taiwan s preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. Results and Discussion From June 2020 until the current outbreak, life in Taiwan was pre-pandemic, except for the mandatory mask and temperature checks in the public transportation introduced on March 31st1 and at large events. Large events with tens of thousands of visitors have been held, such as the Mayday New Year’s Eve pop concert at Taoyuan International Baseball Stadium on December 1 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint 31st2. Both the 2nd and 3rd outbreak involved the alpha variant (B.1.1.7), which Hsu et al.3 estimated to have a 1.44-fold higher infection probability and 57% higher basic reproduction number based on household transmissions during the 1st and 2nd outbreak, in agreement with previous estimates of 43-90%4. Evidently, the measures in place to reduce transmissibility and prevent a large scale outbreak were not suﬃcient, so Taiwan should have had a surge in cases if community transmission existed during the two periods without local infections. In addition, the National Health Insurance Administration (NHIA) has proactively been seeking out patients with severe respiratory symptoms in its database and allowing all hospitals, clinics, and pharmacies to see the patients’ travel history obtained from the National Immigration Agency from February 18th, 2020 onwards5. The current outbreak has been traced to a cluster of infected China Airline pilots and the Novotel at Taoyuan International Airport violating the quarantine rules by housing quarantined ﬂight crews and local guests in the same building6,7. This initial failure of the mandatory quarantine at the boarder and contact tracing during the current outbreak combined with the success of contact tracing during the 2nd outbreak and the transmissibility reduction measures remaining the same, implies that Taiwan’s success is due to border control and tracing of contacts upon suspicion. Thus we can ﬁnally with conﬁdence say that Taiwan has ended two community outbreaks thanks to contact tracing, testing, and isolation. Results and Discussion y g, g, The Taiwanese COVID-19 control strategy implemented by the Central Epidemic Command Center (CECC), activated on January 20th, 20205, is based on six main pilars: (a) border control with quarantine upon arrival, (b) self-health monitoring when having visited a place with known cases, (c) testing when showing symptoms and seeking medical care, (d) mandatory supervised quarantine of conﬁrmed infected and individuals at high-risk of having been infected, (e) contact tracing, and (f) a four-level system of measures to suppress community spreading. These are devised to control the eﬀective reproduction number, i.e. the expected number of people an infected individual will transmit the disease to while infectious. The reproduction number can be seen as the transmission risk per contact (transmissibility), times the number of contacts per day, times the number of days the person is infectious. From June 7th until May 11th no restrictions on the size of gatherings, i.e. curbing of the number of contacts, existed8,7. The current level 3 measures in place since May 19th, consist of mandatory wearing of masks at all times outside private spaces and social distancing, which reduce the transmissibility; indoor gatherings limited to ﬁve people and closure of certain businesses, partly including schools and preschools, which reduce the number of contacts; and mandatory COVID-19 testing in areas where community transmission has taken place, which reduces the number of days an infected person can transmit the disease before being quarantined9,10. As demonstrated by numerous countries, such as the United Kingdom and United States, community suppression measures, such as lockdowns, alone do not end a community outbreak. As soon as these non-pharmacological interventions (NPIs) are lifted the reproduction number will increase again, so why do we claim that Taiwan is on track to ending this community outbreak? In short, the ratio of the 7-day moving average of conﬁrmed infected to suspected has already fallen to less than one third of the peak value of 0.029 on May 28th, while the number of daily suspected has increased to above 25 000, and the ratio of the 7-day moving average of excluded to suspected is slightly above one, which is needed to resolve the backlog from when it was down at 0.37. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Results and Discussion In other words, the contact tracing, testing, and isolation of infected has, after initial failure and challenges, successfully been scaled up to the required volume of around 25 000 suspected per day and the number of conﬁrmed infected is decreasing. The same pattern has been repeated during all three outbreaks: ﬁrst the number of conﬁrmed infected increase rapidly and the contact tracing, testing, and exclusion of cases lags, then focus on contact tracing brings up the number of suspected and investment into expanding testing clears the backlog, see Figure 1b-g. Thus the key to ending an outbreak is to focus on contact tracing to bring up 2 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint the number of suspected and increase testing and isolation/care capacity. The focus on contact tracing and bringing up the number of suspected is key because one need to prevent every infected person from infecting a new person to end the outbreak. It makes the ratio of conﬁrmed infected to suspected the essential metric to follow. The Lancet Commission Task Force on public health measures to suppress the pandemic also report on the eﬀectiveness of contact tracing and quarantine measures, albeit not on its use to end outbreaks11. Since May 19th, Taiwan has implemented an SMS text message contact tracing system based on each person upon entry in each business scanning a place speciﬁc QR code that generates a unique SMS sent to 1922–the Taiwan Center for Disease Control hotline12, which helps scale the contact tracing. This data tells us that the contact tracing, testing, and isolation capacity has been scaled to the required level. Therefore, it is not a question of if the outbreak can be ended but a question of when it will be ended. Results and Discussion It is a matter of keeping up the work until this outbreak also is brought to an end. To keep up the work requires will–will from everyone to contribute to the contact tracing, self-health monitoring, testing, and caring of each other. To have will, one need to believe that it can be done, therefore it is vital to get this message to everyone: Together we can and will also end this outbreak. The two extensions of the level 3 measures have made some ask if vaccination is necessary to end this outbreak? Of the only three countries, with a population over 1 million, that have vaccinated over half their population, both Bahrain and Mongolia in the past week reported over 5 000 conﬁrmed infected, while Israel reported a mere 117 infected13. Actually, both Bahrain and Mongolia have set new records in the weekly number of conﬁrmed infected within the previous three weeks. Israel only recently, on June 1st, lifted most restrictions, including limitations on the number of people at gatherings and proof of vaccination to enter e.g. gyms, theatres, hotels, and synagogues, and have kept border controls14. In a recent simulation study, Moghadas et al.15 predict that if all unvaccinated individuals in the United States revert to pre-pandemic life on July 4th, then it would result in the worst daily incidence of COVID-19 cases despite assuming close to 70% being vaccinated, but if only the vaccinate individuals revert, then it result in a minimal increase even if they revert two weeks after the ﬁrst dose. Based on the recent estimate of the basic reproduction number without NPIs in Taiwan during the second outbreak at 6.73, 85% of the Taiwanese population would need to be vaccinated to reach herd immunity. Considering the diﬃculties the Taiwanese government has faced in purchasing COVID-19 vaccines, it is unlikely that Taiwan could implement a population scale vaccination program and reach herd immunity any faster than other nations. A population wide vaccination campaign is likely to take a year before it could end this community outbreak, while the proven method of contact tracing, testing, and isolation is likely to end it around Aug. 16th. We forecast this end date based on ﬁtting an exponential model to the daily ratios of the 7-day moving average of conﬁrmed infected to suspected from the peak of 0.029 on May 28th to 0.0077 on June 16th. Results and Discussion The calculated non- simultaneous prediction bounds on a new observation with 95% conﬁdence puts the earliest end date on July 2nd and the latest indeﬁnitely into the future, see Figure 2. The predicted half- time is 9.9 days (95% conﬁdence interval: 9.1-10.8). We therefore encourage all Taiwanese to be hopeful and remain vigilant; the end around Aug. 16th is only two months away. Methods To limit the inﬂuence of reporting diﬀerences between the week days and ﬂuctuations in when cases are reported to the Taiwan CDC, we use the 7-day moving mean calculated over the past seven days. It is worth noting that our moving mean cover the mean serial interval, which was estimated to 6.2 days among the 42 infector-infectee pairs during the 1st outbreak18. To forecast when the current third community outbreak will end, we assumed that the de- crease in the ratio of local & unknown conﬁrmed to suspected cases can be described as an exponential growth process. More precisely, we assumed the model f(x) = a · 2−b·x, (1) (1) where a is the initial value and the reciprocal of b is the half-time. We ﬁtted this model to the ratio of the 7-day moving mean of local & unknown conﬁrmed to the 7-day moving mean of suspected cases from the peak on 2021-05-28 to 2021-06-16, i.e. 19 time points, using the Curve Fitting Toolbox in Matlab R2015b by MathWorks using the ”NonlinearLeastSquares” estimator. The root mean squared error of the model ﬁtted to the data points was 0.0011 and adjusted R- squared 0.97, indicating a good ﬁt that explains the data well. The estimated parameters are a 0.029 (95% conﬁdence bounds 0.028 - 0.030) and b 0.10 (95% conﬁdence bounds 0.09 - 0.11). The later corresponds to a halving of the ratio every 9.9 days (95% conﬁdence bounds 9.1 - 10.8 days). The previous two outbreaks have ended after the ratio has been brought down to 1.2 · 10−4, see Figure 2, so we assume that the same is required to end this outbreak. Note that the length of the window of our moving mean and the baseline number of suspected aﬀects this ratio. When only one conﬁrmed infected remains, the nominator will be 0.14, i.e. 1/7, and the denominator then need to be around 1200, i.e. on average 1200 suspected each day for the last conﬁrmed infected. The mean 7-day moving mean number of suspected between the 1st and 2nd outbreak was 324 and between the 2nd and 3rd outbreak it was 684. By extrapolation we forecast that this ratio would be reached on 2021-08-16 after required six halvings. This forecast depends on the contact tracing, testing, and isolation being equally eﬀective as during the past three weeks and no new local clusters emerging from people infected abroad. Funding This study was supported by Ministry of Science and Technology, Taiwan (MOST 109-2224-E- 006-003, 109-2740-B-006-001). The funding has covered a stipend to Yu-Heng Wu and general lab expenses. The funding institution has not been involved in this study. Methods The calculated non-simultaneous prediction bounds on a new observation with 95% conﬁdence, drawn as black dashed lines in Figure 2, puts the earliest end date on July 2nd and the latest indeﬁnitely into the future. The later in agreement with a failure to execute the contact tracing, testing, and isolation or new local clusters emerging from people infected abroad. Conclusion As soon as this outbreak is ended, we hope the world would take notice and learn from the Taiwanese strategy, so unnecessary suﬀering and deaths can be avoided. Like Wang et al.5, we think “Taiwan is an example of how a society can respond quickly to a crisis and protect the interests of its citizens.” 3 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Acknowledgements The authors acknowledges valuable discussions, help with presentation, and comments from Dr Akram Ashyani, Dipesh Dhayfule, and Jose Chang. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint The authors have no conﬂicts of interest relevant to this article. The authors have no conﬂicts of interest relevant to this article. Data All COVID-19 data were collected from the Taiwan Centers for Disease Control (Taiwan CDC), which provided an online platform for downloading datasets, including respiratory syndrome coronavirus-2 (SARS-CoV-2) infection data16. Both data downloaded from the online platform and manually extracted from their News Bulletins17 were used. All data used is for the readers convenience also included in the Appendix. Disclosure statement The authors have nothing to disclose. 4 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Contribution statement TEMN did conceptualisation, funding acquisition, methodology, project administration, re- sources, supervision, and writing of the original draft. YHW did investigation, data curation, formal analysis, and software development. Both did data validation, visualisation, and review & editing of the ﬁnal manuscript. Bibliography [1] Hsiao YH, Hetherington W. Virus Outbreak: Masks required for train passengers. Taipei, Taiwan: The Taipei Times; 2020. Available from: https://www.taipeitimes.com/News/ taiwan/archives/2020/04/01/2003733786. [2] Taiwan News. 5 Taiwanese attend Mayday concert despite self-monitoring for COVID. Taipei, Taiwan: Taiwan News; 2021. Available from: https://www.taiwannews.com.tw/ en/news/4091789. [3] Hsu CY, Wang JT, Huang KC, Fan ACH, Yeh YP, Chen SLS. Household transmission but without the community-acquired outbreak of COVID-19 in Taiwan. Journal of the Formosan Medical Association = Taiwan yi zhi. 2021 may;. [4] Davies NG, Abbott S, Barnard RC, Jarvis CI, Kucharski AJ, Munday JD, et al. Esti- mated transmissibility and impact of SARS-CoV-2 lineage B. 1.1. 7 in England. Science. 2021;372(6538). [5] Wang CJ, Ng CY, Brook RH. Response to COVID-19 in Taiwan. JAMA. 2020 apr;323(14):1341. Available from: https://jamanetwork.com/journals/jama/ fullarticle/2762689. [6] Zennie M. How a False Sense of Security, and a Little Secret Tea, Broke Down Taiwan’s COVID-19 Defenses. Taipei, Taiwan: Time; 2021. Available from: https://time.com/ 6050316/taiwan-covid-19-outbreak-tea/. [7] Yeh J. CORONAVIRUS/Worried by rise in COVID-19 cases, Taiwan bans large scale gatherings. Taipei, Taiwan: Central News Agency; 2021. Available from: https: //focustaiwan.tw/society/202105110007. 5 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint [8] Lee IC. Virus Outbreak: CECC urges vigilance as curbs eased. Taipei, Taiwan: The Taipei Times; 2020. Available from: https://www.taipeitimes.com/News/front/archives/ 2020/06/08/2003737829. [9] Chiang Hc, Chen Cl, Yeh J. CORONAVIRUS/Level 3 COVID-19 alert introduced across Taiwan (Update). Taipei, Taiwan: Central News Agency; 2021. Available from: https: //focustaiwan.tw/society/202105190017. [10] Everington K. Taiwan’s 4 epidemic warning levels. Taipei, Taiwan: Taiwan News; 2021. Available from: https://www.taiwannews.com.tw/en/news/4203460. [11] Lee JK, Bullen C, Ben Amor Y, Bush SR, Colombo F, Gaviria A, et al. Institutional and behaviour-change interventions to support COVID-19 public health measures: a review by the Lancet Commission Task Force on public health measures to suppress the pandemic. International health. 2021 may;. [12] Hu C. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Bibliography How to use SMS contact tracing system, powered by Audrey Tang, to enter every store; 2021. Last visited on 2021-06-15. Available from: https://meet-global.bnext. com.tw/articles/view/47383. [13] Johns Hopkins University of Medicine CRC. Understanding vaccination progress; 2021. Last visited on 2021-06-15. Available from: https://coronavirus.jhu.edu/vaccines/ international. [14] Staﬀ T. Back to normal: Israel lifts nearly all COVID restraints as virus fades away. Jerusalem, Israel: The Times of Israel; 2021. Available from: https://www.timesofisrael.com/back-to-normal-israel-lifts-nearly-all- covid-restraints-as-virus-fades-away/. [15] Moghadas SM, Sah P, Vilches TN, Galvani AP. Can the USA return to pre-COVID-19 normal by July 4? The Lancet Infectious Diseases. 2021 jun;Available from: https:// linkinghub.elsevier.com/retrieve/pii/S1473309921003248. [16] Taiwan Centers of Disease Control. Taiwan Centers of Disease Control - Datasets; 2021 Last visited on 2021-06-17. Available from: https://data.cdc.gov.tw/en/dataset. [17] Taiwan Centers of Disease Control. Taiwan Centers of Disease Control - News Bulletin; 2021. Last visited on 2021-06-17. Available from: https://www.cdc.gov.tw/Bulletin/ List/MmgtpeidAR5Ooai4-fgHzQ. [18] Chen YC, Yang HP, Li HC, Huang PY, Chen CL, Chiu CH. Features and transmission dynamics of SARS-CoV-2 superspreading events in Taiwan: Implications for eﬀective and sustainable community-centered control. Pediatrics and neonatology. 2021 may;. 6 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint 2020-01-27 2020-02-24 2020-03-23 2020-04-20 2020-05-18 2020-06-15 2020-07-13 2020-08-10 2020-09-07 2020-10-05 2020-11-02 2020-11-30 2020-12-28 2021-01-25 2021-02-22 2021-03-22 2021-04-19 2021-05-17 2021-06-14 Date 10 1 100 101 102 103 No. local & unknown confirmed cases a 7-day moving average Daily number 0.8 1.0 1.2 1.4 Ratio of excluded to suspected cases 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 Ratio of local & unknown confirmed to suspected cases 16/2 11/4 09/3 16/3 23/3 30/3 06/4 b 250 500 750 1000 1250 1500 No. suspected cases 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 No. Bibliography local & unknown confirmed cases 16/2 11/4 17/2 24/2 02/3 09/3 16/3 23/3 30/3 06/4 e 0.8 1.0 1.2 1.4 1.6 Ratio of excluded to suspected cases 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 0.0012 0.0014 12/1 09/2 13/1 20/1 27/1 03/2 c 250 500 750 1000 1250 1500 1750 No. suspected cases 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 12/1 09/2 13/1 20/1 27/1 03/2 f 0.4 0.6 0.8 1.0 1.2 1.4 Ratio of excluded to suspected cases 0.000 0.005 0.010 0.015 0.020 0.025 0.030 20/4 16/6 26/4 03/5 10/5 17/5 24/5 31/5 07/6 14/6 d 0 5000 10000 15000 20000 25000 30000 No. suspected cases 0 100 200 300 400 500 600 20/4 16/6 17/5 24/5 31/5 07/6 14/6 g Figure 1: Reported number of local and unknown infected and 7-day moving average in Taiwa from the ﬁrst local case 2020-01-28 until 2021-06-16 (a). The three community outbreaks ar marked in blue (1st), green (2nd), and red (3rd). The cases between are not connected. The rat of local and unknown conﬁrmed to suspected cases versus ratio of excluded to suspected case reported for the three outbreaks (b-d). Number of local and unknown conﬁrmed cases versu number of suspected cases reported for the three outbreaks (e-g). The 7-day moving average each quantity is used in b-g. 7 2020-01-27 2020-02-24 2020-03-23 2020-04-20 2020-05-18 2020-06-15 2020-07-13 2020-08-10 2020-09-07 2020-10-05 2020-11-02 2020-11-30 2020-12-28 2021-01-25 2021-02-22 2021-03-22 2021-04-19 2021-05-17 2021-06-14 10 1 100 101 102 103 No. local & unknown confirmed cases a 7-day moving average Daily number b d a 020-01-27 020-02-24 020-03-23 020-04-20 020-05-18 020-06-1 0.8 1.0 1.2 1.4 Ratio of excluded to suspected cases 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 Ratio of local & unknown confirmed to suspected cases 16/2 11/4 09/3 16/3 23/3 30/3 06/4 b 0 0 0 0 0 0 0 0 07-13 020-08-10 020-09-07 020-10-05 020-11-02 020-11-30 02 Date 0.8 1.0 1.2 1.4 1.6 Ratio of excluded to suspected cases 0 2 4 6 8 0 2 4 12/1 09/2 13/1 20/1 27/1 03/2 c 2-28 1-25 2-22 3-22 4-19 5-17 6-1 0.4 0.6 0.8 1.0 1.2 1.4 Ratio of excluded to suspected cases 0.000 0.005 0.010 0.015 0.020 0.025 0.030 20/4 16/6 26/4 03/5 10/5 17/5 24/5 31/5 07/6 14/6 d b d Ratio of excluded to suspected cases 250 500 750 1000 1250 1500 1750 No. Bibliography suspected cases 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 12/1 09/2 13/1 20/1 27/1 03/2 f p 250 500 750 1000 1250 1500 No. suspected cases 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 No. local & unknown confirmed cases 16/2 11/4 17/2 24/2 02/3 09/3 16/3 23/3 30/3 06/4 e 0 0 5000 10000 15000 20000 25000 30000 No. suspected cases 0 100 200 300 400 500 600 20/4 16/6 17/5 24/5 31/5 07/6 14/6 g g No. suspected cases Figure 1: Reported number of local and unknown infected and 7-day moving average in Taiwan from the ﬁrst local case 2020-01-28 until 2021-06-16 (a). The three community outbreaks are marked in blue (1st), green (2nd), and red (3rd). The cases between are not connected. The ratio of local and unknown conﬁrmed to suspected cases versus ratio of excluded to suspected cases reported for the three outbreaks (b-d). Number of local and unknown conﬁrmed cases versus number of suspected cases reported for the three outbreaks (e-g). The 7-day moving average of each quantity is used in b-g. 7 7 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. r review) The copyright holder for this preprint this version posted June 27, 2021. ; 10.1101/2021.06.20.21259178 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Bibliography ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint 2020-01-20 2020-02-17 2020-03-16 2020-04-13 2020-05-11 2020-06-08 2020-07-06 2020-08-03 2020-08-31 2020-09-28 2020-10-26 2020-11-23 2020-12-21 2021-01-18 2021-02-15 2021-03-15 2021-04-12 2021-05-10 2021-06-07 2021-07-05 2021-08-02 2021-08-30 Date 10-5 10-4 10-3 10-2 10-1 Ratio of local & unknown confirmed to suspected cases f(x) = 0.03"2-0.10"x, adj R2 0.97 2020-01-20 2020-02-17 2020-03-16 2020-04-13 2020-05-11 2020-06-08 2020-07-06 2020-08-03 2020-08-31 2020-09-28 2020-10-26 2020-11-23 2020-12-21 2021-01-18 2021-02-15 2021-03-15 2021-04-12 2021-05-10 2021-06-07 2021-07-05 2021-08-02 2021-08-30 Date 10-5 10-4 10-3 10-2 10-1 Ratio of local & unknown confirmed to suspected cases f(x) = 0.03"2-0.10"x, adj R2 0.97 Figure 2: Evolution of the ratio of the 7-day moving mean of local & unknown conﬁrmed to the 7-day moving mean of suspected cases and our forecast of the end of the third outbreak. The three community outbreaks are marked in blue (1st), green (2nd), and red (3rd). The cases between in grey are not connected. 2 2 Date 2 2 Date Figure 2: Evolution of the ratio of the 7-day moving mean of local & unknown conﬁrmed to the 7-day moving mean of suspected cases and our forecast of the end of the third outbreak. The three community outbreaks are marked in blue (1st), green (2nd), and red (3rd). The cases between in grey are not connected. 8 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Appendix Here we provide details on the data collection, cleaning, and analysis to enable veriﬁcation and reproduction of the results. Data collection All data is publicly available and was collected from the Taiwan Centers for Disease Con- trol (Taiwan CDC). We collected the cumulative number of conﬁrmed infected with SARS- CoV-2 with a know local source and unknown source, as well as the cumulative reported suspected and excluded cases, from the Taiwan CDC News Bulletins, available in Chinese at https://www.cdc.gov.tw/Bulletin/List/MmgtpeidAR5Ooai4-fgHzQ (last visited 2021-06- 17). Unfortunately, these news bulletins have not been issued every day, nor at the same time of the day through out the pandemic, which means that the daily increase derived by subtracting the value in the previous report is inﬂuenced by the varying time between these reports. The way of counting the number of suspected and excluded was changed on 2020-03-06, which contain a large correction to these cumulative numbers. We therefore also downloaded the ”Daily Num- ber of Cases Suspected SARS -CoV-2 Infection Tested” dataset from the Taiwan CDC Open Data portal https://data.cdc.gov.tw/en/dataset/daily-cases-suspected-sars-cov-2- infection_tested (last visited 2021-06-17). This dataset contains the daily number of cases suspected to have SARS-CoV-2 from three sources: (i) notiﬁcations of infectious diseases, (ii) home quarantine and inspection, and (iii) expanded monitoring, as well as the total daily number. Data cleaning and preprocessing cases CDC News Bulletin suspected CDC Open Data suspected CDC News Bulletin (corrected) suspected 7-day mean of News (corrected) suspected 7-day mean of Open suspected 7-day mean of News excluded 7-day mean of Open (estimated) excluded Figure 3: Comparison of the change in number of suspected between CDC News Bulletins (red circles), the total daily number of suspected in the CDC Open Data (blue x), and the change in number of suspected between CDC News Bulletins corrected for the number of days in between (red dot), and the 7-day moving mean of the later two (blue and red lines), and the 7-day moving mean of the change in number of excluded between CDC News Bulletins corrected for the number of days in between (magenta line), and the 7-day moving mean of our estimate of the daily number of excluded based on the total daily number of suspected in the CDC Open Data (cyan line). 2020-01-20 2020-02-17 2020-03-16 2020-04-13 2020-05-11 2020-06-08 2020-07-06 2020-08-03 2020-08-31 2020-09-28 2020-10-26 2020-11-23 2020-12-21 2021-01-18 2021-02-15 2021-03-15 2021-04-12 2021-05-10 2021-06-07 Date 10-1 100 101 102 103 104 105 No. cases CDC News Bulletin suspected CDC Open Data suspected CDC News Bulletin (corrected) suspected 7-day mean of News (corrected) suspected 7-day mean of Open suspected 7-day mean of News excluded 7-day mean of Open (estimated) excluded Date Figure 3: Comparison of the change in number of suspected between CDC News Bulletins (red circles), the total daily number of suspected in the CDC Open Data (blue x), and the change in number of suspected between CDC News Bulletins corrected for the number of days in between (red dot), and the 7-day moving mean of the later two (blue and red lines), and the 7-day moving mean of the change in number of excluded between CDC News Bulletins corrected for the number of days in between (magenta line), and the 7-day moving mean of our estimate of the daily number of excluded based on the total daily number of suspected in the CDC Open Data (cyan line). consecutive News Bulletins and the change is therefore larger than expected from one day to the next. We have veriﬁed that the cumulative number of excluded always remain smaller than the cumulative number of suspected. Data cleaning and preprocessing We ﬁrst removed the ﬁrst CDC News Bulletin for the days when more than one was issued, thus keeping the later report with the larger cumulative numbers. The we corrected for the number of days in between the report for all cases with more than one day in between by evenly distributing the change in cumulative number over the days in between and ensuring that the cumulative sum equals the reported one. Due to the change in the way of counting the number of suspected and excluded on 2020-03-06, the number of suspected cases is unreasonably low before it and a jump correcting for the change occur on that day. These artefacts are clearly seen in Figure 3 as a dip in the number of suspected before and a value slightly above 10 000 on this day. These artefacts clearly inﬂuence the 7-day moving mean of the number of suspected that we need for getting a stable estimate of the ratio of conﬁrmed infected to suspected cases. The total daily number of suspected cases in the CDC Open Data portals dataset does not contain this error, so we decided to use it instead. Since the number of excluded cases only was available from in the CDC News Bulletins and contain a similar change in the way fo counting on 2020-03-06, we assumed that the ratio of excluded to suspected was correct and used the ratio to estimate the number of daily excluded based on the total number of daily suspected from the CDC Open Data dataset. To ensure that the cumulative sum of daily excluded matched the reported values in the News Bulletins after March 6th, we proportionally to the daily suspected added or removed excluded cases from the days between two reports iteratively from the later report day to the former to avoid errors from rounding to whole integers later. This resulted in an estimate of the daily total number of excluded cases that on each day with a CDC News Bulletin report match the reported number exactly. The result of our preprocessing is visualised in Figure 3, where the number of suspected cases from the corrected News Bulletin and Open Data closely match after 2020-03-06. Note that a red circle without a dot in it indicate that more than one day had passed between the 9 . Data cleaning and preprocessing CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint 2020-01-20 2020-02-17 2020-03-16 2020-04-13 2020-05-11 2020-06-08 2020-07-06 2020-08-03 2020-08-31 2020-09-28 2020-10-26 2020-11-23 2020-12-21 2021-01-18 2021-02-15 2021-03-15 2021-04-12 2021-05-10 2021-06-07 Date 10-1 100 101 102 103 104 105 No. cases CDC News Bulletin suspected CDC Open Data suspected CDC News Bulletin (corrected) suspected 7-day mean of News (corrected) suspected 7-day mean of Open suspected 7-day mean of News excluded 7-day mean of Open (estimated) excluded Figure 3: Comparison of the change in number of suspected between CDC News Bulletins (red circles), the total daily number of suspected in the CDC Open Data (blue x), and the change in number of suspected between CDC News Bulletins corrected for the number of days in between (red dot), and the 7-day moving mean of the later two (blue and red lines), and the 7-day moving mean of the change in number of excluded between CDC News Bulletins corrected for the number of days in between (magenta line), and the 7-day moving mean of our estimate of the daily number of excluded based on the total daily number of suspected in the CDC Open Data (cyan line). 2020-01-20 2020-02-17 2020-03-16 2020-04-13 2020-05-11 2020-06-08 2020-07-06 2020-08-03 2020-08-31 2020-09-28 2020-10-26 2020-11-23 2020-12-21 2021-01-18 2021-02-15 2021-03-15 2021-04-12 2021-05-10 2021-06-07 Date 10-1 100 101 102 103 104 105 No. Data cleaning and preprocessing Since our analysis if dependent on the 7-day moving mean of suspected and excluded we also evaluated the relative diﬀerence in the 7-day moving mean daily values obtained from the CDC News Bulletin and Open Data, relative to the later. The relative diﬀerence in number of suspected remains below 41% every day after 2020-03-06, below 10% 81% of the days, and below 1% 33% of the days. The large relative diﬀerences occur on separated days with small relative diﬀerences in between and are caused by diﬀerences in on which day extreme numbers of suspected are reported, see Figure 3. This is to be expected, since the Open Data most probably contains all suspected cases on each day, while the News Bulletins contain the cases added since the last bulletin. Thus these diﬀerences may shift values by one day, but no more. We cannot tell which of these two data sources that is more reliable, but considering that the Open Data at least is corrected for the change in the way of calculating the number of suspected we decided to use it and our estimate of the total daily number of excluded based on it. This does not aﬀect the ratio of excluded suspected cases, but it aﬀects the ratio of local & unknown conﬁrmed to suspected cases by up to 41% on individual days, which in practise means that speciﬁc values may be shifted in time by one day. Since our forecast of when the third outbreak will end is based on 19 time points any eﬀects of this diﬀerence is well within the estimated conﬁdence intervals. 10 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint 2020-01-20 2020-02-17 2020-03-16 2020-04-13 2020-05-11 2020-06-08 2020-07-06 2020-08-03 2020-08-31 2020-09-28 2020-10-26 2020-11-23 2020-12-21 2021-01-18 2021-02-15 2021-03-15 2021-04-12 2021-05-10 2021-06-07 Date 10-4 10-3 10-2 10-1 100 101 Relative difference in no. Data cleaning and preprocessing cases 7-day mean of News (corrected) suspected vs. 7-day mean of Open suspected 7-day mean of News excluded vs. 7-day mean of Open (estimated) excluded Figure 4: Relative diﬀerence in the 7-day moving mean of the change in number of suspected between CDC News Bulletins corrected for the number of days in between and the 7-day moving mean of the total daily number of suspected in the CDC Open Data (red line), and the 7-day moving mean of the change in number of excluded between CDC News Bulletins corrected for the number of days in between and the 7-day moving mean of our estimate of the daily number of excluded based on the total daily number of suspected in the CDC Open Data (magenta line). 2020-01-20 2020-02-17 2020-03-16 2020-04-13 2020-05-11 2020-06-08 2020-07-06 2020-08-03 2020-08-31 2020-09-28 2020-10-26 2020-11-23 2020-12-21 2021-01-18 2021-02-15 2021-03-15 2021-04-12 2021-05-10 2021-06-07 Date 10-4 10-3 10-2 10-1 100 101 Relative difference in no. cases 7-day mean of News (corrected) suspected vs. 7-day mean of Open suspected 7-day mean of News excluded vs. 7-day mean of Open (estimated) excluded 7-day mean of News (corrected) suspected vs. 7-day mean of Open suspected 7-day mean of News excluded vs. 7-day mean of Open (estimated) excluded 2 Date Date Figure 4: Relative diﬀerence in the 7-day moving mean of the change in number of suspected between CDC News Bulletins corrected for the number of days in between and the 7-day moving mean of the total daily number of suspected in the CDC Open Data (red line), and the 7-day moving mean of the change in number of excluded between CDC News Bulletins corrected for the number of days in between and the 7-day moving mean of our estimate of the daily number of excluded based on the total daily number of suspected in the CDC Open Data (magenta line). Data used The data we used after cleaning and preprocessing is included in Table 1-4. 11 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Table 1: The data used in this study. Table 1: The data used in this study. Date Susp. Excl. Conf. Date Susp. Excl. Conf Date Susp. Excl. Data used CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Table 2: The data used in this study (continued). Date Susp. Excl. Conf. Date Susp. Excl. Conf Date Susp. Excl. Data used Conf 2020-06-07 108 71 0 2020-07-25 95 85 0 2020-09-11 186 205 0 2020-06-08 129 84 0 2020-07-26 123 112 0 2020-09-12 126 123 0 2020-06-09 152 101 0 2020-07-27 201 182 0 2020-09-13 210 205 0 2020-06-10 152 101 0 2020-07-28 341 143 0 2020-09-14 154 152 0 2020-06-11 135 141 0 2020-07-29 220 320 0 2020-09-15 160 137 0 2020-06-12 142 148 0 2020-07-30 203 232 0 2020-09-16 282 244 0 2020-06-13 118 125 0 2020-07-31 235 271 0 2020-09-17 680 177 0 2020-06-14 111 117 0 2020-08-01 104 120 1 2020-09-18 351 793 0 2020-06-15 145 152 0 2020-08-02 132 196 0 2020-09-19 95 216 0 2020-06-16 147 134 0 2020-08-03 273 178 0 2020-09-20 98 204 0 2020-06-17 146 134 0 2020-08-04 192 126 0 2020-09-21 283 89 0 2020-06-18 135 164 0 2020-08-05 212 215 0 2020-09-22 175 174 0 2020-06-19 159 152 0 2020-08-06 214 182 0 2020-09-23 257 256 0 2020-06-20 114 109 0 2020-08-07 164 200 0 2020-09-24 198 293 0 2020-06-21 104 99 0 2020-08-08 173 212 0 2020-09-25 137 203 0 2020-06-22 156 149 0 2020-08-09 142 194 0 2020-09-26 275 235 0 2020-06-23 126 121 0 2020-08-10 247 230 0 2020-09-27 204 175 0 2020-06-24 173 166 0 2020-08-11 147 137 0 2020-09-28 259 222 0 2020-06-25 217 129 0 2020-08-12 190 178 0 2020-09-29 211 176 0 2020-06-26 176 188 0 2020-08-13 229 235 0 2020-09-30 260 218 0 2020-06-27 154 164 0 2020-08-14 198 204 0 2020-10-01 152 176 0 2020-06-28 127 135 0 2020-08-15 133 137 0 2020-10-02 392 187 0 2020-06-29 184 198 0 2020-08-16 124 180 0 2020-10-03 109 121 0 2020-06-30 210 225 0 2020-08-17 260 116 0 2020-10-04 167 187 0 2020-07-01 141 151 0 2020-08-18 165 216 0 2020-10-05 356 398 0 2020-07-02 129 143 0 2020-08-19 222 204 0 2020-10-06 170 174 0 2020-07-03 140 157 0 2020-08-20 214 236 0 2020-10-07 432 242 0 2020-07-04 119 118 0 2020-08-21 217 239 0 2020-10-08 590 457 0 2020-07-05 151 149 0 2020-08-22 128 142 0 2020-10-09 339 420 0 2020-07-06 177 175 0 2020-08-23 117 111 0 2020-10-10 114 184 0 2020-07-07 136 135 0 2020-08-24 242 230 0 2020-10-11 123 199 0 2020-07-08 141 140 0 2020-08-25 155 147 0 2020-10-12 305 492 0 2020-07-09 188 144 0 2020-08-26 182 173 0 2020-10-13 271 316 0 2020-07-10 165 128 0 2020-08-27 204 210 0 2020-10-14 345 267 0 2020-07-11 118 126 0 2020-08-28 177 183 0 2020-10-15 589 282 0 2020-07-12 126 133 0 2020-08-29 148 153 0 2020-10-16 382 365 0 2020-07-13 126 134 0 2020-08-30 157 166 0 2020-10-17 132 195 0 2020-07-14 155 166 0 2020-08-31 215 229 0 2020-10-18 176 262 0 2020-07-15 164 175 0 2020-09-01 144 153 0 2020-10-19 314 467 0 2020-07-16 191 152 0 2020-09-02 152 162 0 2020-10-20 223 209 0 2020-07-17 145 196 0 2020-09-03 232 181 0 2020-10-21 215 180 0 2020-07-18 111 107 0 2020-09-04 242 189 0 2020-10-22 329 214 0 2020-07-19 135 129 0 2020-09-05 112 186 0 2020-10-23 177 353 0 2020-07-20 161 155 0 2020-09-06 173 204 0 2020-10-24 97 195 0 2020-07-21 143 137 0 2020-09-07 188 91 0 2020-10-25 118 103 0 2020-07-22 156 151 0 2020-09-08 187 221 0 2020-10-26 293 256 0 2020-07-23 163 158 0 2020-09-09 155 185 0 2020-10-27 206 181 0 2020-07-24 135 130 0 2020-09-10 224 146 0 2020-10-28 198 174 0 13 Table 2: The data used in this study (continued). Data used Conf 2020-01-15 0 0 0 2020-03-03 506 466 0 2020-04-20 1835 931 0 2020-01-16 4 2 0 2020-03-04 485 454 1 2020-04-21 1371 1526 0 2020-01-17 0 0 0 2020-03-05 297 283 1 2020-04-22 1154 1469 0 2020-01-18 1 0 0 2020-03-06 381 393 0 2020-04-23 1014 1163 0 2020-01-19 1 0 0 2020-03-07 307 382 0 2020-04-24 827 1132 0 2020-01-20 2 1 0 2020-03-08 298 313 0 2020-04-25 619 1091 0 2020-01-21 12 5 0 2020-03-09 476 359 1 2020-04-26 495 738 0 2020-01-22 44 7 0 2020-03-10 332 376 0 2020-04-27 740 588 0 2020-01-23 70 14 0 2020-03-11 445 395 0 2020-04-28 694 792 0 2020-01-24 57 20 0 2020-03-12 677 685 1 2020-04-29 484 705 0 2020-01-25 91 42 0 2020-03-13 439 454 0 2020-04-30 495 513 0 2020-01-26 117 58 0 2020-03-14 379 405 0 2020-05-01 374 477 0 2020-01-27 123 64 0 2020-03-15 306 341 0 2020-05-02 380 436 0 2020-01-28 95 62 1 2020-03-16 730 388 0 2020-05-03 342 365 0 2020-01-29 106 74 1 2020-03-17 873 600 2 2020-05-04 528 442 0 2020-01-30 100 74 0 2020-03-18 1197 786 1 2020-05-05 632 660 0 2020-01-31 93 82 0 2020-03-19 1363 1095 3 2020-05-06 451 507 0 2020-02-01 87 76 0 2020-03-20 1371 1236 0 2020-05-07 417 457 0 2020-02-02 64 57 0 2020-03-21 1047 880 3 2020-05-08 402 408 0 2020-02-03 131 116 0 2020-03-22 811 823 1 2020-05-09 269 386 0 2020-02-04 103 91 0 2020-03-23 1139 734 0 2020-05-10 267 293 0 2020-02-05 157 147 0 2020-03-24 924 697 1 2020-05-11 362 307 0 2020-02-06 77 73 0 2020-03-25 938 603 2 2020-05-12 349 343 0 2020-02-07 199 191 0 2020-03-26 929 802 0 2020-05-13 277 317 0 2020-02-08 225 221 0 2020-03-27 864 881 2 2020-05-14 274 285 0 2020-02-09 128 121 0 2020-03-28 527 592 1 2020-05-15 323 306 0 2020-02-10 205 195 0 2020-03-29 835 456 1 2020-05-16 220 283 0 2020-02-11 168 162 0 2020-03-30 1023 679 2 2020-05-17 185 214 0 2020-02-12 128 127 0 2020-03-31 924 708 0 2020-05-18 269 235 0 2020-02-13 115 117 0 2020-04-01 1009 990 2 2020-05-19 223 236 0 2020-02-14 72 73 0 2020-04-02 822 775 2 2020-05-20 226 253 0 2020-02-15 89 91 1 2020-04-03 880 854 1 2020-05-21 236 247 0 2020-02-16 112 115 1 2020-04-04 864 894 0 2020-05-22 187 221 0 2020-02-17 813 827 2 2020-04-05 912 850 1 2020-05-23 170 200 0 2020-02-18 832 841 1 2020-04-06 1814 894 0 2020-05-24 183 183 0 2020-02-19 696 706 1 2020-04-07 1685 1507 1 2020-05-25 222 198 0 2020-02-20 796 808 2 2020-04-08 1628 1725 1 2020-05-26 299 204 0 2020-02-21 714 728 0 2020-04-09 1576 1526 0 2020-05-27 183 286 0 2020-02-22 444 459 2 2020-04-10 1564 1613 0 2020-05-28 170 174 0 2020-02-23 328 340 2 2020-04-11 1091 1288 1 2020-05-29 156 169 0 2020-02-24 698 725 1 2020-04-12 664 1026 0 2020-05-30 174 168 0 2020-02-25 609 632 1 2020-04-13 1340 2183 0 2020-05-31 237 160 0 2020-02-26 521 541 0 2020-04-14 1204 1439 0 2020-06-01 186 197 0 2020-02-27 520 541 1 2020-04-15 952 1078 0 2020-06-02 178 245 0 2020-02-28 371 386 4 2020-04-16 903 1273 0 2020-06-03 188 185 0 2020-02-29 511 513 0 2020-04-17 838 1046 0 2020-06-04 170 175 0 2020-03-01 430 415 1 2020-04-18 557 893 0 2020-06-05 184 201 0 2020-03-02 600 559 1 2020-04-19 624 600 0 2020-06-06 138 177 0 12 12 . Data used 13 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Table 3: The data used in this study (continued). Date Susp. Excl. Conf. Date Susp. Excl. Conf Date Susp. Excl. Data used Conf 2020-10-29 569 192 0 2020-12-16 757 844 0 2021-02-02 3243 1000 0 2020-10-30 156 244 0 2020-12-17 790 715 0 2021-02-03 1167 2029 0 2020-10-31 93 208 0 2020-12-18 577 573 0 2021-02-04 927 1082 0 2020-11-01 116 191 0 2020-12-19 240 502 0 2021-02-05 877 2127 1 2020-11-02 352 237 0 2020-12-20 183 559 0 2021-02-06 517 1918 0 2020-11-03 288 262 0 2020-12-21 760 432 0 2021-02-07 793 825 0 2020-11-04 325 259 0 2020-12-22 800 455 1 2021-02-08 1107 457 0 2020-11-05 272 262 0 2020-12-23 670 851 0 2021-02-09 784 844 1 2020-11-06 394 230 0 2020-12-24 728 560 0 2021-02-10 379 729 0 2020-11-07 114 209 0 2020-12-25 759 586 0 2021-02-11 275 639 0 2020-11-08 118 217 0 2020-12-26 324 678 0 2021-02-12 276 369 0 2020-11-09 320 180 0 2020-12-27 365 624 0 2021-02-13 324 0 0 2020-11-10 292 262 0 2020-12-28 1017 356 0 2021-02-14 334 0 0 2020-11-11 287 388 0 2020-12-29 810 770 0 2021-02-15 325 0 0 2020-11-12 380 363 0 2020-12-30 674 778 0 2021-02-16 320 0 0 2020-11-13 204 296 0 2020-12-31 507 745 0 2021-02-17 759 2001 0 2020-11-14 98 201 0 2021-01-01 200 621 0 2021-02-18 698 632 0 2020-11-15 102 149 0 2021-01-02 251 402 0 2021-02-19 839 821 0 2020-11-16 382 182 0 2021-01-03 159 302 0 2021-02-20 371 839 0 2020-11-17 215 238 0 2021-01-04 1185 180 0 2021-02-21 231 0 0 2020-11-18 287 286 0 2021-01-05 597 654 0 2021-02-22 736 0 0 2020-11-19 429 277 0 2021-01-06 807 987 0 2021-02-23 487 0 0 2020-11-20 199 265 0 2021-01-07 699 603 0 2021-02-24 546 2445 0 2020-11-21 210 325 0 2021-01-08 597 728 0 2021-02-25 544 525 0 2020-11-22 126 195 0 2021-01-09 249 539 0 2021-02-26 418 0 0 2020-11-23 341 200 0 2021-01-10 276 176 0 2021-02-27 221 669 0 2020-11-24 342 240 0 2021-01-11 985 628 0 2021-02-28 197 307 0 2020-11-25 329 232 0 2021-01-12 612 697 2 2021-03-01 276 0 0 2020-11-26 425 320 0 2021-01-13 758 862 0 2021-03-02 434 0 0 2020-11-27 638 392 0 2021-01-14 967 0 0 2021-03-03 288 789 0 2020-11-28 479 359 0 2021-01-15 1174 1446 0 2021-03-04 675 278 0 2020-11-29 241 693 0 2021-01-16 543 836 0 2021-03-05 706 0 0 2020-11-30 337 377 0 2021-01-17 447 854 2 2021-03-06 349 1225 0 2020-12-01 363 280 0 2021-01-18 1518 610 1 2021-03-07 176 771 0 2020-12-02 184 262 0 2021-01-19 951 657 4 2021-03-08 1093 233 0 2020-12-03 898 301 0 2021-01-20 1244 1178 1 2021-03-09 357 551 0 2020-12-04 283 280 0 2021-01-21 1188 1202 0 2021-03-10 490 880 0 2020-12-05 146 605 0 2021-01-22 1324 1341 2 2021-03-11 626 0 0 2020-12-06 137 352 0 2021-01-23 1007 1081 1 2021-03-12 641 799 0 2020-12-07 242 141 0 2021-01-24 593 1293 2 2021-03-13 212 0 0 2020-12-08 291 170 0 2021-01-25 1738 0 0 2021-03-14 142 1034 0 2020-12-09 300 211 0 2021-01-26 1516 1867 0 2021-03-15 536 152 0 2020-12-10 434 78 0 2021-01-27 1629 1639 0 2021-03-16 650 0 0 2020-12-11 780 330 0 2021-01-28 1602 1290 0 2021-03-17 485 865 0 2020-12-12 423 540 0 2021-01-29 1878 1609 0 2021-03-18 360 479 0 2020-12-13 216 1065 0 2021-01-30 1359 1362 4 2021-03-19 432 713 0 2020-12-14 847 270 0 2021-01-31 872 1277 0 2021-03-20 219 424 0 2020-12-15 801 594 0 2021-02-01 1210 1220 0 2021-03-21 163 391 0 14 Table 3: The data used in this study (continued). Data used 14 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Table 4: The data used in this study (continued). Date Susp. Excl. Conf. Date Susp. Excl. Conf Date Susp. Excl. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. review) The copyright holder for this preprint this version posted June 27, 2021. ; 10.1101/2021.06.20.21259178 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 27, 2021. ; https://doi.org/10.1101/2021.06.20.21259178 doi: medRxiv preprint Data used Conf 2021-03-22 803 0 0 2021-04-20 526 413 2 2021-05-19 17528 8746 265 2021-03-23 437 771 0 2021-04-21 837 577 0 2021-05-20 17892 12403 283 2021-03-24 569 706 0 2021-04-22 670 520 0 2021-05-21 21653 15358 311 2021-03-25 716 556 0 2021-04-23 832 920 2 2021-05-22 14450 17597 721 2021-03-26 628 763 0 2021-04-24 543 656 1 2021-05-23 12865 14176 457 2021-03-27 252 637 0 2021-04-25 614 479 2 2021-05-24 22926 12381 590 2021-03-28 164 405 0 2021-04-26 1099 488 2 2021-05-25 24383 15368 537 2021-03-29 850 212 0 2021-04-27 752 1089 1 2021-05-26 20871 18701 633 2021-03-30 834 604 0 2021-04-28 764 1018 3 2021-05-27 19811 22596 666 2021-03-31 964 807 0 2021-04-29 1144 685 3 2021-05-28 28993 15799 552 2021-04-01 604 623 0 2021-04-30 854 1056 3 2021-05-29 16141 29381 484 2021-04-02 275 693 0 2021-05-01 383 766 1 2021-05-30 13535 27136 354 2021-04-03 290 412 0 2021-05-02 418 625 4 2021-05-31 27502 11967 347 2021-04-04 158 362 0 2021-05-03 1393 435 2 2021-06-01 30905 22678 326 2021-04-05 303 171 0 2021-05-04 988 993 2 2021-06-02 29598 35689 548 2021-04-06 1159 421 0 2021-05-05 1094 1194 0 2021-06-03 27796 31670 583 2021-04-07 658 478 0 2021-05-06 1006 1001 1 2021-06-04 31416 29217 470 2021-04-08 669 0 0 2021-05-07 1067 939 0 2021-06-05 18505 29470 510 2021-04-09 846 1716 0 2021-05-08 413 926 2 2021-06-06 13167 20020 342 2021-04-10 230 658 0 2021-05-09 680 698 0 2021-06-07 33272 14079 190 2021-04-11 159 553 0 2021-05-10 1237 556 4 2021-06-08 31728 26995 203 2021-04-12 755 344 0 2021-05-11 1218 1003 7 2021-06-09 33580 41180 273 2021-04-13 538 638 0 2021-05-12 2432 1136 17 2021-06-10 35025 29801 251 2021-04-14 598 721 0 2021-05-13 3893 1647 13 2021-06-11 36148 34517 277 2021-04-15 526 570 0 2021-05-14 6177 0 29 2021-06-12 22323 40622 245 2021-04-16 548 507 0 2021-05-15 6497 7736 180 2021-06-13 15821 24841 174 2021-04-17 139 511 0 2021-05-16 8425 4755 206 2021-06-14 19907 16906 185 2021-04-18 128 319 0 2021-05-17 15447 6322 333 2021-06-15 30647 17792 132 2021-04-19 719 133 0 2021-05-18 18087 8306 238 2021-06-16 25689 33366 165 Table 4: The data used in this study (continued). 15
https://openalex.org/W2092389010	https://www.frontiersin.org/articles/10.3389/fncir.2013.00163/pdf	English	null	Application of FRET probes in the analysis of neuronal plasticity	Frontiers in neural circuits	2,013	cc-by	19,151	INTRODUCTION The efﬁciency of FRET depends on two main factors, the distance between the two ﬂuorophores and their relative orientation. This feature enables the change in distance and angle between two ﬂu- orophores to be calculated, leading Lubert Stryer to call FRET a “molecular ruler” (Stryer, 1978). Using this property of FRET, various optical probes have been designed to detect aspects of different cellular functions in vitro and in vivo. The efﬁciency of FRET depends on two main factors, the distance between the two ﬂuorophores and their relative orientation. This feature enables the change in distance and angle between two ﬂu- orophores to be calculated, leading Lubert Stryer to call FRET a “molecular ruler” (Stryer, 1978). Using this property of FRET, various optical probes have been designed to detect aspects of different cellular functions in vitro and in vivo. The brain is a highly interconnected functional network com- prised of billions of neurons that communicate with each other at synapses. Throughout life, the neuronal connectivity that subserves brain function is modiﬁed and reﬁned in an activity- dependent manner, a phenomenon termed neuronal plasticity. Plasticity mechanisms can inﬂuence neuronal function and struc- ture through modiﬁcations at the level of synapses, dendrites and axons (Citri and Malenka, 2008; Holtmaat and Svoboda, 2009). The sensitivity and compatibility of FRET imaging with live imaging are critical for analyzing the molecular mechanisms of neuronal circuit plasticity. In particular, much progress has been made in recent years regarding the analysis of synaptic plasticity of excitatory synapses in excitatory neurons, which are typically formed on dendritic spines (Hayashi and Majewska, 2005; Bosch and Hayashi, 2012). FRET imaging is now being applied in vivo and offers a unique opportunity to study how and when neurons or synapses change and which signaling events contribute to such changes in response to stimuli in the intact brain. Different forms of plasticity are tightly regulated by a com- plex network of signal transduction cascades, which are the results of protein-protein interaction, posttranslational modiﬁca- tion, subcellular translocation of proteins, protein synthesis, etc. Therefore, the temporal and spatial precision of these events is critical to support proper brain function in the developing and mature brain. The development of probes that offer spatiotem- poral detection of these cellular events is vital to our ability to examine these important molecular mechanisms in biological systems. INTRODUCTION For this purpose, optical microscopic imaging enables complex and varied neuronal signals to be captured with high temporal and spatial resolution from live biological samples. In this article, we will provide an overview of the basic and practical aspects of FRET imaging, summarize currently available FRET-based probes and then discuss how these probes advanced our understanding of the molecular mechanisms underlying neuronal plasticity, mainly hippocampal long-term potentiation (LTP). Technological advances in the past two decades have made a signiﬁcant contribution to our ability to extend ﬂuorescent imaging techniques beyond that of simple morphological anal- ysis. One of the key developments is Förster resonance energy transfer (FRET). First reported by Förster (1946), the technique describes how energy from a “donor” ﬂuorophore can excite an “acceptor” ﬂuorophore, resulting in light emission from the latter. Application of FRET probes in the analysis of neuronal plasticity Yoshibumi Ueda 1†, Showming Kwok 2 and Yasunori Hayashi 1,3* 1 Brain Science Institute, RIKEN, Wako, Saitama, Japan 2 Department of Brain and Cognitive Sciences, The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA, USA 3 Brain Science Institute, Saitama University, Saitama, Japan Breakthroughs in imaging techniques and optical probes in recent years have revolutionized the ﬁeld of life sciences in ways that traditional methods could never match. The spatial and temporal regulation of molecular events can now be studied with great precision. There have been several key discoveries that have made this possible. Since green ﬂuorescent protein (GFP) was cloned in 1992, it has become the dominant tracer of proteins in living cells. Then the evolution of color variants of GFP opened the door to the application of Förster resonance energy transfer (FRET), which is now widely recognized as a powerful tool to study complicated signal transduction events and interactions between molecules. Employment of ﬂuorescent lifetime imaging microscopy (FLIM) allows the precise detection of FRET in small subcellular structures such as dendritic spines. In this review, we provide an overview of the basic and practical aspects of FRET imaging and discuss how different FRET probes have revealed insights into the molecular mechanisms of synaptic plasticity and enabled visualization of neuronal network activity both in vitro and in vivo. †Present address: Keywords: optical probes, synaptic plasticity, Förster resonance energy transfer, ﬂuorescence lifetime imaging microscopy NEURAL CIRCUITS REVIEW ARTICLE published: 10 October 2013 doi: 10.3389/fncir.2013.00163 Frontiers in Neural Circuits Edited by: Masanobu Kano, The University of Tokyo, Japan Masanobu Kano, The University of Tokyo, Japan y , p Reviewed by: Akihiro Yamanaka, Nagoya University, Japan Michiyuki Matsuda, Kyoto University, Japan Correspondence: Yasunori Hayashi, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan e-mail: yhayashi@brain.riken.jp; yhayashi-tky@umin.ac.jp †Present address: Yoshibumi Ueda, Department of Hematology and Immunology, Kanazawa Medical University, Kahoku, Ishikawa, Japan Reviewed by: Akihiro Yamanaka, Nagoya University, Japan Michiyuki Matsuda, Kyoto University, Japan Correspondence: Yasunori Hayashi, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan e-mail: yhayashi@brain.riken.jp; yhayashi-tky@umin.ac.jp †Present address: Yoshibumi Ueda, Department of Hematology and Immunology, Kanazawa Medical University, Kahoku, Ishikawa, Japan Reviewed by: Akihiro Yamanaka, Nagoya University, Japan Michiyuki Matsuda, Kyoto University, Japan REVIEW ARTICLE published: 10 October 2013 doi: 10.3389/fncir.2013.00163 REVIEW ARTICLE published: 10 October 2013 doi: 10.3389/fncir.2013.00163 Correspondence: Correspondence: Yasunori Hayashi, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan e-mail: yhayashi@brain.riken.jp; yhayashi-tky@umin.ac.jp *Correspondence: Yasunori Hayashi, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan e-mail: yhayashi@brain.riken.jp; yhayashi-tky@umin.ac.jp Cleavage-based approach The ﬁrst reported GFP-based probe detecting Factor Xa activity employed the cleavage-based approach (Figure 1A) (Mitra et al., 1996). In this type of probe, a protease cleavage sequence was ﬂanked by donor and acceptor ﬂuorophores. Under basal condi- tions, FRET occurs between the ﬂuorophores. However, cleavage of the target sequence causes a resultant separation of donor and acceptor molecules, leading to a decrease in FRET efﬁciency. The same approach was used to study other proteases including cas- pases (Xu et al., 1998; Onuki et al., 2002; Li et al., 2006; Joseph et al., 2011). One thing to note when using this type of probe is that the protease cleavage is irreversible. Therefore, it is not suit- able for detecting a protease with high basal activity. Also, the measurement cannot be repeated multiple times as the uncleaved fraction decreases and the cleaved fraction accumulates over time. However, FRET approaches using small molecular weight ﬂu- orescent compounds are technically demanding. For example, generation of the cAMP probe requires the cumbersome pro- cess of protein puriﬁcation, in vitro chemical coupling with dyes and introduction into cells. The success of the oxonol-based probe largely owed to the identiﬁcation of oxonol as a ﬂuorescent molecule that travels across the plasma membrane upon a change in membrane voltage. The emergence of genetically encoded FRET probes in the late 1990s dramatically changed the situation. This largely owes to the development and expansion of green ﬂuorescent pro- tein (GFP) and its color variants (Shaner et al., 2005). In a landmark study of genetically encoded FRET probes, Miyawaki et al. developed the ﬁrst GFP-based calcium indicator, cameleon using cyan ﬂuorescent protein (CFP) as a donor and yellow ﬂu- orescent protein (YFP) as an acceptor (Miyawaki et al., 1997). Cameleon consists of a calmodulin (CaM) protein fused with a M13 sequence (a 26-residue CaM binding peptide from myosin light-chain kinase), ﬂanked by CFP and YFP. The gly-gly motif between CaM and the M13 peptide gives this probe its con- formational ﬂexibility. In the absence of calcium, CaM and the M13 sequence do not interact with each other. However, in the presence of calcium, they form a complex, which short- ens the distance between the donor and acceptor ﬂuorophores, allowing FRET to occur. Using this probe, they observed cal- cium dynamics in living cells and demonstrated the poten- tial of FRET for the analysis of neuronal circuit dynamics. MONITORING in situ BIOCHEMICAL PROCESSES USING FRET-BASED PROBES In 1991, Tsien’s group made the ﬁrst attempt to image live cellu- lar functions using FRET (Adams et al., 1991; Zhang et al., 2002). October 2013 \| Volume 7 \| Article 163 \| 1 www.frontiersin.org FRET analysis of neuronal circuit Ueda et al. Ueda et al. genetically-encoded FRET probes are now widely used standard tools in biological systems. They attempted to visualize the intracellular dynamics of adeno- sine 3′, 5′- cyclic monophosphate (cAMP) by designing a probe based on cAMP-dependent protein kinase, in which the regu- latory and catalytic subunits were labeled with ﬂuorescein and rhodamine, respectively. Upon binding of cAMP, the regulatory subunit dissociates from the catalytic subunit, thereby eliminating FRET. STRATEGIES OF PROBE DESIGN Multiple genetically-encoded FRET probes have been developed for use in neuronal and non-neuronal cells. These probes can be classiﬁed into several categories depending on the approach used to detect different types of biological phenomena (Table 1, Figure 1). Subsequently, they also reported a voltage sensing FRET probe utilizing ﬂuorescein-labeled lectin as a donor and oxonol, an anionic ﬂuorescent compound, as an acceptor in living cells (Gonzalez and Tsien, 1995). At resting membrane potential, both dyes are localized on the outer leaﬂet of the plasma mem- brane and FRET occurs. Upon depolarization, negatively charged oxonol translocates to the inner leaﬂet of the plasma membrane and increases the distance from the donor, leading to a reduction in the efﬁciency of FRET. Cleavage-based approach Since then, probes for other molecules such as cAMP, guano- sine 3′, 5′- cyclic monophosphate (cGMP), and Cl−, small GTP-binding protein (small G-protein), phosphoinositide and signaling events e.g., phosphorylation have been developed (Table 1). Frontiers in Neural Circuits www.frontiersin.org Intermolecular FRET approach The interaction between proteins can be monitored by inter- molecular FRET, where one party of the protein complex is tagged by a donor and the other by an acceptor (Figure 1B). The interac- tion can be a heteromer of two different proteins or a homomer of the same protein. Application of this approach includes, small G-protein activity (Yasuda et al., 2006), 3-phosphoinositide- dependent protein kinase 1 (PDK)-Akt (Calleja et al., 2007), phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-myosin V (van Diepen et al., 2009), and protein-tyrosine phosphatase 1B (PTP1B)-receptor tyrosine kinases (RTKs) (Haj et al., 2002) interaction (Table 1). A variant of this approach is homomultimer FRET where a monomer in a polymeric pro- tein complex is labeled with both donor and acceptor molecules (Figure 1C), which allows the polymerization status of the pro- tein to be monitored. This was employed to detect actin polymer- ization/depolymerization (Okamoto et al., 2004). p y The quantitative aspect of FRET is difﬁcult to control in inter- molecular FRET (when compared with intramolecular FRET) because the expression level of donor and acceptor molecules often varies amongst cells. In contrast, in an intramolecular FRET probe, the donor and acceptor are on the same molecule and thus, the ratio of the donor to acceptor is always constant. Also, endoge- nous proteins may participate in forming protein complexes and this can decrease intermolecular FRET efﬁciency. Therefore, with intermolecular FRET, the efﬁciency must be measured as an aver- age of multiples cells or compared before and after a treatment (e.g., induction of synaptic plasticity) in the same cell. In prac- tice, a donor which does not interact with an acceptor increases the background of the measurement, whereas excess levels of Compared to small molecular weight ﬂuorescent molecule- based FRET probes, genetically encoded FRET probes offer a number of advantages. They can be constructed easily with standard molecular biological techniques, thus making probe design simple and ﬂexible. They can be expressed in cells by simply introducing vector DNA into neurons without protein puriﬁcation and chemical labeling. Use of an appropriate DNA transduction method or a promoter to express the probe allow cell-type speciﬁc labeling. Due to these technical advantages, the October 2013 \| Volume 7 \| Article 163 \| 2 www.frontiersin.org www.frontiersin.org www.frontiersin.org FRET analysis of neuronal circuit Ueda et al. Table 1 \| A list of genetically encoded FRET probes. Intermolecular FRET approach Table 1 \| A list of genetically encoded FRET probes. Classiﬁcation Target Name of probe Year Probe design Small molecule Calcium Cameleon 1997 3-2 Small molecule Cyclic guanosine monophosphate (cGMP) CGY, Cygnet, pGES-DE2, cGi 2000, 2001, 2006, 2013 3-1 Small molecule Cyclic adenosine monophosphate (cAMP) Epac 2000, 2004 2, 3-1 Small molecule Inositol trisphosphate (IP3) LIBRA, Fretino, FIRE 2004, 2005, 2006 3-1 Small molecule Nitric oxide (NO) NOA-1, Piccell 2005, 2006 3-1 Small molecule Adenosine triphosphate (ATP) A Team 1.03-nD/nA 2012 3-1 Small molecule Estrogen SCCoR 2004 3-3 Small molecule Androgen Ficaro 2006 3-3 Small molecule Glucocorticoid receptor ligands GLUCOCOR 2007 3-3 Small molecule Neurotrophic factor ECaus 2008 3-3 Small molecule Nuclear receptor conpro 2007 3-2 Small molecule O-N-acetylglucosamine (O-GlcNAc) 2006 3-3 Small molecule Vitamin A (Retinoic acid) GEPRAS 2013 3-1 Small molecule Molybdate MolyProbe 2013 3-1 Small molecule Glutamate FLIPE 2005 3-1 Small molecule Zn2+ eCALWY-1 2009 2 Small molecule Cl− Clomeleon 2000 other Small molecule pH GFpH, YFpH 2001 other Small molecule Glucose FLIPglu 2003 3-1 Small molecule Maltose FLIPmal 2002 3-1 Small molecule Ribose FLIPrib 2003 3-1 Kinase Calcium/Calmodulin-dependent protein kinase II (CaMKII) Camui α, green-Camui α, Camk2a reporter 2005, 2009, 2011, 2013 3-1 Kinase Src Srcus 2001, 2005, 2007 3-3 Kinase Protein kinase C (PKC) CKAR, CY-PKCdelta 2003, 2005 3-3, 3-1 Kinase Protein kinase D (PKD) DKAR 2007 3-3 Kinase Protein kinase A (PKA) ART, AKAR 2000, 2001 3-3 Kinase Abl Picchu 2001 3-3 Kinase Bcr-Abl Bcr-Abl activity sensor 2010 3-3 Kinase c-Raf Prin-cRaf 2005 3-1 Kinase PAK1 Pakabi 2009 3-1 Kinase B-raf Prin-Braf 2006 3-1 Kinase ZAP-70 ROZA 2008 3-3 Kinase Akt Aktus, BKAR, Akind 2003, 2005, 2007 3-3 Kinase ERK Miu2, Erkus, EKAR 2006, 2007, 2008 3-1, 3-3, 3-3 Frontiers in Neural Circuits October 2013 \| Volume 7 \| Article 163 \| 3 www.frontiersin.org FRET analysis of neuronal circuit Ueda et al. Frontiers in Neural Circuits Intermolecular FRET approach FRET analysis of neuronal circuit Table 1 \| Continued Classiﬁcation Target Name of probe Year Probe design References Protein interaction Protein tyrosine phosphatase 1B-receptor tyrosine kinases (PTP 1B-RTKs) interaction 2002 2 Haj et al., 2002 Protein interaction Breast cancer resistance protein/ATP-binding cassette sub-family G member (BCRP/ABCG) 2010 2 Ni et al., 2010 Protein interaction Coﬁlin-actin interaction 2008 2 Homma et al., 2008 Protein interaction PTEN-Myosin V interaction 2009 2 van Diepen et al., 2009 Protease Caspase-3 EGFP-DEVD-EBFP 1998 1 Xu et al., 1998 Protease Caspase-8 CFP-c3-YFP-c6-mRFP 2002 1 Onuki et al., 2002 Protease Caspase-9 SCAT9 2011 1 Joseph et al., 2011 Protease Caspase-7 VDEVDc 2006 1 Li et al., 2006 Protease Matrix Metalloproteinase (MMP) YFP-MSS-CFPdisplay, MTI-MMP-FRET biosensor 2007, 2008 1 Yang et al., 2007; Ouyang et al., 2008 Protease Protease activity (Factor Xa) 1996 1 Mitra et al., 1996 Protease Calpain activity pYSCS 2000 1 Vanderklish et al., 2000 Protease Presenilin GFP-PSI-RFP 2009 3-1 Uemura et al., 2009 Other Strain sensor stFRET 2008 3-1 Meng et al., 2008 Other Membrane potential VSFP, Mermaid, ArcLight, VSFP-Butterﬂy 2001, 2008, 2012, 2013 3-1 Sakai et al., 2001; Tsutsui et al., 2008; Jin et al., 2012; Akemann et al., 2013 Other Myosin II GSldCB 1998, 2006 3-1 Suzuki et al., 1998; Zeng et al., 2006 Other HIV Rev protein YRGnC-11ad 2005 3-1 Endoh et al., 2005 Other Redox Redoxﬂuor, Gaskins 2010, 2011 3-1 Yano et al., 2010; Kolossov et al., 2011 The numbers in the Probe Design column correspond to the section number in the “Strategies of probe design” chapter of the main text. Names of probes are shown. See the webpage by Dr. Michiyuki Matsuda http://www.lif.kyoto-u.ac.jp/labs/fret/e-phogemon/unifret.htm for updated information. Table 1 \| Continued The numbers in the Probe Design column correspond to the section number in the “Strategies of probe design” chapter of the main text. Names of probes are shown. See the webpage by Dr. Michiyuki Matsuda http://www.lif.kyoto-u.ac.jp/labs/fret/e-phogemon/unifret.htm for updated information. acceptor molecules usually does not cause a problem (Okamoto and Hayashi, 2006). Therefore, whenever possible, excess acceptor molecules should be used. Intrinsic conformation change of protein. If a protein of inter- est changes its conformation by activation/inactivation, one can design a probe to detect the conformational change as a way of monitoring the activity level (Figure 1D). Intermolecular FRET approach This may be accom- plished by ﬂanking the protein with a donor and an acceptor or inserting one or both of the ﬂuorophore(s) between the domains. This approach has been successfully employed for Ca2+/CaM- dependent protein kinase II (CaMKII) (Takao et al., 2005; Kwok et al., 2008; Fujii et al., 2013), calcineurin (Fujii et al., 2013), c-raf (Terai and Matsuda, 2005), p21 protein-activated kinase 1 (PAK1) (Parrini et al., 2009), B-raf (Terai and Matsuda, 2006), regula- tor of chromosome condensation 1 (RCC1) (Hao and Macara, 2008), vitamin A receptor (Shimozono et al., 2013) and to moni- tor changes in membrane potential (Tsutsui et al., 2008; Akemann et al., 2012). X-ray crystal structure is a useful guide to identify locations on a protein where the donor and acceptor pair can be placed. Intermolecular FRET approach Table 1 \| Continued Table 1 \| Continued Classiﬁcation Target Name of probe Year Probe design References Kinase Insulin receptor Phocus 2002 3-3 Sato et al., 2002 Kinase Epidermal Growth factor receptor (EGFR) 2001 3-3 Ting et al., 2001 Kinase Ataxia telangiectasia mutated (ATM) 2007 3-3 Johnson et al., 2007 Kinase Aurora B kinase 2008 3-3 Fuller et al., 2008 Kinase Cyclin B1-CDK1 2010 3-3 Gavet and Pines, 2010 Kinase Myosine light chain kinase MLCK-FIP 2002 3-1 Chew et al., 2002 Kinase JNK JNKAR1, JUNKAR1EV 2010, 2011 3-3 Fosbrink et al., 2010; Komatsu et al., 2011 Kinase RSK Eevee-RSK 2011 3-3 Komatsu et al., 2011 Kinase S6K Eevee-S6K 2011 3-3 Komatsu et al., 2011 Kinase Focal Adhesion Kinase (FAK) CYFAK413, FERM-sensor 2008, 2009 2, 3-1 Cai et al., 2008; Papusheva et al., 2009 Kinase PLK1 2008 3-3 Macurek et al., 2008 Kinase SAP3K 2009 3-3 Tomida et al., 2009 Kinase DAPK1 DAPK1(334)-F40 2011 3-1 Piljic et al., 2011 Phosphatase Calcineurin CaNAR1 2008, 2013 3-1 Newman and Zhang, 2008; Fujii et al., 2013 Small G-protein Ras Raichu-Ras, Fras 2001, 2006 3-2, 2 Yasuda et al., 2006; Mochizuki et al., 2001 Small G-protein Rap Raichu-Rap 2001 3-2 Mochizuki et al., 2001 Small G-protein Rac Raichu-Rac1 2004 3-2 Aoki et al., 2004 Small G-protein Rab5 Raichu-Rab5 2008 3-2 Kitano et al., 2008 Small G-protein Rho Raichu-RhoA 2003, 2011 3-2, 2 Yoshizaki et al., 2003; Murakoshi et al., 2011 Small G-protein Cdc42 Raichu-cdc42 2004, 2011 3-2, 2 Aoki et al., 2004; Murakoshi et al., 2011 Small G-protein Ral Raichu-Ral 2004 3-3 Takaya et al., 2004 Small G-protein TC10 Raichu-TC10 2006 3-2 Kawase et al., 2006 Signal transduction RCC1 (GEF of Ran) CFP-RCC1-YFP 2008 3-1 Hao and Macara, 2008 Signal transduction CrkII phosphorylation Picchu 2001 3-1 Kurokawa et al., 2001 Signal transduction N-WASP Stinger 2004 3-1 Lorenz et al., 2004; Ward et al., 2004 Signal transduction Adrenergic receptor α2AAR-cam 2003 3-1 Vilardaga et al., 2003 Signal transduction Parathyroid hormone receptor PTHR-cam 2003 3-1 Vilardaga et al., 2003 Signal transduction Plasma membrane Calcium pump BFP-PMCA-GFP 2007 3-1 Corradi and Adamo, 2007 Acetylation Histone acetylation Histac 2004, 2009 3-3 Lin et al., 2004; Sasaki et al., 2009 Lipid Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) Fllip, FLIMPA 2003, 2013 3-4 Sato et al., 2003; Ueda and Hayashi, 2013 Lipid Phosphatidylinositol (4,5)-bisphosphate (PIP2) Pippi-PI(4,5)P2 2008 3-4 Nishioka et al., 2008 Lipid Phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2) Pippi-PI(3,4)P2 2008 3-4 Nishioka et al., 2008 Lipid Phosphatidylinositol 4-phosphate (PI4P) Pippi-PI(4)P 2008 3-4 Nishioka et al., 2008 Lipid Phosphatidic acid Pii 2010 3-4 Nishioka et al., 2010 Lipid Diacylglycerol (DAG) Daglas, DIGDA 2006, 2008 3-4 Sato et al., 2006a; Nishioka et al., 2008 Protein interaction Actin 2004, 2008 2 Okamoto et al., 2004; Murakoshi et al., 2008 Protein interaction PDK1-Akt interaction 2007 2 Calleja et al., 2007 (Continued) October 2013 \| Volume 7 \| Article 163 \| 4 Frontiers in Neural Circuits www.frontiersin.org Ueda et al. Intramolecular FRET approach This approach detects the conformational change of the probe via a change in the distance and angle of donor and acceptor pro- teins located on the same molecule. Because both ﬂuorophores are on the same molecule, complications such as the differential redistribution of the donor and acceptor proteins and hetero- geneity in the expression level of donor and acceptor among cells can be eliminated. Using this approach, many different probes have been generated to enable the detection of covalent modiﬁca- tions of proteins, membrane voltage, small biological molecules, and signal transduction (Table 1). One can design a probe to detect conformational change that is intrinsic to the protein of interest or design a fusion protein that changes its conformation upon the occurrence of a speciﬁed biological event. Advantage of intramolecular FRET is relative ease of constructing probe which shows FRET. But it is sometimes difﬁcult to ﬁnd right posi- tion of the ﬂuorophore so that external stimuli change the FRET efﬁciency. Conformation change induced by a speciﬁc protein interaction. Conformation change induced by a speciﬁc protein interaction. Activation or inactivation of a protein can trigger an interac- tion with a speciﬁc target protein. By using such an interaction, one can design a FRET probe to detect the activation of a protein October 2013 \| Volume 7 \| Article 163 \| 5 Frontiers in Neural Circuits www.frontiersin.org FRET analysis of neuronal circuit Ueda et al. FIGURE 1 \| Strategies of probe design. Light blue, donor; yellow, acceptor. (A) Protease. (B) Intermolecular protein interaction. (C) Polymerization status. (D) Intrinsic conformation change of protein, which can be used to detect activation of a protein if it accompanies conformation change of the structure. (E) Conformation change of fusion protein induced by activation/inactivation. An example of detection of small GTPase activation (green) by small GTPase binding protein (red) is shown. (F) Conformation change of fusion protein induced by covalent modiﬁcation/inactivation. Here an example of detection of kinase activity by substrate sequence (gray) and phosphoprotein binding domain (orange) is depicted. (G) Small molecule on membrane lipid. An example of detection of small GTPase activation (green) by small GTPase binding protein (red) is shown. (F) Conformation change of fusion protein induced by covalent modiﬁcation/inactivation. Here an example of detection of kinase activity by substrate sequence (gray) and phosphoprotein binding domain (orange) is depicted. (G) Small molecule on membrane lipid. FIGURE 1 \| Strategies of probe design. Light blue, donor; yellow, acceptor. (A) Protease. (B) Intermolecular protein interaction. (C) Polymerization status. (D) Intrinsic conformation change of protein, which can be used to detect activation of a protein if it accompanies conformation change of the structure. (E) Conformation change of fusion protein induced by activation/inactivation. which ﬂank a substrate domain that can be covalently modiﬁed by the protein of interest and a protein domain that specif- ically recognizes the covalently modiﬁed protein (Figure 1F). When the protein is covalently modiﬁed, it binds to the adja- cent recognition domain, leading to a conformational change in the entire molecule, resulting in a change in FRET. By making use of speciﬁc kinase substrate and phosphor-protein recog- nition domains, this strategy has been applied to the design of FRET sensors for kinases and phosphatases including PKA (Zhang et al., 2001), C (Violin et al., 2003), and D (Kunkel et al., 2007), Akt (Sasaki et al., 2003), and Src (Ting et al., 2001). Ratiometric FRET detection In ratiometric FRET detection, the acceptor and donor images are acquired separately and the ratio of ﬂuorescent intensity between the two images is subsequently calculated. When FRET occurs, the acceptor/donor ratio increases. Because any ﬂuo- rescent microscopy (e. g., wide ﬁeld, confocal, two-photon) can be used for this measurement, ratiometric FRET measure- ment is often used, though it is not best for several reasons. When performing this type of imaging, maximum care must be taken to minimize spectral bleed-through, to properly sub- tract background and to take into account the ﬂuorophore relocalization. These factors make imaging in small structures particularly challenging. For example, CFP, a donor ﬂuorophore that is often paired with YFP as an acceptor, can bleed into the YFP channel, thereby decreasing the signal/noise ratio. Hence to minimize bleed-through, a suitable band-pass ﬁlter should be used, even if the overall brightness of the signal is com- promised. Also, background subtraction has to be performed with great care, as a subtle change in background can have a signiﬁcant effect on the signal ratio. The issue of probe relo- calization should also be carefully considered. This may be particularly problematic when measuring intermolecular FRET between two different molecules, which may differentially relo- calize during neuronal plasticity. For example, if donor moves while the acceptor does not, it will cause an apparent change in ﬂuorescent ratio without an actual change in protein inter- action. This situation can be circumvented by using a probe with intramolecular FRET, where both donor and acceptor are on the same molecule or intermolecular FRET between homo- mers, where both are expected to move in parallel (Ni and Zhang, 2010). It is also possible to mathematically correct the FRET by separately measuring the amount of local acceptor. But in such cases, it is better to employ ﬂuorescent life-time imag- ing microscopy, which relies only on donor ﬂuorescence (see below). py There are largely two different methods of FLIM, time and fre- quency domain measurements (Yasuda, 2006). The time domain measures the ﬂuorescence decay after a brief (< picoseconds) excitation pulse, while frequency domain measures lifetime by modulating the excitation light intensity and the detector gain dif- ferently (heterodyning) at high frequency (Yasuda, 2006). Both imaging systems are costly because FLIM requires a dedicated light-source and time-resolved detection. However, if one already has a two-photon microscope, adding components onto the exist- ing system is straightforward. Conformation change induced by a speciﬁc protein interaction. It should be noted that this type of probe actually detects a temporal integration of both kinase and phosphatase activity. Also, there may be kinases or phosphatases other than the tar- get protein, which also phosphorylate or dephosphorylate the probe. (Figure 1E). The cameleon probe mentioned above falls into this category. Another example is the Raichu series of probes that were developed to observe the activity of small G-proteins (Mochizuki et al., 2001). The basic structure of Raichu probes is comprised of four modules; a donor, an acceptor, a G-protein, and a G- protein-binding domain from its binding partner (Figure 1E). The inactive GDP-bound form does not interact with each other the G-protein-binding domain. Upon binding with GTP, the G- protein and G-protein-binding domain interact with each other to bring the two ﬂuorophores into close proximity, thereby lead- ing to FRET. This probe design strategy has been applied to Ras, Rho family protein, and other small G-proteins (Hao and Macara, 2008; Kiyokawa et al., 2011). Conformation change induced by a covalent modiﬁcation of protein. This type of probe consists of a donor and an acceptor, October 2013 \| Volume 7 \| Article 163 \| 6 Frontiers in Neural Circuits www.frontiersin.org Ueda et al. Ueda et al. FRET analysis of neuronal circuit Acceptor bleaching Small molecules on membranes. Using a similar strategy, small molecules on membranes can also be measured (Figure 1G). In this case, one of the ﬂuorophores is tethered to the membrane through rigid α-helical linkers whereas the other ﬂuorophore retains its ﬂexibility via a gly-gly hinge. A spe- ciﬁc lipid-binding domain is inserted in-between. When a small molecule binding domain interacts with its target of interest, a conformational change occurs through the hinge, resulting in an increase in FRET efﬁciency. This strat- egy has mainly been used to design probes for lipid sec- ond messengers such as phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), phosphatidylinositol 3,4,5-trisphosphate (PIP3), phosphatidyli- nositol 4-monophosphate (PI(4)P), and diacylglycerol (DAG) (Sato et al., 2003, 2006a; Nishioka et al., 2008; Ueda and Hayashi, 2013). When the acceptor is photobleached with an appropriate wave- length, the donor ﬂuorescence is dequenched and increased. This maneuver, called acceptor bleaching, gives a quantitative reading of FRET as it depends only on the donor ﬂuorescence inten- sity. Excitation light wavelength, intensity, and duration must be carefully chosen to photobleach only the acceptor ﬂuorophore. The photobleaching of the donor ﬂuorophore will underesti- mate the FRET. This can be done by simply illuminating the donor protein without an acceptor and making sure that donor ﬂuorescence does not photobleach. It should be noted that the photobleaching of an acceptor is irreversible and therefore, accep- tor photobleaching is a terminal experiment where only a single, speciﬁc and accurate static measure of FRET efﬁciency is needed (Miyawaki, 2003). Obviously, for this reason, acceptor bleaching is not compatible with time-lapse imaging. DETECTION OF FRET Several imaging methods for FRET detection are used in typi- cal biological laboratory settings (Miyawaki, 2003; Yasuda, 2006, 2012). The third approach to quantifying FRET relies on a parameter of ﬂuorescence, called ﬂuorescence lifetime (Yasuda, 2006). When a ﬂuorescent molecule is excited, it emits ﬂuorescence in a decay- ing manner from the time of activation, typically in exponential fashion. When FRET occurs, the donor ﬂuoresecence lifetime is shortened. Because ﬂuorescence lifetime is unaffected under a wide range of concentrations and does not depend on accep- tor ﬂuorescence, it is less prone to artifact caused by a change in the local concentration of donor and acceptor, which is espe- cially important in heterooligomer FRET. In contrast, ratiometric measurement can show a pseudopositive signal caused by bleed- through between ﬂuorescence channels, which can be an issue when measuring FRET from a structure where protein com- position can change. Therefore, ﬂuorescence lifetime imaging microscopy (FLIM) is the ideal choice for FRET detection. Ratiometric FRET detection This will not give absolute proportion of component showing FRET but by comparing the average lifetime over time, will give sufﬁ- cient information even from a noisy decay curve not suitable for ﬁtting (Lee et al., 2009; Murakoshi et al., 2011). to complicate the situation. When endogenous counterpart exists, often the case in a cell, the dimer can be either between two exoge- nous donor molecules or between one donor and one endogenous counterpart, in addition to the dimer made of two endogenous molecules. As a result, the acceptor interacts with either two, one or zero ﬂuorescent molecules. Mathematically, it is possible to perform triple (or more) exponential ﬁtting. However, such mea- surement requires (1) bright sample, (2) capability of hardware that captures high photon counts over a large number of pixels rapidly, and (3) ease of sophisticated data analysis. Cellular aut- oﬂuorescence also complicates the analysis (Colyer et al., 2012). To circumvent this, one can calculate average lifetime of the pho- tons, which theoretically gives lifetime in single exponential. This will not give absolute proportion of component showing FRET but by comparing the average lifetime over time, will give sufﬁ- cient information even from a noisy decay curve not suitable for ﬁtting (Lee et al., 2009; Murakoshi et al., 2011). EGFP has a weak tendency to dimerize (Zacharias et al., 2002), which can lead to issues with protein aggregation, depending on the protein it is fused with (Lantsman and Tombes, 2005). Therefore, monomerized versions of EGFP, such as the A206K mutant (the amino acid numbering is based on wild type GFP) is preferred for FRET experiments as it will reduce any pseudopos- itive FRET signal caused by non-speciﬁc aggregation. However, in certain cases, such as in cleavage-based protease sensors, the dimerization of donor and acceptor molecules can be beneﬁ- cial to increase the difference in FRET efﬁciency before and after cleavage. In fact, a random mutagenesis study to enhance FRET efﬁciency of caspase probe lead to the identiﬁcation of a CyPet-YPet pair (Nguyen and Daugherty, 2005), which was sub- sequently shown to form a dimer between donor and acceptor (Ohashi et al., 2007). For comprehensive review on ﬂuorescence proteins, please refer to Shaner et al. (2005) and Newman et al. (2011). Another issue of the time domain measurement is the “dead zone” of the sampling. Ratiometric FRET detection For example, in a system set up on a Ti- sapphire laser based two-photon microscope, the repetition rate of the laser is at 80 MHz or every 12.5 ns. There is always a dead zone of sampling between each cycle, where the acquisition system must reset for the next cycle. Given that many ﬂuores- cent proteins have lifetime of 2–5 ns range, the dead zone can limit the effective range of ﬁtting and underestimate especially the component with longer lifetime. Recent studies that intro- duced wideﬁeld photon-counting detector and phasor analysis might provide a new approach to perform FLIM experiments, alleviating these shortfalls (Kwok et al., 2008; Colyer et al., 2012). APPLICATION OF FRET PROBES TO STUDY NEURONAL CIRCUIT DYNAMICS Numbers of FRET probes have been developed and tested in various cell types. Here we list some of the recent research accom- plishments using FRET probes in neuronal circuits. See Table 1 for an extended list of various FRET probes. CHOICE OF FLUORESCENCE PROTEINS Ca2+ Ca2+ To effectively measure the change in the distance and angle between two ﬂuorophores in a FRET construct, it is critical to start with a suitable pair of ﬂuorescent molecules with efﬁcient FRET. The efﬁciency of FRET (E) depends on several param- eters characteristic to each pair of ﬂuorescent proteins. Förster distance (R0), the distance at which the energy transfer efﬁciency is 50%, depends on the overlap of donor emission and acceptor excitation (J), quantum yield of the donor (Q0), and acceptor molar extinction coefﬁcient (εA). As the values for J, Q0, and εA increase, so does the value of R0, which in turn produces a larger E value. So far, CFP (or an improved version such as Cerulean or K26R/N164H mutant of ECFP) and YFP (such as Venus) is the most commonly for ratiometric FRET measurements. A CFP- YFP pair gives a R0 of 4.8–5.2 nm, depending on the variants used (Rizzo et al., 2006; Kwok et al., 2008; Lam et al., 2012). Recently, it was reported that the Clover and mRuby2 offers Förster radius of 6.3 nm and is currently considered to be the best FRET pair available to date (Lam et al., 2012). Intracellular Ca2+ plays an important role in regulating vari- ous cellular functions such as signaling, gene regulation, cell death, and survival. Under basal conditions, the intracellular Ca2+ concentration is maintained at low levels by various Ca2+- extrusion and sequestration mechanisms. Upon neuronal acti- vation, local intracellular Ca2+ concentration increases through inﬂux from the extracellular ﬂuid or efﬂux from the intracel- lular pool (Hayashi and Majewska, 2005). Different sources of Ca2+ can have distinct kinetics, subcellular localization and func- tions. Therefore, it is not very surprising that a Ca2+-sensing FRET probe was one of the ﬁrst genetically encoded FRET sen- sors ever made (Miyawaki et al., 1997). A popular use of this type of probe is to detect neuronal circuit activity through a detection of action potentials as Ca2+ inﬂux into cells via voltage depen- dent Ca2+ channels. The activity of hundreds of neurons can be simultaneously monitored (Wallace et al., 2008). Intracellular Ca2+ plays an important role in regulating vari- ous cellular functions such as signaling, gene regulation, cell death, and survival. Under basal conditions, the intracellular Ca2+ concentration is maintained at low levels by various Ca2+- extrusion and sequestration mechanisms. Ratiometric FRET detection Current systems allow the detection of FRET signals at second order time resolution from single den- dritic spines (Murakoshi et al., 2011), which is still slower than the ratiometric imaging that can go to video rate. For the time domain measurement, time correlated single pho- ton counting is currently widely used. This method measures the time elapses between an excitation pulse and an emitted single photon, which is binned into a histogram. The data will then be ﬁtted to exponential curve (Yasuda, 2006). When two states are expected, such as in the case where both bound and unbound FRET pair coexist, it is possible to do double exponential ﬁtting to obtain the ratio of two components (Yasuda, 2006). However, whether ﬁtting double exponential is appropriate or not to a given FRET pair should be carefully considered based on the protein structure. For example, if donor forms a homodimer, it is enough Frontiers in Neural Circuits October 2013 \| Volume 7 \| Article 163 \| 7 www.frontiersin.org FRET analysis of neuronal circuit Ueda et al. Ueda et al. which is the case for EGFP. The original enhanced CFP (ECFP) is not optimal as it shows two lifetime components, in addition to its relatively weak ﬂuorescence. Cerulean and mTurquoise2 are both brighter and have mono exponential decay, therefore, can be used when the cyan range is needed (Rizzo et al., 2004; Goedhart et al., 2012). to complicate the situation. When endogenous counterpart exists, often the case in a cell, the dimer can be either between two exoge- nous donor molecules or between one donor and one endogenous counterpart, in addition to the dimer made of two endogenous molecules. As a result, the acceptor interacts with either two, one or zero ﬂuorescent molecules. Mathematically, it is possible to perform triple (or more) exponential ﬁtting. However, such mea- surement requires (1) bright sample, (2) capability of hardware that captures high photon counts over a large number of pixels rapidly, and (3) ease of sophisticated data analysis. Cellular aut- oﬂuorescence also complicates the analysis (Colyer et al., 2012). To circumvent this, one can calculate average lifetime of the pho- tons, which theoretically gives lifetime in single exponential. Frontiers in Neural Circuits Ca2+ Upon neuronal acti- vation, local intracellular Ca2+ concentration increases through inﬂux from the extracellular ﬂuid or efﬂux from the intracel- lular pool (Hayashi and Majewska, 2005). Different sources of Ca2+ can have distinct kinetics, subcellular localization and func- tions. Therefore, it is not very surprising that a Ca2+-sensing FRET probe was one of the ﬁrst genetically encoded FRET sen- sors ever made (Miyawaki et al., 1997). A popular use of this type of probe is to detect neuronal circuit activity through a detection of action potentials as Ca2+ inﬂux into cells via voltage depen- dent Ca2+ channels. The activity of hundreds of neurons can be simultaneously monitored (Wallace et al., 2008). Since Miyawaki et al. characterized cameleon, the ﬁrst Ca2+ sensing FRET probe, various probes with different afﬁnities to Ca2+ have been reported (Miyawaki, 2005). Cameleon was expanded into the yellow cameleon series, which had greater sensitivity to Ca2+ and better signal/noise ratio (Nagai et al., 2004; Horikawa et al., 2010). Griesbeck et al. utilized troponin C and I to generate the Tn series Ca2+ sensor protein (Heim and Griesbeck, 2004). Cameleon has been mainly applied to zebraﬁsh (Mizuno et al., 2013) and C. elegans (Haspel et al., 2010). Recently For FLIM, enhanced GFP (EGFP) is often used as a donor, and paired with either monomeric red ﬂuorescent protein (mRFP) or mCherry as an acceptor. The acceptor brightness is not an issue in FLIM as it relies solely on the donor ﬂuorescence measurement. Therefore, non-ﬂuorescent, quencher proteins such as REACh (Ganesan et al., 2006), darkVenus (Kwok et al., 2008), and super REACh (Lee et al., 2009) may also be used as acceptors to donor EGFP. Ideally, the donor should show a single lifetime with FLIM, October 2013 \| Volume 7 \| Article 163 \| 8 Frontiers in Neural Circuits www.frontiersin.org www.frontiersin.org Ueda et al. Ueda et al. FRET analysis of neuronal circuit was proposed as a memory molecule, which can be used to store long term information after a synapse undergoes LTP (Lisman et al., 2002). YC-Nano 140, new version of cameleon, was expressed to barrel cortex of mice using adeno-associated virus vector and showed different responses between two groups of neurons which are projected to different regions in neocortex (Chen et al., 2013). Ca2+ However, direct demonstration of the persistent activation of CaMKII after the induction of LTP was lacking because of a deﬁciency in effective methods to detect the spatial and tempo- ral activation of CaMKII at the single spine level. To circum- vent this, a FRET probe, Camui, was engineered by employing the intramolecular FRET approach to detect the conformational change associated with CaMKII activation by fusing donor and acceptor ﬂuorophores to both termini of CaMKII (Takao et al., 2005; Kwok et al., 2008). Camui shows FRET in its basal inac- tive state. Addition of ATP, CaM, and Ca2+ leads to a rapid and persistent decrease in FRET. The conformational change due to binding of Ca2+/CaM and autophosphorylation is account- able for the change in FRET. This persistent, Ca2+-independent change in FRET is absent when ATP is omitted or when a kinase dead mutant is used. Furthermore, a phosphoblocking mutant (T286A) stops the persistent change in FRET, whereas a phospho- mimicking mutant (T286D) shows decreased FRET without Ca2+ stimulation. Hence, Camui detects the collective activation of CaMKII by the binding of Ca2+/CaM and the autophosphoryla- tion at T286. Using a FLIM version of Camui, green-Camuiα, Lee et al. discovered that CaMKII activity is only transient (<2 min) after the induction of structural LTP (sLTP) even though CaMKII activation is required for sustaining structural synaptic plastic- ity. This is much shorter than what had been believed (Lee et al., 2009). Using a separate approach not involving FRET for its prin- ciple mode of detection, Nakai et al. generated G-CaMP (Nakai et al., 2001). G-CaMP was engineered to express CaM and a M13 peptide inserted in the β-barrel wall of GFP, which ultimately dis- torts its overall structure of GFP and quenches its ﬂuorescence. An increase in Ca2+ concentration induces CaM and M13 pep- tide to interact, which then leads to a conformation change in the β-barrel. This in turn changes the protonation status of the ﬂuorophore and dequenches the ﬂuorescence. A related Ca2+ sensor termed pericam also utilizes a similar strategy (Nagai et al., 2001). Recently, B-GECO and R-GECO, a blue and red version of G-CaMP were developed to allow the simultaneous detection of calcium in more than one subcellular compartments or cell types (Zhao et al., 2011). Ca2+ With improvements in the sensitivity of probes and detection methods, it is now possible to visual- ize the Ca2+-inﬂux in single dendritic spines evoked by unitary excitatory postsynaptic potential (epsp) (Ohkura et al., 2012). Currently G-CaMP is becoming the ﬁrst choice for Ca2+ imag- ing, especially in vivo because it is convenient to detect the Ca2+ responses with one channel. However, a recent report compar- ing the sensitivity between G-CaMP3 and YCs in Purkinje cells of acute cerebellar slice from mice (Yamada et al., 2011) showed that YC exhibited better response than G-CaMP3, indicating that optimal probes need to be carefully chosen in a given brain region of interest. We investigated the spatial and temporal regulation of CaMKII in rapid ocular dominance (OD) plasticity in layer II/III of fer- ret visual cortex in vivo, a paradigmatic model for studying the role of sensory experience in shaping cortical neural cir- cuits (Mower et al., 2011). By taking advantage of the superﬁcial location of layer II/III pyramidal neurons for optical detection of Camui signals (Figure 2A), we found that brief monocular deprivation (MD, 4 h) leads to activation of CaMKII at most synapses in the deprived eye domains (Figure 2B). However, a change in CaMKII activity was not observed in the spines located in binocular and non-deprived eye domains following the same visual manipulation. Four hours of MD also lead to the elimination of a small fraction of spines in the deprived eye domain, whose basal CaMKII activity was lower than the aver- age CaMKII activity in the same cortical site. The spines that persisted after MD had either high basal CaMKII activity or increased activity. Therefore, the emerging picture of the role of CaMKII activity in vivo is that (1) the eliminated spines have low CaMKII activity (although not all spines with low activity are removed) and (2) high CaMKII activity might have a protective role for spines and these preserved spines could potentially serve as a substrate for the reorganization of intracortical presynaptic partners. Frontiers in Neural Circuits Small G-protein FIGURE 2 \| In vivo imaging of CaMKII activity using Camui. (A) Expression of Camui in ferret visual cortex allowed for visualization of CaMKII activity in dendrites and spines of a neuron in a speciﬁc ocular dominance (OD) domain. Blood vessel and OD maps were acquired using intrinsic signal optical imaging (Upper panel: A, anterior; M, medial). Gray scale indicates ocular dominance index (white, ipsilateral eye dominated; black, contralateral eye dominated). Blood vessels map and the low magniﬁcation two-photon microscopic image were cross referenced to identify the two-photon images (Lower panel) in a given OD map. A dendritic segment (red box) is magniﬁed (Right) and displayed as channel separated images (CFP and YFP) as well as a ratiometric image in intensity-modulated display mode, indicating the CFP/YFP ratio. Warm hue represents high CaMKII activity. (B) Sample images of spines with decreased (left) or increased (right) CaMKII activity after 4 h of monocular deprivation. Numbers below the images indicate the normalized CFP/YFP ratio, a measurement of FRET signal. From Mower et al. (2011). Small G-protein family, including Ras, Rho, Ran, Rab, Sar/Arf subfamilies, is a large group of signaling molecules that con- trol various cellular functions (Saneyoshi and Hayashi, 2012). The activity of small G-protein is controlled by intrinsic GTPase activity and by the type of guanine nucleotide it is bound with. GTP-bound form consists active form, which is con- verted into GDP-bound form by the GTPase activity. The cycle between GDP-bound inactive and GTP-bound active forms is reg- ulated by three classes of proteins, guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and gua- nine nucleotide dissociation inhibitors (GDIs) (Saneyoshi and Hayashi, 2012). GEFs exchange GDP bound on a small G-protein with GTP, which leads to an activation of signaling activity of the small G-protein. The GTP-bound forms of G-protein lapse into inactive forms when GAP induces activation of GTPase activity that converts the bound GTP to GDP. GDI removes GDP-bound inactive forms of G-proteins from cell membranes and therefore maintains them in inactive forms. Two family member of Ras family, Ras itself and Rap are impli- cated in synaptic plasticity. Zhu et al. showed that Ras relays the NMDA-R and CaMKII signaling that drives synaptic delivery of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type glutamate receptors (AMPA-Rs) during LTP (Zhu et al., 2002). In contrast, Rap mediates NMDA-R-dependent removal of synap- tic AMPA-Rs that occurs during LTD. A CaMKII activity sensor, Camui CaMKII is a member of the serine/threonine protein kinase family that is highly expressed in the brain, especially at the postsynaptic density (PSD) of excitatory synapses (Kennedy et al., 1983; Chen et al., 2005). CaMKII has been highly implicated in both induc- tion and maintenance of functional and structural LTP (Lisman et al., 2002; Matsuzaki et al., 2004). The activation of CaMKII pre- cedes the structural enlargement of stimulated spines, suggesting that CaMKII is a molecular trigger of downstream processes that lead to structural changes. In addition, the CaMKII has struc- tural role at the synapse through its capacity to bundle F-actin (Okamoto et al., 2007, 2009). Under basal conditions, CaMKII is kept inactive by intrasub- unit steric block of the substrate-binding site (S site) in the kinase domain by a pseudosubstrate region within the autoinhibitory domain (Lisman et al., 2002). Binding of Ca2+/CaM to the reg- ulatory domain (adjacent to the autoinhibitory domain) alters its conformation and disrupts the inhibitory interaction at the S site. This disruption releases the kinase domain from autoinhi- bition and allows it to rapidly self-phosphorylate threonine 286 (T286) of CaMKII, as well as other substrates. CaMKII autophos- phorylation at T286 prevents the autoinhibitory domain from binding with the T site of the catalytic domain and from blocking the kinase activity, thereby allowing the kinase to retain substan- tial activity even in the absence of Ca2+. Thus, this holoenzyme remains active for a prolonged period of time, signiﬁcantly out- lasting that of a Ca2+ spike. Based on these observations, CaMKII At ﬁrst, this result is seemingly at odds with the study by Lee et al., where they observed a transient activation of CaMKII by LTP induction with glutamate uncaging. However, this result most likely reﬂects the ability of CaMKII to respond to dif- ferent neuronal activity patterns (De Koninck and Schulman, 1998; Fujii et al., 2013). In the study by Lee et al. (2009), October 2013 \| Volume 7 \| Article 163 \| 9 www.frontiersin.org www.frontiersin.org FRET analysis of neuronal circuit Ueda et al. Small G-protein Thus, Ras and Rap serve as independent regulators for potentiating and depressing central synapses. Ras is also implicated in spine formation. The expres- sion of a constitutively active Ras in neocortex neurons lead to an increase in spine density (Gartner et al., 2005). Conversely, a loss of SynGAP, a Ras-GAP that expresses speciﬁcally in the brain, leads to an increase in spine formation and enlargement of spine size (Vazquez et al., 2004). In order to elucidate the Ras activity during LTP in the spines of hippocampal neurons, Yasuda et al. designed an intermolecu- lar FLIM-based probe to detect Ras activity, in which momomeric EGFP was tagged to the N-terminus of Ras, and two momo- meric RFPs were attached to the N- and C-termini of the Ras binding domain (RBD) of Raf (Yasuda et al., 2006). When Ras at the plasma membrane is activated, RBD is recruited to the membrane and binds to Ras, resulting in an increase in FRET. Using this probe, they investigated the activity of G-proteins in single dendritic spines in CA1 pyramidal neurons during sLTP. After the induction of sLTP, Ras was activated, which was then maintained for 30 min (Yasuda et al., 2006). Interestingly, the Ras signaling is not restricted to spines but spreads over 10 μm into dendritic shafts and eventually reaches neighboring spines, which can subsequently undergo sLTP with only weak stimulation (a stimulation that would normally induce only temporary poten- tiation) (Harvey et al., 2008b). These data suggest that the spread of Ras-dependent signaling is necessary for the local regulation of the LTP induction threshold. FIGURE 2 \| In vivo imaging of CaMKII activity using Camui. (A) FIGURE 2 \| In vivo imaging of CaMKII activity using Camui. (A) Expression of Camui in ferret visual cortex allowed for visualization of CaMKII activity in dendrites and spines of a neuron in a speciﬁc ocular dominance (OD) domain. Blood vessel and OD maps were acquired using intrinsic signal optical imaging (Upper panel: A, anterior; M, medial). Gray scale indicates ocular dominance index (white, ipsilateral eye dominated; black, contralateral eye dominated). Blood vessels map and the low magniﬁcation two-photon microscopic image were cross referenced to identify the two-photon images (Lower panel) in a given OD map. Small G-protein A dendritic segment (red box) is magniﬁed (Right) and displayed as channel separated images (CFP and YFP) as well as a ratiometric image in intensity-modulated display mode, indicating the CFP/YFP ratio. Warm hue represents high CaMKII activity. (B) Sample images of spines with decreased (left) or increased (right) CaMKII activity after 4 h of monocular deprivation. Numbers below the images indicate the normalized CFP/YFP ratio, a measurement of FRET signal. From Mower et al. (2011). FIGURE 2 \| In vivo imaging of CaMKII activity using Camui. (A) Expression of Camui in ferret visual cortex allowed for visualization of CaMKII activity in dendrites and spines of a neuron in a speciﬁc ocular dominance (OD) domain. Blood vessel and OD maps were acquired using intrinsic signal optical imaging (Upper panel: A, anterior; M, medial). Gray scale indicates ocular dominance index (white, ipsilateral eye dominated; black, contralateral eye dominated). Blood vessels map and the low magniﬁcation two-photon microscopic image were cross referenced to identify the two-photon images (Lower panel) in a given OD map. A dendritic segment (red box) is magniﬁed (Right) and displayed as channel separated images (CFP and YFP) as well as a ratiometric image in intensity-modulated display mode, indicating the CFP/YFP ratio. Warm hue represents high CaMKII activity. (B) Sample images of spines with decreased (left) or increased (right) CaMKII activity after 4 h of monocular deprivation. Numbers below the images indicate the normalized CFP/YFP ratio, a measurement of FRET signal. From Mower et al. (2011). FIGURE 2 \| In vivo imaging of CaMKII activity using Camui. (A) Expression of Camui in ferret visual cortex allowed for visualization of CaMKII activity in dendrites and spines of a neuron in a speciﬁc ocular dominance (OD) domain. Blood vessel and OD maps were acquired using intrinsic signal optical imaging (Upper panel: A, anterior; M, medial). Gray scale indicates ocular dominance index (white, ipsilateral eye dominated; black, contralateral eye dominated). Blood vessels map and the low magniﬁcation two-photon microscopic image were cross referenced to identify the two-photon images (Lower panel) in a given OD map. A dendritic segment (red box) is magniﬁed (Right) and displayed as channel separated images (CFP and YFP) as well as a ratiometric image in intensity-modulated display mode, indicating the CFP/YFP ratio. Warm hue represents high CaMKII activity. (B) Sample images of spines with decreased (left) or increased (right) CaMKII activity after 4 h of monocular deprivation. Phosphatidylinositol 3,4,5-Trisphosphate (PIP3) PIP3 is a phosphoinositide that plays an important role in a vari- ety of cellular functions. PIP3 is produced from phosphatidylinos- itol 4,5-bisphosphate (PIP2) by phosphoinositide 3-kinase (PI3K) in response to hormone and neurotransmitter while PTEN con- verts PIP3 back to PIP2. In hippocampal pyramidal neurons, PIP3 is crucial for maintaining AMPA-R clustering during LTP (Arendt et al., 2010). PIP3 also regulates neuronal polarity, dendritic arborization, and nerve growth factor-induced axonal ﬁlopodia formation (Jaworski et al., 2005; Ketschek and Gallo, 2010). In order to exert these functions, local PIP3 accumula- tion leads to the recruitment of effector proteins such as Akt (Thomas et al., 2001), WASP family Verprolin-homologous pro- tein (WAVE) (Oikawa et al., 2004) and GEF of small G proteins to speciﬁc subcellular compartments (Han et al., 1998; Shinohara et al., 2002; Innocenti et al., 2003). In order to obtain information about the spatiotemporal dynamics of ERK activity in neuronal cells, several FRET-based probes have been developed. Miu2 detects the conformational change of ERK activation by ﬂanking ERK with CFP and YFP (Fujioka et al., 2006). Erkus is based on the detection of sub- strate protein phosphorylation (Sato et al., 2007) (Figure 1F). The ERK substrate sequence was obtained from EGFR and fused to the phospho-binding domain from FHA2 by a ﬂexible pep- tide linker. The D domain, a sequence that selectively binds to ERK was attached to increase the speciﬁcity and efﬁciency of phosphorylation. This fusion protein was ﬂanked by CFP and YFP. When phospho-substrate peptide is phosphorylated by active ERK, the phosphoprotein-binding domain interacts with the phospho-substrate peptide, leading to a change in overall con- formation, which can be detected by a change in FRET efﬁciency. EKAR uses a similar approach but with a different substrate and a phosphoprotein-binding domain (Harvey et al., 2008a). In order to investigate PIP3 function and regulation in spines, we developed a FLIM-based PIP3 FRET probe, FLIMPA3, by concatenating a donor, a speciﬁc PIP3-binding domain, ﬂexi- ble di-glycine hinge, and an acceptor tethered to the membranes through rigid α-helical linkers (Sato et al., 2003; Murakoshi et al., 2008; Ueda and Hayashi, 2013) (Figure 1G). When FLIMPA3 was expressed in hippocampal CA1 pyramidal neurons, we found that PIP3 showed greater accumulation in spines than in den- dritic shafts under basal conditions (Ueda and Hayashi, 2013). Small G-protein Numbers below the images indicate the normalized CFP/YFP ratio, a measurement of FRET signal. From Mower et al. (2011). CaMKII is activated by local N-methyl-D-aspartate type glu- tamate receptor (NMDA-R) activation. However, in the visual cortex, it likely detects an integration of complex local and global activity patterns that encompass both Hebbian and home- ostatic mechanisms. Further studies are required to fully eluci- date the role of CaMKII in synaptic plasticity both in vitro and in vivo. Rho family G-proteins, including ras homolog family mem- ber (Rho), ras-related C3 botulinum toxin substrate (Rac), and cell division control protein 42 homolog (Cdc42), are small GTP binding proteins that control the actin cytoskeleton (Komatsu et al., 2011; Saneyoshi and Hayashi, 2012). Because actin is the October 2013 \| Volume 7 \| Article 163 \| 10 Frontiers in Neural Circuits www.frontiersin.org FRET analysis of neuronal circuit Ueda et al. major cytoskeletal protein in dendritic spines, the role of the Rho family G-proteins on the maintenance and rearrangement of spine morphology has been investigated (Saneyoshi and Hayashi, 2012). The expression of a constitutively active form of Rac1 in hippocampal pyramidal neurons leads to an increase in the num- ber (Tashiro et al., 2000), length and width of spines (Zhang and Macara, 2006), while a dominant negative had the opposite effect (Nakayama et al., 2000; Zhang and Macara, 2006; Impey et al., 2010). In contrast, a constitutively active form of RhoA reduces the density of spines (Tashiro et al., 2000; Impey et al., 2010) and causes a simpliﬁcation of dendritic branch pattern (Nakayama et al., 2000). Inhibition of RhoA activity leads to an increase in the number of spines in some neurons (Tashiro et al., 2000; Impey et al., 2010). Cdc42 is also implicated in spine morphogenesis (Tashiro et al., 2000; Irie and Yamaguchi, 2002). sLTP, PIP3 in spines was reduced. Application of a PTEN inhibitor did not signiﬁcantly change the reduction in PIP3. Additionally, the reduction of PIP3 after sLTP was highly correlated with PIP3 enrichment before sLTP induction. Therefore, the reduction in PIP3 during sLTP is likely to be due to the addition of mem- brane from the dendritic shaft. Interestingly, whilst PIP3 globally decreases in spines during sLTP, we observed a speciﬁc accumu- lation of PIP3 in spinules, ﬁlopodia-like protrusions found on spines. Extracellular Signal-regulated Kinase (ERK) ERK is a serine/threonine protein kinase that belongs to the mitogen-activated protein kinase (MAPK) family, which plays important roles in a variety of cellular functions such as cell dif- ferentiation, proliferation, and survival (Chang and Karin, 2001). In neuronal circuits, ERK is involved in a wide range of functions including the regulation of dendritic protein synthesis (Impey et al., 1998a,b; Roberson et al., 1999; Davis et al., 2000; Patterson et al., 2001; Waltereit et al., 2001), morphological changes in dendritic spines (Wu et al., 2001; Goldin and Segal, 2003) and hippocampal LTP and memory formation in vivo (Giovannini et al., 2001). Abnormal ERK signaling is associated with mental retardation (Costa et al., 2002). Small G-protein When PIP3 in spinules was blocked by a PI3K inhibitor that reduces PIP3 levels, the number of spinules after sLTP were diminished, indicating that PIP3 in spinules regulates spinule formation. Electron microscopic studies found that spinules could be trans-synaptically endocytosed by presynaptic terminals as sepa- rate vesicles from the postsynaptic side (Spacek and Harris, 2004). Therefore, the trans-endocytosis of spinules may serve as a mech- anism for retrograde signaling or may aid postsynaptic membrane remodeling by removing excess membrane (Spacek and Harris, 2004). Accumulated PIP3 in spinules that trafﬁc to the presy- naptic side may act as a retrograde signal or contribute to the formation of new synapses with functional presynaptic boutons. Murakoshi also applied the same Ras probe design strategy to construct probes for Rho family protein (Murakoshi et al., 2011). The temporal and spatial extent of activity spreading over the dendritic shaft was investigated (Murakoshi et al., 2011). Activity of both RhoA and Cdc42 was maintained for up to 30 min, which is consistent with the observation that the ﬁlamentous (F-) actin/globular (G-) actin equilibrium moves toward F-actin after LTP induction (Okamoto et al., 2004, see below). RhoA spreads with a length constant of 4.5 μm along the dendrite. On the other hand, Cdc42 activity was restricted only in the stimulated spine, whose length constant is 1.9 μm. Frontiers in Neural Circuits Chloride sensor l l Cl−ion regulates neuronal properties such as intracellular pH, cell volume, and ﬂuid secretion (Duran et al., 2010). More importantly, Cl−is a major carrier of electrical current in inhibitory synaptic transmission mediated by GABA and glycine receptors. The basal level of intracellular chloride ions (Cl−) is maintained by a number of mechanisms including chloride transporter system that consist of Na+-Cl−, Na+-K+-2Cl−, and K+-Cl−transporters, and the activation of tonic GABA receptors, calcium-activated Cl−channels, cAMP-activated Cl−channels, cell-volume regulated anion channels, and transporters local- ized within subcellular organelles (Duran et al., 2010). Since all these factors sum up to determine the intracellular Cl−con- centration, it is of a great interest to visualize the dynamics of intracellular Cl−. Actin exists in equilibrium between F-actin/G-actin but it was not known how the F-actin/ G-actin equilibrium changes dur- ing synaptic plasticity. This is because the dendritic spine is too small and does not show discrete F-actin structure that is observ- able with light microscopy. To circumvent this, an intermolecular FRET approach was used to monitor the F-actin/ G-actin equi- librium (Okamoto et al., 2004). The distance between actin monomers in F-actin is 55 Å, which is within the appropriate range to be detected with FRET. Actin was tagged with CFP and YFP as a donor and an acceptor, respectively. Using this approach, Okamoto et al. observed actin dynamics in hippocam- pal CA1 pyramidal neuronal cells during bidirectional plasticity (Okamoto et al., 2004). Upon tetanic stimulation, the equilibrium of F-actin/G-actin shifted toward F-actin, which was accompa- nied by spine enlargement (Figure 3). In contrast, prolonged low-frequency stimulation, typically inducing LTD, lead to spine shrinkage and actin depolymerization. This evidence suggests that the equilibrium of F-actin/ G-actin regulates bidirectional structural plasticity. The chloride sensor, Clomeleon, consists of CFP, a ﬂexible pep- tide linker, and a Cl−sensitive YFP (with S65G, S72A, K79R, T203Y, H231L mutations) (Kuner and Augustine, 2000). YFP intensity is quenched in the presence of Cl−, thereby chang- ing FRET efﬁciency in a Cl−concentration-dependent manner. Using this probe, in hippocampal dissociated cultures of neurons and glial cells, the developmental time course of Cl−concentra- tion was investigated (Kuner and Augustine, 2000). Phosphatidylinositol 3,4,5-Trisphosphate (PIP3) PI3K inhibitor treatment decreased PIP3 accumulation in spines, indicating that PIP3 accumulation is largely due to basal PI3K activity in spines. This result is consistent with a previous report in which PI3K is ubiquitously localized in neuronal cells, but only becomes active after AMPA-R binding (Man et al., 2003). During Using EKAR in hippocampal pyramidal neurons, Harvey et al. observed ERK activity induced by back-propagating action potentials (Harvey et al., 2008a). Stimulated bursts of action potentials caused global Ca2+ inﬂux through voltage-gated Ca2+ channels, leading to Ras activation, an upstream molecule of October 2013 \| Volume 7 \| Article 163 \| 11 www.frontiersin.org FRET analysis of neuronal circuit Ueda et al. molecular basis of motility and morphological changes of spines (Okamoto et al., 2004, 2009; Matus, 2005; Honkura et al., 2008). ERK (Yasuda et al., 2006; Harvey et al., 2008b). After stimula- tion, ERK activity reached a peak by around 5 min, then gradually decreased, and ﬁnally returned to basal levels by 30 min. The time course of ERK activation was longer than that of Ras, consis- tent with the idea that ERK is the downstream effector of Ras. They also investigated ERK activity in the somatic cytoplasm and nucleus of neuronal cells. After theta-burst stimulation, ERK activity in both regions was up-regulated in a parallel manner, indicating that global Ca2+ inﬂux through VGCCs can diffuse rapidly between these two compartments (Harvey et al., 2008a). As in non-neuronal cells, F-actin in dendritic spines undergoes a unique directional treadmilling as revealed with experiments using a photoactivatable (PA)-GFP-actin or a photoconvertable ﬂuorescent protein (Honkura et al., 2008; Frost et al., 2010). G-actin is added to the barbed end of F-actin at the periphery of dendritic spines and at the base of the dendritic spine, F-actin is continuously disassembled to G-actin at the pointed end of actin. Taken together, there is an overall directional movement of F-actin from the periphery toward the spine base (Honkura et al., 2008; Frost et al., 2010). Another way to look at this is to divide the actin population into different pools. The ﬁrst pool of F-actin, found at the periphery, has a relatively high turnover of about 40 s (Honkura et al., 2008). The second pool is the pop- ulation that resides at the base of spines, with a turnover time of 17 min (Honkura et al., 2008). Phosphatidylinositol 3,4,5-Trisphosphate (PIP3) These two pools are relatively static and help to maintain the overall spine shape and size. In addition, there is a third pool that appears after LTP induc- tion (Honkura et al., 2008). The turnover time of this pool is 2–15 min and it spreads all over the spine. This pool is required to maintain dendritic spine enlargement upon sLTP induction. If this pool extrudes into the dendritic shafts then sLTP was not maintained. Chloride sensor l l While the Cl−concentration in glia cells was low throughout embryonic and postnatal stages, the concentration in neurons was higher at embryonic stages, and then decreased during postnatal develop- ment, consistent with the observation that activation of GABA receptors in immature neurons leads to neuronal excitation rather than inhibition (Kuner and Augustine, 2000). Using this probe, it was also possible to observe Cl−inﬂux through GABA receptors in hippocampal CA1 pyramidal neurons following interneuron stimulation (Berglund et al., 2006). However, at this point, the sensitivity of the Cl−sensor is not as good as to visualize Cl− inﬂux induced by unitary inhibitory postsynaptic current (ipsc). This would require further elaboration of the probe. FIGURE 3 \| Dendritic spine expressing actin FRET probe. Single dendritic spine that was subjected to local tetanic stimulation is accompanied by the enlargement of spine and actin polymerization. From Okamoto et al. (2004). circuit dynamics with a voltage- sensitive ﬂuorescent protein. J. Neurophysiol. 108, 2323–2337. doi: 10.1152/jn.00452.2012 Voltage sensors explain long-term potentiation?” (Sanes and Lichtman, 1999), well represents the sentiment around that time on the never- ending debate on the mechanism of LTP. Fortunately, the recent introduction of technologies to optically measure the activity of molecules involved in synaptic plasticity has drastically changed the ﬁeld and successfully clariﬁed a number of points that remained unsolved before and provided new concepts of synaptic plasticity. Electrophysiological recordings are considered to be the “gold standard” technique for measuring neuronal membrane poten- tials. However, several drawbacks to this method exist, such as the invasive nature of the technique and limitations in the number of neurons that can be measured simultaneously. To circumvent these issues, small molecular weight voltage-sensitive ﬂuorescent dyes have been used with some success. The main disadvantage of using small molecular weight voltage-sensitive ﬂuorescent dyes is the lack of cell-type speciﬁcity, because the dyes are generally bogus loaded and taken up by cells in a non-speciﬁc manner. It is also important to note that the dyes can diminish over time or cause toxicity. Therefore, these dyes are mostly suited for use in acute experiments. In the quest to understand the molecular mechanisms under- pinning neuronal circuit plasticity, FRET has played a critical role in revealing important insights into the spatiotemporal dynam- ics of the key players. However, limiting its further application, it has been empirically known that it is difﬁcult to establish trans- genic mice expressing FRET probes (Hara et al., 2004). This may be due to the repeat of very similar DNA sequence (CFP and YFP) within transgene (Kamioka et al., 2012). It is also possi- ble that probe proteins work as a gain-of-function mutant that hampers the function of endogenous proteins (Hara et al., 2004). Nonetheless, number of transgenic animals expressing FRET probes has been increasing (Hara et al., 2004; Berglund et al., 2006; Zhang et al., 2010; Yamaguchi et al., 2011; Kamioka et al., 2012; Wang et al., 2012; Thunemann et al., 2013). Additional difﬁculty lies in the detection of FRET, especially in the in vivo preparation. In practice, the animal’s heartbeat and breathing introduce not only the movement of the cells during imag- ing but also the hemodynamic noise and therefore changes the absorbance in the optical path of the excitation and emission of ﬂuorescence (Akemann et al., 2012). This will affect the accuracy of FRET data acquisition. Actin Actin is the major cytoskeletal protein in dendritic spines (Matus, 2005; Okamoto et al., 2009). It exists in equilibrium between two forms, globular (G-actin) and ﬁlamentous actin (F-actin) (Okamoto et al., 2009; Saneyoshi and Hayashi, 2012). Actin has a rapid turnover time within the dendritic spine. An experiment using ﬂuorescence recovery after photobleaching (FRAP) of GFP- fused actin revealed that over 85% of actin in dendritic spines is dynamically turning over, with an average time constant of 44 s (Star et al., 2002). This dynamic turnover is the underlying FIGURE 3 \| Dendritic spine expressing actin FRET probe. Single dendritic spine that was subjected to local tetanic stimulation is accompanied by the enlargement of spine and actin polymerization. From Okamoto et al. (2004). October 2013 \| Volume 7 \| Article 163 \| 12 Frontiers in Neural Circuits www.frontiersin.org FRET analysis of neuronal circuit Ueda et al. CONCLUDING REMARKS We thank Takeo Saneyoshi, Akihiro Goto, Ryan A. Colyer, and Lily Yu for comments on the manuscript. This work was supported by RIKEN, NIH grant R01DA17310, Grant-in- Aid for Scientiﬁc Research (A) and Grant-in-Aid for Scientiﬁc Research on Innovative Area “Foundation of Synapse and Neurocircuit Pathology” from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Yasunori Hayashi). In 1990s, the readout of synaptic plasticity was mostly lim- ited to the size of electrical response of synapse. The data were analyzed by applying to mathematical model of synaptic trans- mission established from studies on neuromuscular junction, which later turned out to be not compatible to the central synapse and caused a huge confusion in the ﬁeld. The title of a review written by Sanes and Lichtman “Can molecules AUTHOR CONTRIBUTIONS h b d h Yoshibumi Ueda, Showming Kwok, and Yasunori Hayashi jointly wrote the manuscript. Voltage sensors With continued technological advances, it will be possible to apply FRET to increasingly complex preparations, even in vivo, to fully understand the complicated neuronal signaling processes that occur in the ever-changing brain. Genetically-encoded membrane potential sensors, VSFP2 (Sakai et al., 2001) and Mermaid (Tsutsui et al., 2008), allow us to visualize the membrane voltage of a large number of individual neurons with high temporal resolution. Both probes are based on a membrane embedded phosphatase that senses voltage, Ci-VSP, a protein derived from tunicate, Ciona intesti- nalis. Ci-VSP is composed of a voltage-sensor domain (VSD) and phosphatase domain (Murata et al., 2005). The phos- phatase domain on the C-terminus was replaced with a ﬂuo- rophore pair fused in tandem. Membrane depolarization causes a conformational change in the overall structure, leading to a decrease in the distance between ﬂuorophores, and ultimately a change in FRET efﬁciency. In cultured cortical neurons express- ing Mermaid, a stimulated burst (30 pulses at 100 Hz) of spikes could be observed (Tsutsui et al., 2008). Recently, VSFP- butterﬂy and ArcLight were developed, where the acceptor was moved from the C-terminus to the N-terminus (Akemann et al., 2012, 2013; Jin et al., 2012). VSFP-butterﬂy has been used to visualize changes in membrane voltage elicited by the stimu- lation of a single whisker in layer 2/3 pyramidal neurons in the mouse barrel cortex (Akemann et al., 2012). The authors also succeeded in visualizing spontaneous slow brain oscilla- tions traveling over the somatosensory cortex (Akemann et al., 2012). PIP3 controls synaptic function by maintaining AMPA receptor clus- tering at the postsynaptic mem- brane. Nat. Neurosci. 13, 36–44. doi: 10.1038/nn.2462 Aoki, K., Nakamura, T., and Matsuda, M. (2004). Spatio-temporal regu- lation of Rac1 and Cdc42 activity during nerve growth factor-induced neurite outgrowth in PC12 cells. J. Biol. 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Copyright © 2013 Ueda, Kwok and Hayashi. This is an open-access arti- cle distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or repro- duction in other forums is permitted, provided the original author(s) or licen- sor are credited and that the original publication in this journal is cited, in accordance with accepted academic prac- tice. No use, distribution or reproduction is permitted which does not comply with these terms. 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. Zhu, J. J., Qin, Y., Zhao, M., Van Aelst, L., and Malinow, R. (2002). Ras and Rap control AMPA recep- tor trafﬁcking during synaptic plasticity. Cell 110, 443–455. doi: 10.1016/S0092-8674(02)00897-8 Zhang, J., Ma, Y., Taylor, S. S., and Tsien, R. Y. (2001). Genetically encoded reporters of protein kinase A activity reveal impact of sub- strate tethering. Proc. Natl. Acad. Sci. U.S.A. 98, 14997–15002. doi: 10.1073/pnas.211566798 Received: 30 July 2013; accepted: 23 September 2013; published online: 10 October 2013. Citation: Ueda Y, Kwok S and Hayashi Y (2013) Application of FRET probes in the analysis of neuronal plasticity. Front. Neural Circuits 7:163. doi: 10.3389/fncir. 2013.00163 Received: 30 July 2013; accepted: 23 September 2013; published online: 10 October 2013. Citation: Ueda Y, Kwok S and Hayashi Y (2013) Application of FRET probes in the analysis of neuronal plasticity. Front. Neural Circuits 7:163. doi: 10.3389/fncir. 2013.00163 Conﬂict of Interest Statement: Yasunori Hayashi is partly supported by Takeda Pharmaceuticals Co. Ltd. and Fujitsu Laboratories. The other Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y. F., Nakano, M., et al. 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https://openalex.org/W2625707410	https://www.jstage.jst.go.jp/article/hptf/24/0/24_14123/_pdf	Japanese	null	Magnetically Controlled Nitric Oxide Release	Hosokawa Funtai Kogaku Shinko Zaidan nenpo	2,016	cc-by	861	抄録一酸化窒素（NO）は免疫・呼吸器・循環器系等の多くの生体システムの制御に重要な役割を果たす生理活性物質として知られる反面，有毒ガスとしても認知されており，作用させるタイミングや暴露量を誤れば重大な副作用をもたらす．いわば「諸刃の剣」であるNO の医科学的な利活用には，「必要な時」に「必要な分量」だけNO を作用させるNO の制御放出技術の確立が必要となる．本研究は，非侵襲的に生体透過能の優れる磁場の特長を活かした全く新しいNO 制御放出技術として，熱分解してNO を放出する化合物と磁性ナノ粒子を均一に複合化・適度な大きさとしたナノ複合体から，外部磁場をトリガーとしてNO を放出させる技術の開発をめざした．NO 放出化合物のスクリーニング，溶媒蒸発法を用いたナノ複合体合成，NO 制御放出実験，および細胞毒性評価試験を行うことにより，交番磁場の印加時のみにNO を放出する低細胞毒性なNO 放出ナノ複合体の合成に成功した． Magnetically Controlled Nitric Oxide Release 研究代表者Research leader: ABSTRACT Nitric oxide (NO) is a major signalling molecule in neural, circulatory and immune systems. The biological effects of NO have been shown to be highly site, concentration and dosage dependent. This project aims to develop a system which enables controlled release of NO by an external magnetic ﬁeld, to which tissues are almost transparent. イミングや暴露量を誤れば重大な副作用をもたらす[1]．いわば「諸刃の剣」であるNO の医科学的な利活用には，「必要な時」に「必要な分量」だけNO を作用させる，NO の制御放出技術の確立が必要となる．本研究の目的は，NO が実際に作用している生体深部で，「必要な時」に「必イミングや暴露量を誤れば重大な副作用をもたらす[1]．いわば「諸刃の剣」であるNO の医科学的な利活用には，「必要な時」に「必要な分量」だけNO を作用させる，NO の制御放出技術の確立が必要となる．本研究の目的は，NO が実際に作用している生体深部で，「必要な時」に「必イミングや暴露量を誤れば重大な副作用をもたらす[1]．いわば「諸刃の剣」であるNO の医科学的な利活用には，「必要な時」に「必要な分量」だけNO を作用させる，NO の制御放出技術の確立が必要となる．本研究の目的は，NO が実際に作用している生体深部で，「必要な時」に「必 Hosokawa Powder Technology Foundation ANNUAL REPORT No.24(2016) 133–137 /Doi:10.14356/hptf.14123 Hosokawa Powder Technology Foundation ANNUAL REPORT No.24(2016) 133–137 /Doi:10.14356/hptf.14123 1．Fe3O4 ナノ粒子の合成 1．Fe3O4 ナノ粒子の合成ナノ複合体を構成する磁性ナノ粒子である平均粒径22 nm のFe3O4 ナノ粒子は，オレイン酸の共存下，ステアリン酸鉄（III）の熱分解反応をトリ-n-オクチルアミン溶液中で行うことにより合成した．具体的には，300 ml の三ツ口フラスコに，ステアリン酸鉄（III）（7.2 g），オレイン酸（1.2 g）およびトリ-n-オクチルアミン（32.26 g）を加え，380°C で30 分間反応させることにより合成した．生成したFe3O4 ナノ粒子は遠心分離により回収した．交番磁場下で磁性ナノ粒子が発熱する現象自体は古くから知られているが，その熱エネルギーをトリガーとして生体内で化学反応を行わ研究背景と目的一酸化窒素（NO）は免疫・呼吸器・循環器系等の多くの生体システムの制御に重要な役割を果たす生理活性物質として知られる反面，有毒ガスとしても認知されており，作用させるタ一酸化窒素（NO）は免疫・呼吸器・循環器系等の多くの生体システムの制御に重要な役割を果たす生理活性物質として知られる反面，有毒ガスとしても認知されており，作用させるタ一酸化窒素（NO）は免疫・呼吸器・循環器系等の多くの生体システムの制御に重要な役割を果たす生理活性物質として知られる反面，有毒ガスとしても認知されており，作用させるタ – 133 – Copyright © 2017 The Author. Published by Hosokawa Powder Technology Foundation. This is an open access article under the CC BY 2.1 JP license (http://creativecommons.org/licenses/by/2.1/jp/). Research Grant Report Hosokawa Powder Technology Foundation ANNUAL REPORT No.24(2016) 133–137 せる研究例はこれまでにない．なぜならば，ナノ粒子は非常に大きな比表面積を有し周辺への熱散逸が著しいため，周辺組織の加熱損傷を引き起こすことなくナノ粒子だけを加熱することが極めて困難なためである．この問題を解決するのが，熱分解してNO を放出する化合物と磁性ナノ粒子を均一に複合化・適度な大きさとしたナノ複合体という独創的なマテリアルデザインである．NO 放出に必要な熱分解反応を行う高温度領域を複合体内部のみに限定することで，周囲への熱散逸の低減をはかる（図1 参照）．また，発生したNO はガス状態にあるため，複合体外部へ容易に拡散するであろう．要な分量」だけNO を放出させるシステムの基盤となる技術を確立することである．光をトリガーとして機能性分子を放出する光ケージ化合物は「必要な時」に「必要な分量」だけ物質を放出させる有効な手法であり，光をトリガーとするNO の制御放出も既に報告されているが[2]，光の生体組織への侵入長は最大でも数cm 程度であるため，NO が実際に作用している生体深部で光ケージ化合物を用いることは極めて難しい．核磁気共鳴画像診断で生体深部の情報が得られることからも分かるように，磁場の生体透過能は極めて高い．本研究はこの点に着目し，非侵襲的に生体透過能の優れる磁場の特長を活かした全く新しい技術の開発をめざす．生体透過性に優れる利点を持つ磁場であるが，有機分子との相互作用が皆無に近いという問題がある．そのため，磁場をトリガーとして用いるには，磁場の持つエネルギーをより使いやすい状態に変換せねばならない．その有効な手段のひとつが，交番磁場下で磁性ナノ粒子が発熱する現象[3] を利用して磁場のエネルギーを熱エネルギーに変換することである．本研究では，交番磁場下における磁性ナノ粒子の発熱を利用して NO を放出するナノ複合体（図1）を創製することを目指す．研究方法 1．Fe3O4 ナノ粒子の合成 1．NO 放出化合物のスクリーニング 1．NO 放出化合物のスクリーニング NO 放出化合物とFe3O4 ナノ粒子からなる NO 放出ナノ複合体において，交番磁場下での Fe3O4 ナノ粒子の発熱を利用してNO を遊離させるためには，適切な分解温度を有するNO 放出化合物の選択が鍵となる．本研究の最終的な目的は，NO が実際に作用している生体深部で「必要な時」に「必要な分量」だけNO を放出させることであり，具体的には交番磁場が印加されたときのみNO を遊離するナノ複合体を創製することである．そのためには，人体の通常体温程度の温度領域（40°C 以下程度）における熱分解反応は無視できるくらい少なくなければならない．その一方で，周辺組織の加熱損傷を起こすことなくNO 放出化合物を熱分解・ NO を遊離させるためにはNO 放出化合物の熱分解温度は高すぎてはならない．質量分析装置が付属した熱重量分析装置を持ちいて，これらの条件を満足するNO 放出化合物のスクリーニングを行った結果，40°C 以上でのみNO を放出する化合物を見いだすことに成功した（図2）． 4．交流磁場を用いたNO 放出実験ナノ複合体分散水溶液をガラス瓶にとり，交番磁場装置（5005，第一高周波）を用いて交番磁場（330 G, 100 kHz）を印加した．ナノ複合体から周辺への熱散逸の目安となるナノ複合体分散水溶液の温度は，ガラスファイバー温度計（FL-2000，安立計器）を用いて測定した． 5．NO 発生量の定量交番磁場下でのFe3O4 ナノ粒子の発熱により NO 放出化合物から遊離したNO は，ナノ複合体の分散媒である水に溶解するため，その濃度を測定することにより放出NO 量を評価した．磁場印加終了後すぐにナノ複合体分散水溶液を遠心分離し，NO 指示薬（DAF-2）を含むPBS 緩衝溶液で上清を希釈・測定溶液を調整した．波長490 nm における発光強度は，マイクロプレートリーダ（Fluoroskan Asent FL，サーモフィッシャー）を用いて測定した．別途測定しておいた発光強度-NO 濃度検量線を用いて測定溶液中のNO 濃度を見積もった． 3．ナノ複合体の細胞毒性試験ナノ複合体の細胞毒性は，CellTiter-GloTM Luminescent Cell Viability Assay（プロメガ）を用いて，T24，H460 およびCHO 細胞に対してアデノシン3 リン酸（ATP）測定法[5] により行った．96 ウェルプレートに細胞密度が1 × 104 個となるように播種・24 時間培養した後に，所定濃度のナノ複合体分散液をウェルに添加した．更に1 日培養した後に100 μl の試薬を直接ウェルに添加した．37°C で15 分間培養した後に，マイクロプレートリーダ（（Fluoroskan Asent FL，サーモフィッシャー）を用いて波長 490 nm における発光強度を測定した．すべての実験は3 回繰り返した．別途測定しておいた発光強度-ATP 濃度検量線を用いて細胞中の ATP 濃度を見積もった．細胞生存率（%）はナノ複合体を加えていないウェルを基準として計算した． NO 放出化合物の熱分解実験は質量分析装置が付属した熱重量分析装置（TG-DTA2000SA/ MS9610，ブルカーAXS）により行った．透過型電子顕微鏡（TEM）観察はJEM-1400（JEOL）により行った．TEM 試料は，ナノ粒子分散液をカーボン被膜で被覆されたCu グリッドに滴下することにより調製した． NO 放出化合物の熱分解実験は質量分析装置が付属した熱重量分析装置（TG-DTA2000SA/ MS9610，ブルカーAXS）により行った．透過型電子顕微鏡（TEM）観察はJEM-1400（JEOL）により行った．TEM 試料は，ナノ粒子分散液をカーボン被膜で被覆されたCu グリッドに滴下することにより調製した． 2．ナノ複合体の作製熱分解してNO を放出する化合物（NO 放出化合物）およびFe3O4 ナノ粒子から構成されるナノ複合体（図1 参照）は溶媒蒸発法[4] により合成した．Fe3O4 ナノ粒子とNO 放出化合物を含むクロロホルム溶液，界面活性剤，および水から構成されるマイクロエマルションを室温で48 時間放置することによりクロロホルムを完全に蒸発させ，NO 放出化合物とFe3O4 ナノ粒子から構成されるナノ複合体を得た．得られたナノ複合体は磁気分離と水による洗浄を2 回繰り返して精製した．図1　交番磁場下でNO を放出するナノ複合体の概念図．熱分解してNO を放出する化合物と交番磁場下で発熱する磁性ナノ粒子を均一に複合化・適度な大きさとした凝集体であり，NO 放出に必要な熱分解反応を行う高温度領域を複合体内部のみに限定することで，周囲への熱散逸を低減している．図1　交番磁場下でNO を放出するナノ複合体の概念図．熱分解してNO を放出する化合物と交番磁場下で発熱する磁性ナノ粒子を均一に複合化・適度な大きさとした凝集体であり，NO 放出に必要な熱分解反応を行う高温度領域を複合体内部のみに限定することで，周囲への熱散逸を低減している． – 134 – Research Grant Report Hosokawa Powder Technology Foundation ANNUAL REPORT No.24(2016) 133–137 6．その他の分析方法研究成果研究成果 2．NO 放出ナノ複合体の合成と特性評価 NO 放出化合物とFe3O4 ナノ粒子を均一に複合化・適度な大きさのナノ複合体とすることにより，周囲への熱散逸を低減しつつ，内部の高温度領域のみからNO を放出させる事を目指した（図1 参照）．ナノ複合体の合成は，NO 放出化合物およびFe3O4 ナノ粒子を含むクロロホ – 135 – Research Grant Report Hosokawa Powder Technology Foundation ANNUAL REPORT No.24(2016) 133–137 ルム溶液を用いて溶媒蒸発法[4] により行った． NO 放出実験はナノ複合体分散水溶液に対して交番磁場を印加することにより行った．なお，交番磁場の印加に際しては，ナノ複合体分散水溶液の温度をNO 放出化合物の熱分解が無視できる40°C 未満に保つようにした．このことにより，交番磁場印加時に発熱したFe3O4 ナノ粒子が遊離させたNO のみを評価することができる．ルム溶液を用いて溶媒蒸発法[4] により行った． NO 放出実験はナノ複合体分散水溶液に対して交番磁場を印加することにより行った．なお，交番磁場の印加に際しては，ナノ複合体分散水溶液の温度をNO 放出化合物の熱分解が無視できる40°C 未満に保つようにした．このことにより，交番磁場印加時に発熱したFe3O4 ナノ粒子が遊離させたNO のみを評価することができる．ノ複合体が放出可能なNO 量は0.75 nmol/mg 程度であり，交番磁場によるNO の放出速度は 0.13 nmol/mg・分程度であることが分かった（図 3（b））． T24，H460 およびCHO 細胞を用いてナノ複合体の細胞毒性を評価した（図4）．いずれの細胞種に対しても，50 μg/ml 程度の比較的高濃度な領域まで細胞毒性はなく，ナノ複合体の細胞毒性が低いことが確認された．以上の実験を，Fe3O4 ナノ粒子およびNO 放出化合物の分率等のナノ複合体作製条件，および交流磁場印加条件を様々に変化させて行うことにより，外部磁場印加時のみにNO を放出するナノ複合体を実現することに成功した（図 3）．得られたナノ複合体は大きさが100 ～ 150 nm 程度の比較的均一な形状を有しており，水溶液に安定分散する（図3（a）挿入図）．ナ以上の実験を，Fe3O4 ナノ粒子およびNO 放出化合物の分率等のナノ複合体作製条件，および交流磁場印加条件を様々に変化させて行うことにより，外部磁場印加時のみにNO を放出するナノ複合体を実現することに成功した（図 3）．得られたナノ複合体は大きさが100 ～ 150 nm 程度の比較的均一な形状を有しており，水溶液に安定分散する（図3（a）挿入図）．ナ 3．結論と展望 NO 放出化合物のスクリーニング，溶媒蒸発法を用いたナノ複合体合成，NO 制御放出実験，および細胞毒性評価試験を行うことにより，交番磁場の印加時のみにNO を放出する低細胞毒性なNO 放出ナノ複合体の合成に成功した．従来，「必要な時」に「必要な分量」だけNO を供給できないことが狭心症以外の疾患・障害治療への問題となっていた．磁場をトリガーとするNO の制御放出技術の確立は，NO が実際に作用している生体深部での制御放出実現に向けた画期的なブレイクスルーであり，医科学的見地から極めて重要である．生活習慣病や虚血・図2　昇温過程におけるNO 放出化合物のNO 放出プロファイル．40°C 以上の温度領域でのみ NO が放出されていることが分かる．図3　（a）溶媒蒸発法により合成されたナノ複合体のTEM 像．挿入図はナノ複合体の水分散液．（b）外部磁場の総印加時間とNO 総発生量の関係．図4　NO 放出ナノ複合体の細胞毒性試験の結果． 3．結論と展望 NO 放出化合物のスクリーニング，溶媒蒸発法を用いたナノ複合体合成，NO 制御放出実験，および細胞毒性評価試験を行うことにより，交番磁場の印加時のみにNO を放出する低細胞毒性なNO 放出ナノ複合体の合成に成功した．従来，「必要な時」に「必要な分量」だけNO を供給できないことが狭心症以外の疾患・障害治療への問題となっていた．磁場をトリガーとするNO の制御放出技術の確立は，NO が実際に作用している生体深部での制御放出実現に向けた画期的なブレイクスルーであり，医科学的見地から極めて重要である．生活習慣病や虚血・図2　昇温過程におけるNO 放出化合物のNO 放出プロファイル．40°C 以上の温度領域でのみ NO が放出されていることが分かる．るNO の制御放出技術の確立は，NO が実際に作用している生体深部での制御放出実現に向けた画期的なブレイクスルーであり，医科学的見地から極めて重要である．生活習慣病や虚血・図2　昇温過程におけるNO 放出化合物のNO 放出プロファイル．40°C 以上の温度領域でのみ NO が放出されていることが分かる．図3　（a）溶媒蒸発法により合成されたナノ複合体のTEM 像．挿入図はナノ複合体の水分散液．（b）外部磁場の総印加時間とNO 総発生量の関係．図4　NO 放出ナノ複合体の細胞毒性試験の結果．図4　NO 放出ナノ複合体の細胞毒性試験の結果．図4　NO 放出ナノ複合体の細胞毒性試験の結果．図2　昇温過程におけるNO 放出化合物のNO 放出プロファイル．40°C 以上の温度領域でのみ NO が放出されていることが分かる．図4　NO 放出ナノ複合体の細胞毒性試験の結果．図4　NO 放出ナノ複合体の細胞毒性試験の結果．図3　（a）溶媒蒸発法により合成されたナノ複合体のTEM 像．挿入図はナノ複合体の水分散液．（b）外部磁場の総印加時間とNO 総発生量の関係．図3　（a）溶媒蒸発法により合成されたナノ複合体のTEM 像．挿入図はナノ複合体の水分散液．（b）外部磁場の総印加時間とNO 総発生量の関係． – 136 – Research Grant Report Hosokawa Powder Technology Foundation ANNUAL REPORT No.24(2016) 133–137 参考文献再灌流障害をはじめとする既存のNO 供与体では対処できない疾患治療に向けた重要なマイルストーンになると期待される．再灌流障害をはじめとする既存のNO 供与体では対処できない疾患治療に向けた重要なマイルストーンになると期待される．本技術では外部刺激として磁場を用いるが， NO 放出の直接的なトリガーはナノ複合体が持つ熱エネルギーである．磁性ナノ粒子や金ナノ粒子を用いた温熱療法に代表されるように，ナノ粒子の持つ熱エネルギーは周辺組織を加熱する目的で使用されることが一般的であり，周辺に影響を与えないトリガーとして用いられることは皆無と言ってもよい．しかし，周辺への熱散逸を効果的に抑制するナノ複合体という独創的なマテリアルデザインがその状況を一変させるであろう．比較的低温度で開裂する化学結合様式は多く知られており，NO 以外の薬物・生理活性物質への本技術コンセプトの展開・発展も期待できよう． [1] Bogdan C., Nitric oxide and the immune response, Nature Immunology, 2 (2001) 907–916. ストーンになると期待される．本技術では外部刺激として磁場を用いるが， NO 放出の直接的なトリガーはナノ複合体が持つ熱エネルギーである．磁性ナノ粒子や金ナノ粒子を用いた温熱療法に代表されるように，ナノ粒子の持つ熱エネルギーは周辺組織を加熱する目的で使用されることが一般的であり，周辺に影響を与えないトリガーとして用いられることは皆無と言ってもよい．しかし，周辺への熱散逸を効果的に抑制するナノ複合体という独創的なマテリアルデザインがその状況を一変させるであろう．比較的低温度で開裂する化学結合様式は多く知られており，NO 以外の薬物・生理活性物質への本技術コンセプトの展開・発展も期待できよう． [2] Sortino S., Light-controlled nitric oxide delivering mo- lecular assemblies, Chemical Society Reviews, 39 (2010) 2903–2913. [3] Pankhurst Q.A., Thanh N.T.K., Jones S.K., Dobson J., Progress in applications of magnetic nanoparticles in biomedicine, Journal of Physics D: Applied Physics, 42 (2009) 224001. [4] Lu Z., Yin Y., Colloidal nanoparticle clusters: function- al materials by design, Chemical Society Reviews, 41 (2012) 6874–6887. [5] Crouch S.P.M., Kozlowski R., Slater K.J., Fletcher J., The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity, Journal of Immunologi- cal Methods, 160 (1993) 81–88. – 137 – – 137 –
https://openalex.org/W4379878685	https://zenodo.org/records/8017878/files/A%20Woman%20in%20Crop%20Science%20Conversation%20with%20Beatrice%20Ifie.pdf	English	null	A Women in Crop Science Conversation with Beatrice Ifie	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	988	Background Beatrice Elohor Ifie, born in Nigeria, obtained her first degree in Botany at the University of Ibadan, Nigeria. Because of her interest in helping farmers through crop improvement, she proceeded to study Agriculture and obtained a Master’s degree in Plant Breeding at the Department of Agronomy from the same university. After her master’s she achieved her Doctoral degree in Plant Breeding at the West Africa Centre for Crop Improvement (WACCI), University of Ghana (UG). Currently she is leading the Miscanthus Breeding Programme at the Institute of Biological, Environmental and Rural Sciences (IBERS) at the Aberystwyth University in the UK. Before joining IBERS, she was a Senior Lecturer at the University of Ghana and the Leader of the Maize Breeding Programme at WACCI. Currently she is leading the Miscanthus Breeding Programme at the Institute of Biological, Environmental and Rural Sciences (IBERS) at the Aberystwyth University in the UK. Before joining IBERS, she was a Senior Lecturer at the University of Ghana and the Leader of the Maize Breeding Programme at WACCI. 1International Maize and Wheat Improvement Center (CIMMYT), Texcoco, Mexico 1International Maize and Wheat Improvement Center (CIMMYT), Texcoco, Mexico Contact: j.vanvlassealaer@cgiar.org Contact: j.vanvlassealaer@cgiar.org Lab or field? Field f Conference or stakeholder meeting? Both, it really depends on the relevance of the theme of the meeting. Literature review or project report? Literature review Conventional or molecular methods? Both methods are complementary! Hybrid, inbred or vegetative? It depends on the crop. Qualitative or quantitative research? It depends on the traits of interest. GenStat or R? R Favourite crop & why? I do not have a favourite crop. Each crop has its own uniqueness. What do you love the most about your job? The new experience it offers to contribute to science for impact and development. I enjoy making crosses, creating useful variation, and selecting plants with desirable characteristics. Any major hurdles? Limited grant opportunities for plant breeding. Who has influenced you the most and why? Several people have influenced me in so many ways. To mention a few, I would say my dad, who started the conversation of a PhD before I even thought of it. Then, Prof. Eric Yirenkyi Danquah, the Director of WACCI, a mentor and a visionary leader who put within me the drive for excellence and opportunities to grow professionally. Most important publication or the publication of which you are most proud? The publication from my PhD, which has received the most citations: Ifie, B. E., Badu‐Apraku, B., Gracen, V., & Danquah, E. Y. (2015). Genetic Analysis of Grain Yield o IITA and CIMMYT Early‐Maturing Maize Inbreds under Striga‐Infested and Low–Soil‐Nitrogen Environments. Crop Science, 55(2), 610-623. What is your favourite aspect of your research? Receiving positive feedback from farmers and other stakeholders on potential of our crop Most important publication or the publication of which you are most proud? Can you briefly describe what you do? My research is on improving crop plants for high yield and resilience. I have worked on several food crops, but mostly focused on maize until recently when my interest shifted to the energy crop, Miscanthus. I currently lead the Miscanthus Breeding Programme at the Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University. Before joining IBERS, I was a Senior Lecturer at the University of Ghana (UG) and Leader of the Maize Breeding Programme at the West Africa Centre for Crop Improvement (WACCI) at the University of Ghana. 1 Why did you get into research and especially Crop Science? I would say it is by fate as my initial intent was to study medicine, but I ended up studying Crop Science. As a young undergraduate Plant Science student, the career path was not clear to me, not until I had the opportunity to work as an intern at the International Institute of Tropical Agriculture (IITA). The exposure I got from that internship changed it all for me. The experience stimulated my fascination for research in plant breeding. I saw the opportunities available in the field to contribute to science for impact and development, I went for it and there has been no looking back. Most important publication or the publication of which you are most proud? The publication from my PhD, which has received the most citations: Ifie, B. E., Badu‐Apraku, B., Gracen, V., & Danquah, E. Y. (2015). Genetic Analysis of Grain Yield of IITA and CIMMYT Early‐Maturing Maize Inbreds under Striga‐Infested and Low–Soil‐Nitrogen Environments. Crop Science, 55(2), 610-623. The publication from my PhD, which has received the most citations: Ifie, B. E., Badu‐Apraku, B., Gracen, V., & Danquah, E. Y. (2015). Genetic Analysis of Grain Yield of IITA and CIMMYT Early‐Maturing Maize Inbreds under Striga‐Infested and Low–Soil‐Nitrogen Environments. Crop Science, 55(2), 610-623. What is your favourite aspect of your research? Receiving positive feedback from farmers and other stakeholders on potential of our crop Receiving positive feedback from farmers and other stakeholders on potential of our crop varieties and its socioeconomic and environmental impact. 2 2 Beach or mountain? Beach Beach or mountain? Beach Tea or coffee? I am making some effort to drink tea and coffee. Both are on the same scale for me. Appetizer or dessert? Dessert. I always reserve space for dessert! Instagram or Twitter? Twitter Fame or fortune? Both Beach or mountain? Beach Tea or coffee? I am making some effort to drink tea and coffee. Both are on the same scale for me. Appetizer or dessert? Dessert. I always reserve space for dessert! Instagram or Twitter? Twitter Tea or coffee? I am making some effort to drink tea and coffee. Both are on the same scale fo Tea or coffee? I am making some effort to drink tea and coffee. Both are on the same scale for me. Instagram or Twitter? Twitter Fame or fortune? Both Many thanks to Beatrice for participating in the Women in Crop Science conversations series and we look forward to following your career as it progresses. 3 3
https://openalex.org/W1908120140	https://europepmc.org/articles/pmc4609861?pdf=render	English	null	Live or Die: Choice Mechanisms in Stressed Cells	Mediators of inflammation	2,015	cc-by	1,610	Francesco Cecconi,1,2,3 Laura Soucek,4,5,6 Dennis D. Taub,7 and Elio Ziparo8 1Cell Stress and Survival Unit, Danish Cancer Society Research Center, Strandboulevarden 49, 2100 Copenhagen, Denmark 2IRCCS Fondazione Santa Lucia, 00143 Rome, Italy 3Department of Biology, University of Rome “Tor Vergata”, 00133 Rome, Italy 3Department of Biology, University of Rome “Tor Vergata”, 00133 Rome, Italy Vall d’Hebron Institute of Oncology (VHIO), 08035 Barcelona, Spain 5Department of Biochemistry and Molecular Biology, Universitat Aut`onoma de Barcelona, 08193 Bellaterra, Spain 6Instituci´o Catalana de Recerca i Estudis Avanc¸ats (ICREA), 08010 Barcelona, Spain 5Department of Biochemistry and Molecular Biology, Universitat Aut`onoma de Barcelona, 08193 Bellaterra, Spain 6Instituci´o Catalana de Recerca i Estudis Avanc¸ats (ICREA), 08010 Barcelona, Spain p 7Center for Translational Studies, Medical Services, VA Medical Center, Department of Veteran Affairs, Washington, DC 20422, USA 8Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy 7Center for Translational Studies, Medical Services, VA Medical Center, Department of Veteran Affairs, Washington, DC 20422, USA 8Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy Correspondence should be addressed to Elio Ziparo; elio.ziparo@uniroma1.it Received 6 July 2015; Accepted 6 July 2015 Copyright © 2015 Francesco Cecconi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Plays Opposite Roles in SH-SY5Y Models of Parkinson’s Disease and Amyotrophic Lateral Sclerosis” by S. Rizza and collaborators demonstrates that denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR) modulation has different effects on neuronal viability depending on the applied stimulus and highlights GSNOR as a crucial molecular player of neuronal homeostasis. The review entitled “Redox Signaling in Diabetic Nephropathy: Hypertrophy versus Death Choices in Mesangial Cells and Podocytes” by G. Manda and collaborators emphasizes the role of the oxidative stress in diabetic nephropathy, which may act as trigger, modulator, and linker within the complex network of pathologic events. This review particularly addresses the molecular switches deciding on the renal cells fate in diabetic nephropathy leading to hypertrophy versus death choices in mesangial cells and podocytes. Cells respond actively to the environmental changes, con- stantly adjusting their structure and function in order to maintain cellular homeostasis. Such changes consequent to both physiological stresses and/or pathological stimuli may be modulated in terms of type, degree, and duration and induce multiple cellular responses. Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2015, Article ID 454863, 2 pages http://dx.doi.org/10.1155/2015/454863 Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2015, Article ID 454863, 2 pages http://dx.doi.org/10.1155/2015/454863 Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2015, Article ID 454863, 2 pages http://dx.doi.org/10.1155/2015/454863 Editorial Live or Die: Choice Mechanisms in Stressed Cells Francesco Cecconi,1,2,3 Laura Soucek,4,5,6 Dennis D. Taub,7 and Elio Ziparo8 1Cell Stress and Survival Unit, Danish Cancer Society Research Center, Strandboulevarden 49, 2100 Copenhagen, Denmar 2IRCCS Fondazione Santa Lucia, 00143 Rome, Italy 3Department of Biology, University of Rome “Tor Vergata”, 00133 Rome, Italy 4Vall d’Hebron Institute of Oncology (VHIO), 08035 Barcelona, Spain 5Department of Biochemistry and Molecular Biology, Universitat Aut`onoma de Barcelona, 08193 Bellaterra, Spain 6Instituci´o Catalana de Recerca i Estudis Avanc¸ats (ICREA), 08010 Barcelona, Spain 7Center for Translational Studies, Medical Services, VA Medical Center, Department of Veteran Affairs, Washington, DC 20 8Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy Francesco Cecconi,1,2,3 Laura Soucek,4,5,6 Dennis D. Taub,7 and Elio Ziparo8 Its role in cardiovascular diseases is addressed in the manuscript “Looking for Pyroptosis-Modulating miRNAs as a Therapeutic Target for Improving Myocardium Survival” by S. Lee et al. The authors aim at elucidating the possible role of miRNAs as therapeutic targets for preventing excessive myocardial pyroptosis in cardiovascular diseases. Hypoxia and other environmental factors such as nutri- ent deprivation, pH changes, and reduced vascularization contribute instead to endoplasmic reticulum stress (ER stress) and activate the Unfolded Protein Response (UPR) in tumor cells. The review “Cancer Microenvironment and Endoplasmic Reticulum Stress Response” by C. Giampietri and collaborators describes the molecular mechanisms of UPR and their role in promoting cancer cells survival and proliferation. Progression through the cell cycle is one of the most important decisions during the life of a cell and several kinds of stress are able to influence this choice. The manuscript “Cellular Response upon Stress: p57 Contribution to the Final Outcome” by M. N. Rossi and F. Antonangeli focuses on the contribution of cyclin-dependent kinase inhibitor p57 in regulating cell cycle arrest and apoptosis after cellular stress with particular attention to cancer cells. Drug Induced Liver Injury (DILI) represents an adverse drug reaction leading to severe liver damage. Kupffer cells sense hepatic stress and produce cytokines thus stimulating an immune response. In the manuscript entitled “Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI” V. Kegel and collaborators describe a human Kupffer cell culture model in vitro for the investigation of immune-mediated signaling in hepatic inflammatory stress involved in the pathogenesis of DILI.l Finally, the review entitled “Targeting the mitotic catas- trophe signaling pathway in cancer” by M. M. Mc Gee describes the molecular mechanism of mitotic cell death, a mechanism to remove defective and genomically unstable cells. Mitotic catastrophe is a regulated antiproliferative process that occurs as a consequence of defective mitosis and may precede apoptosis, thus representing a further response under stressful conditions.hi The readers will find in this special issue not only interesting data and updated reviews on possible mechanisms of cellular stress response, but also relevant questions waiting to be answered. The study of multiple pathways activated under stressful conditions has become increasingly com- plex, requiring expertise from all fields of biology. Francesco Cecconi,1,2,3 Laura Soucek,4,5,6 Dennis D. Taub,7 and Elio Ziparo8 Several different signaling pathways can in fact lead to repair, adaptation, or, in some cases, cell death. A prolonged inflammatory response may ultimately lead to several pathological conditions, including cancer. The specific cell response under stressful conditions has therefore crucial effects not only on the single cell fate but also more generally on tissue homeostasis and ultimately on organism health. This special issue, analyzing different experimental models, aims at highlighting some of the key mechanisms involved in cellular stress response.f Topics of this issue include different stressful conditions such as oxidative, hypoxic, endoplasmic reticulum (ER) and inflammatory stress and consequent cell responses such as apoptosis, necroptosis, cell cycle arrest, mitotic cell death, pyroptosis, and survival.f On the other hand, amyotrophic lateral sclerosis is a mul- tifactorial disease caused by motor neuron degeneration and is often associated with mutation of the superoxide dismutase 1 (SOD1) gene (encoding an enzyme that uses copper and zinc to break down toxic, charged oxygen molecules called super- oxide radicals), thus affecting SOD1 antioxidant activity. The most studied SOD1 mutation is the G93A substitution. The manuscript entitled “Postmitotic Expression of SOD1G93A Gene Affects the Identity of Myogenic Cells and Inhibits Myoblasts Differentiation” by M. Martini and collaborators Among different types of stress, oxidative stress is causally implicated in degenerative diseases onset. In particular nitrooxidative stress has been reported to be implicated in the loss of specific neuronal populations in Parkinson’s disease and amyotrophic lateral sclerosis. In the present special issue, the manuscript entitled “S-Nitrosoglutathione Reductase 2 2 Mediators of Inflammation demonstrates that oxidative stress associated with SOD1G93A expression in C2C12 myogenic cells inhibits their myogenic program and promotes a fibroadipogenic phenotype. knockout mice models that serve to define the role of CAS- PASE 8, FLIP, and FADD genes and other major components of necroptotic signaling.i i Hypoxic stress plays a key role in heart disease, cancer, stroke, and other causes of mortality. The review entitled “Hypoxia Inducible Factor Pathway and Physiological Adap- tation: A Cell Survival Pathway?” by H. Kumar and D.-K. Choi describes the role of the Hypoxia Inducible Factors (HIFs) in the survival pathways activated to regulate cellular adaptation to hypoxia. In parallel, pyroptosis is a recently identified type of regulated cell death associated with inflammatory response and with features distinct from both apoptosis and necrop- tosis. Francesco Cecconi,1,2,3 Laura Soucek,4,5,6 Dennis D. Taub,7 and Elio Ziparo8 A better comprehension of such pathways might help elucidation of disease pathogenesis and may be of crucial interest in order to develop new therapeutic strategies. Long-standing inflammatory stress may also promote tumor development, growth, and progression. In the manuscript entitled “Anti-Inflammatory Effects of a Methanol Extract from the Marine Sponge Geodia cydonium on the Human Breast Cancer MCF-7 Cell Line” S. Costantini and collaborators demonstrate that a methanol extract from this marine sponge reduces levels of VEGF and exerts a dose-dependent anti-inflammatory effect. l yf In the manuscript entitled “Multidrug Resistance Protein-4 Influences Aspirin Toxicity in Human Cell Line,” I. Massimi and collaborators investigate aspirin effect on the efflux transporter protein Multidrug Resistance Protein-4 (MRP4) expression in HEK-293 cells. The authors demonstrate that low nontoxic aspirin dosages may lead to MRP-4 overexpression, thus increasing cellular detoxification of aspirin. Since overexpression of efflux transporters in human cells is a mechanism of resistance to drugs, the manuscript by I. Massimi et al. contributes to a better comprehension of molecular mechanisms adopted by cancer cells for survival in stressful conditions during chemotherapy. Francesco Cecconi Laura Soucek Dennis D. Taub Elio Ziparo Various pathological processes such as ischemic brain injury, myocardial infarction, organ transplantation, and virus replication are accompanied by strong inflammatory stress and necroptosis. The manuscript entitled “Necroptotic Cell Death Signaling and Execution Pathway: Lessons from Knockout Mice” by J. Beliz´ario and collaborators describes recent discoveries regarding necroptosis execution pathway. The authors outline and discuss important phenotypes of
https://openalex.org/W1695348064	https://revistaseletronicas.pucrs.br/ojs/index.php/revistafamecos/article/download/2978/2260	Portuguese	null	A Conjuração dos Imbecis	Revista FAMECOS	2,008	cc-by	1,952	A CULTURA NA ERA DA INFORMAÇÃO A CULTURA NA ERA DA INFORMAÇÃO A conjuração dos imbecis Moral da Verdade, do Direito e da boa consciência — grau zero do político e, certamente mesmo, ponto mais baixo na genealogia da moral. Essa mora- lização dos valores equivale a uma derrota histórica da esquerda (e do pensamento). Mesmo a realidade, o princípio de realidade, converteu-se em artigo de fé. Questione-se a realidade de uma guerra; o julgamento não se faz esperar: traição à lei moral. A esquerda está politicamente tão desen- carnada quanto à direita. Onde foi parar o político? Eh, bem, do lado da extrema-direita. Como disse com pertinência Bruno Latour no Monde, o único discurso político na França, hoje, é o de Le Pen. Todos os outros são discursos morais e pedagógicos, discursos de alfabetizadores e de pregadores de sermão, de administradores e de programadores. Fadado ao mal e à imoralidade, Le Pen abocanha toda a perspectiva política, o saldo de tudo o que é esquecido, ou francamente rejeitado, pela política do bem e das Luzes. Quanto mais se endurece a coalizão moral contra ele — signo de impotência política — mais ele tira proveito político da imoralidade, de ser o único do lado do mal. Quando a direita colocou-se ao lado dos valores morais e da ordem estabelecida, a esquerda não hesitou, outrora, em desafiar esses mesmos valores morais em nome de valores políticos. Hoje, a esquerda é vítima do mesmo deslizamento, do mesmo despojamento: investida pela ordem moral, só pode ver cristalizar-se alhures a energia política repelida; e cristalizar-se contra ela. Os antilepenistas, apostando na denúncia unilateral e ignorando tudo da reversibilidade do mal, deixaram o monopólio deste a Le Pen, que goza assim, graças à sua exclusão, de uma posição inexpugnável. A classe política, ao estigmatizá-lo em nome da Virtude, assegura- lhe a posição mais confortável, bastando-lhe recolher toda a carga simbólica de ambi- valência, de denegação do mal e de hipocrisia produzidas espontaneamente em seu bene- fício, como se fossem encomendadas por ele, seus adversários reclamando-se do bom lado e da boa causa. A energia de Le Pen vem dos seus próprios inimigos, que se apressam em canalizar os próprios erros em benefício dele. Ainda não compreenderam que o bem não resulta jamais de uma evicção do mal, que alcança sempre uma revanche explosiva, mas de um tratamento sutil do mal pelo mal. A conjuração dos imbecis AS DUAS SITUAÇÕES são uma tão crítica e insolúvel quanto a outra: a nulidade da arte contemporânea e a impotência do político face a Jean-Marie Le Pen1. Situações que se intercambiam e resolvem por transfusão; a impotência em opor o que quer que seja de político a Le Pen desliza para o terreno da cultura e da Santa Aliança cultural. Apenas um pensamento reacionário e irracional, ou mesmo fascista, poderia pôr em xeque a arte contemporânea... Texto publicado pelo jornal francês Libération em 7 de maio de 1997. Tradução de Juremir Machado da Silva. O que se pode opor a essa conjuração respeitosa de imbecis? Nada, infelizmente, pode corrigir esse mecanismo de perversão intelectual, dado que ele se inspira na má consciência e na impotência de nossas elites “democráticas” para resolver tanto o impasse da arte quanto o impasse político da luta contra a Frente Nacional. A solução mais simples consiste em confundir os dois problemas na mesma vituperação mora- lizante. A verdadeira questão torna-se então: não se pode mais “abrir”, preferir o insólito, o insolente, o heterodoxo ou o paradoxal sem ser automaticamente considerado de extrema- direita (o que é, diga-se de passagem, uma homenagem à extrema-direita)? Por que tudo o que é moral, conforme e conformista, ou tradicionalmente de direita, tornou-se de esquerda? Revisão dolorosa: antes, a direita encarnava os valores morais, e a esquerda, ao contrário, uma certa exigência histórica e política contraditória; hoje, a esquerda, despojada de toda energia política, tornou-se pura jurisdição moral, encarnação dos valores universais, campeã do reino da Virtude e defensora dos valores museais do Bem e do Verdadeiro; jurisdição que pode exigir prestação de contas de todo mundo sem ter de responder diante de ninguém. A ilusão política da esquerda, congelada durante vinte anos na oposição, revelou-se, com a chegada ao poder, portadora, não do sentido da História, mas de Revista FAMECOS • Porto Alegre • nº 7 • novembro 1997 • semestral 7 ser social. Nisso, ele é o espelho da classe política, que exorciza nele os seus próprios males, como nós exorcizamos nela, na classe política, toda corrupção inerente ao funcio- namento social. Mesma função corruptora, mesma função de catarse. Querer extirpar isso, querer purificar a sociedade e moralizar a vida pública, querer liquidar o que encarna o mal, demonstra o desconhecimento total dos mecanismos do mal e, portanto, da própria forma do político. uma moral da História. 8 Revista FAMECOS • Porto Alegre • nº 7 • novembro 1997 • semestral A conjuração dos imbecis Le Pen significa a transcrição visível desse estado viral, sua projeção espetacular. Como nos sonhos, representa a figuração burlesca, alucinatória, desse estado latente, dessa inércia silenciosa feita de integração forçada e de exclusão sistemática em doses iguais. A esperança, nesta sociedade, de reduzir as desigualdades sociais tendo-se (quase) definitivamente afastado, não há razão para surpreender-se ao ver o ressentimento deslocar-se para as desigualdades raciais. A falência do social produz o sucesso do racial (e de todas as formas de estratégias fatais). Nesse sentido, Le Pen é o único analisador selvagem da sociedade. Que ele seja a extrema- direita, é a triste conseqüência da inexistência desde de algum tempo de esquerda e de extrema-esquerda. Desse processo complexo e inextricável de responsabilidade coletiva, somos todos cúmplices e vítimas. É portanto tipicamente mágico conjurar esse vírus, que se dissemina por toda parte em função mesmo de nosso “progresso” social e técnico, exorcizar essa maldição da exclusão, e nossa impotência diante dela, num homem, numa instituição ou num grupo execráveis, quaisquer que sejam, um cancro que bastaria operar por ablação, quando na verdade as metástases já estão por tudo. A Frente Nacional apenas segue as vias rasgadas pelas metástases, com mais violência na medida em que cremos ter extirpado o abscesso e que os gérmenes se espalham por todo o organismo. Sem contar que essa projeção mágica sobre a FN abusa exatamente dos mesmos métodos desta em relação aos imigrantes. É preciso desconfiar dessa astúcia da contaminação que, pela simples transparência do mal, faz com que o positivo se transforme em vírus negativo, e a exigência de liberdade em “despotismo democrático”. Sempre essa reversibilidade, essa espiral sutil do mal, da qual a inteligência racional não desconfia (enquanto toda a patologia moderna tanto nos ensina sobre o corpo físico, nada consideramos quanto ao Certamente não seriam os juizes, nem os intelectuais, mas somente os imigrantes que estariam também em posição de analisadores, mas um certo pensamento correto apropriou- se amplamente deles. Le Pen é único que opera uma redução radical da distinção direita/esquerda — redução à revelia, certo, mas a crítica sem apelação feita nos anos 60, e em 68, infelizmente desapareceu da vida política. Ele recupera assim uma situação de fato que a classe política se recusa a enfrentar (ela faz mesmo tudo para apagá-la através das eleições), mas da qual será necessário um dia tirar as conseqüências extremas. A conjuração dos imbecis Tudo isso para dizer que se Le Pen representa a encarnação da besteira e da nulidade — certamente esta é a dos outros, os que ao denunciá-lo denunciam a própria impotência e a própria besteira; ao mesmo tempo, transparece o absurdo de combatê-lo fron- talmente, sem nada ter compreendido desse jogo de cadeiras diabólicas, alimentando assim os próprios fantasmas, o próprio duplo negativo, numa falta aterradora de lucidez. Quem comanda esse efeito perverso — estando a esquerda bloqueada na denúncia, enquanto Le Pen detém o monopólio da enunciação; um tirando todos os benefícios do crime, a outra, todos os efeitos negativos da recriminação; ele explodindo no mal, e a esquerda aferrando-se ao vitimal. Ao encarnar o reino da Virtude, que é também o da grande hipocrisia, a esquerda alimenta o Mal. Se Le Pen não existisse, seria preciso inventá-lo. É ele que nos libera de toda uma parte maléfica de nós mesmos, da quintes- sência do que há de pior em nós. Desse ponto de vista, deveríamos anatematizá-lo — mas se ele desaparecesse, pobres de nós, entregues a todos os nossos vírus racistas, sexistas, nacionalistas (o quinhão de todos) ou simplesmente à negatividade assassina do 8 Revista FAMECOS • Porto Alegre • nº 7 • novembro 1997 • semestral 8 Uma verdade bastante simples: ao encurralar Le Pen num gueto, é a esquerda democrática que se encerra, que se designa como potência discriminatória e que se exila na sua obsessão. Ela dá automaticamente ao outro o privilégio da injustiça. E Le Pen não deixa de tirar conclusões em seu benefício dessa legalidade republicana, mas é sobretudo no prestígio ilegal, imaginário, mas muito profundo, do perseguido que ele se instala, tão bem que pode gozar ao mesmo tempo dos benefícios da legalidade e da ilegalidade. Desse ostracismo, extrai uma liberdade de expressão, uma insolência de julgamento que a esquerda não se permite. Exemplo desse pensamento mágico, simulacro, na atualidade de pensamento político, aparece na crítica a Le Pen pela exclusão dos imigrantes. Mas esta é apenas uma gota d’água no processo de exclusão social dominante em todos os níveis.2 corpo social). Devemos, para continuar na política, proteger- nos da ideologia e ver as coisas em termos de física social. Nossa sociedade democrática é a stase; Le Pen é a metástase. A sociedade global perece por causa da inércia e da imuno- deficiência. Revista FAMECOS • Porto Alegre • nº 7 • novembro 1997 • semestral 10 Revista FAMECOS • Porto Alegre • nº 7 • novembro 1997 • semestral A conjuração dos imbecis Se um dia a imaginação política, a exigência e a vontade políticas tiverem uma oportunidade de repercutir será baseada na abolição dessa distinção fóssil que se auto-anulou e desautorizou ao longo das décadas e que só se sustenta pela cumplicidade na corrupção. Distinção apagada nos fatos, mas que, através de um revisionismo incurável, tenta-se obsessivamente ressuscitar, fazendo assim de Revista FAMECOS • Porto Alegre • nº 7 • novembro 1997 • semestral 9 Isabela Bidone Delia Le Pen o gerador da única nova ocorrência política. Como se todo mundo fosse cúmplice para levar ao naufrágio o que ainda resta da democracia, certamente para dar a ilusão retrospectiva de que ela de fato existiu. Há uma possibilidade de tirar as conse- qüências dessa situação extrema (mas original) que não seja através do médium alucinatório de Le Pen, isto é, de outra maneira que não por uma conjuração mágica na qual se esgotam todas as energias? Como não sucumbir a essa excrescência viral de nossos próprios demônios a não ser reconsiderando, para além da ordem moral e do revisionismo democrático, essa análise selvagem da qual Le Pen e a FN nos despojaram ? 2 A própria exclusão, ao mesmo tempo que a fratura social, foi excluída pelo decreto de dissolução da Assembléia [Baudrillard refere-se ao decreto do presidente Jacques Chirac que, neste ano, dissolveu a Assembléia francesa, determinando novas eleições legislativas e a formação de um novo Governo, N.T.]. Notas 1 [Líder da extrema-direita francesa e símbolo da negação do cosmopolitismo de uma nação que cultua a imagem de pátria da cultura e dos Direitos do Homem, N.T.]. 2 A própria exclusão, ao mesmo tempo que a fratura social, foi excluída pelo decreto de dissolução da Assembléia [Baudrillard refere-se ao decreto do presidente Jacques Chirac que, neste ano, dissolveu a Assembléia francesa, determinando novas eleições legislativas e a formação de um novo Governo, N.T.]. 10
https://openalex.org/W1907993398	https://espace.curtin.edu.au/bitstream/20.500.11937/18387/2/235914_235914.pdf	English	null	The prognostic value of the stem-like group in colorectal cancer using a panel of immunohistochemistry markers	Oncotarget	2,015	cc-by	6,277	Oncotarget, Vol. 6, No. 14 Oncotarget, Vol. 6, No. 14 www.impactjournals.com/oncotarget/ The prognostic value of the stem-like group in colorectal cancer using a panel of immunohistochemistry markers Chee Wee Ong1, Pei Yi Chong2, Darragh G. McArt1, Jason Yongsheng Chan8, Hwee Tong Tan6, Alan Prem Kumar2,3,4,5, Maxey C. M. Chung6, Marie-Véronique Clément6,7, Richie Soong2, Sandra Van Schaeybroeck1, David J. J. Waugh1, Patrick G. Johnston1, Philip D. Dunne1,, Manuel Salto-Tellez1, Chee Wee Ong1, Pei Yi Chong2, Darragh G. McArt1, Jason Yongsheng Chan8, Hwee Tong Tan6, Alan Prem Kumar2,3,4,5, Maxey C. M. Chung6, Marie-Véronique Clément6,7, Richie Soong2, Sandra Van Schaeybroeck1, David J. J. Waugh1, Patrick G. Johnston1, Philip D. Dunne1,, Manuel Salto-Tellez1, 1Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Northern Ireland Cancer Science Institute of Singapore, National University of Singapore, Singapore 5Department of Biological Sciences, University of North Texas, Denton, Texas, United States of America 6Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 5Department of Biological Sciences, University of North Texas, Denton, Texas, United States of America 6Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Si National University of Singapore Graduate School for Integrative Sciences and Engineering, National Singapore 8Department of Medical Oncology, National Cancer Centre Singapore, Singapore These authors have contributed equally to this work Correspondence to: Manuel Salto-Tellez, e-mail: m.salto-tellez@qub.ac.uk Keywords: colorectal, carcinoma, biomarkers, cancer stem-cell, immunohistochemistry Keywords: colorectal, carcinoma, biomarkers, cancer stem-cell, immunohistochemistry Received: January 22, 2015 Accepted: March 07, 2015 Published: March 26, 2015 Received: January 22, 2015 Accepted: March 07, 2015 Received: January 22, 2015 Accepted: March 07, 2015 Published: March 26 Published: March 26, 2015 ABSTRACT Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the Western world. It is becoming increasingly clear that CRC is a diverse disease, as exemplified by the identification of subgroups of CRC tumours that are driven by distinct biology. Recently, a number of studies have begun to define panels of diagnostically relevant markers to align patients into individual subgroups in an attempt to give information on prognosis and treatment response. We examined the immunohistochemical expression profile of 18 markers, each representing a putative role in cancer development, in 493 primary colorectal carcinomas using tissue microarrays. Through unsupervised clustering in stage II cancers, we identified two cluster groups that are broadly defined by inflammatory or immune-related factors (CD3, CD8, COX-2 and FOXP3) and stem-like factors (CD44, LGR5, SOX2, OCT4). The expression of the stem-like group markers was associated with a significantly worse prognosis compared to cases with lower expression. In addition, patients classified in the stem-like subgroup displayed a trend towards a benefit from adjuvant treatment. The biologically relevant and poor prognostic stem-like group could also be identified in early stage I cancers, suggesting a potential opportunity for the identification of aggressive tumors at a very early stage of the disease. www.impactjournals.com/oncotarget Unsupervised hierarchical clustering of stage II CRC Consensus unsupervised analysis of the immunohistochemistry scoring revealed proteins with co-expression patterns that grouped into two distinct clusters and one heterogeneous cluster (Figure 1b). Expression cluster 1 contained predominately markers associated with stem-like characteristics; CD44, LGR5, SOX2 and OCT4, while cluster 2 contained inflammatory and immune associated markers; CD8, CD3, COX2 and FOXP3. Cluster 3 was a more heterogeneous group containing markers associated with a wider range of characteristics. Given the clear biology emerging at this stage, we focused on the identified distinct clusters 1 and 2. Expression of either a high stem-like or inflammatory pattern was not mutually exclusive and resulted in the appearance of four clearly distinct patient clusters. Using these clusters, we categorized the patients into one of four molecular sub-types (MST1–4) based on their expression pattern of either the stem-like markers or the inflammatory markers. These subtypes were MST1; high stem-like/low inflammatory, MST2; high stem-like/high inflammatory, MST3; low stem-like/high inflammatory, MST4; low stem-like/low inflammatory (Figure 1). The development of meaningful subgrouping based on gene expression data, which related to prognosis, was first pioneered in breast cancer by two landmark papers in 2000 and 2001 [1, 2]. These paradigm-shifting publications emphasized how tumor specific gene expression data, through the use of high throughput microarray technology, could be used to classify patients based on the biology underlying their disease. In the last decade a number of studies have been published outlining prognostic gene signatures in colorectal cancer using various high throughput microarray platforms [3–5]. However, as these prognostic signatures were purely generated for their prognostic value they failed to address the driving biology behind each individual cancer or to relate that to potential treatment strategies. Given the extreme variation in prognosis, particularly in early stage colorectal cancer, these signatures were initially aimed at informing clinical decision around whether adjuvant treatment following surgery would provide any benefit over watchful surveillance. Nevertheless, the publication of three recent studies sought to set a new paradigm for the classification of colorectal cancer based on gene expression profiling [6–8]. While each of these studies generated independent transcriptional signatures, a common theme was the emergence of a stem cell subtype; which defined the poor prognostic group in stage II CRC. The importance of the prognostic value of this stem cell subtype was subsequently validated in the CRC consensus analysis presented at the American Society of Clinical Oncology (ASCO) meeting in 2014 [9]. Prognostic relevance of identified molecular sub-types in stage II CRC To investigate the clinical relevance of our identified subtypes, we carried out survival analysis using overall survival limited to five years in our stage II cohort. While there was no significant difference in survival between MST1 to 4 (Supplementary Figure S1), there appeared to be a trend towards a worse prognosis in MST1 and MST2, which both shared high expression of stem-like markers, compared to MST3 and MST4. We then re-classified the patients into high or low groups for either stem-like or inflammatory markers for survival analysis. A significantly worse prognosis was found in patients categorized in the high stem-like group compared to those with lower expression (Supplementary Table S1, p = 0.0288). The separation of patients using this stem-like classifier (Figure 2b) resulted in a group with a hazard ratio (HR) of 2.150 (CI 1.083 – 4.271). In contrast, overall survival was unaffected by classification using the inflammatory panel of markers. Another notable characteristic of these studies were that apart from defining the biology driving disease progression, they also attempted to develop clinically useful IHC panels to assign patients to each subtype. To date, these IHC panels have yet to be fully defined or validated in retrospective cohorts or prospective trial setting. Hence, in this study, we analyze a well- characterized retrospective CRC cohort in order to test the prognostic value of the stem-like and inflammatory subtypes, using unsupervised clustering in relation to overall survival. This was initially examined in stage II cancers, followed by the other cancer stages. Moreover, our findings are further tested by patient stratification into either in treated and untreated groups, with the aim of validating the prognostic value of the identified subgroups in CRC using routine standard IHC methods. RESULTS to either standard of targeted treatment strategies. The addition of clinicopathological markers such as tumor grade, invasion of lymph nodes, blood vessels or perineural invasion can further be used to identify poor prognostic subgroups of patients within each stage, but no definitive molecular signature has yet been attributed to these subgroups. Unsupervised hierarchical clustering of stage II CRC INTRODUCTION five years. These survival rates decrease to less than 5% for patients with advanced stage IV disease. The heterogeneity of colorectal cancers (CRC) at both the molecular level and in terms of prognosis is apparent; even within each stage, as there exist groups of patients with variation in both overall survival and response To date, the strongest prognostic value is derived from the tumor node metastasis system of staging (TNM). Patients with stage I disease have the best prognosis, with over 90% surviving past www.impactjournals.com/oncotarget Oncotarget 12763 Effect of adjuvant treatment on prognosis of identified subgroups in stage II CRC Within our stage II cohort, we have patients who were either treated with adjuvant chemotherapy www.impactjournals.com/oncotarget Oncotarget 12764 Figure 1: Unsupervised hierarchical clustering analysis of immunohistochemical staining profile. Heatmaps showing appearance of distinct patients clusters in Stage I a. II b. III c. and IV d. colorectal cancer cases. Figure 1: Unsupervised hierarchical clustering analysis of immunohistochemical staining profile. Heatmaps showing appearance of distinct patients clusters in Stage I a. II b. III c. and IV d. colorectal cancer cases. (30%) or untreated (70%). Although the treatment was heterogeneous, it was predominantly fluorouracil based (5FU). Analysis of the patients who received no adjuvant treatment confirmed the true prognostic value of the stem-like classifier (Figure 2c), with an increased HR of 2.345 (CI 1.036 – 5.311; p = 0.0409) in the high- stem patients compared to the low-stem sub-group. More importantly, analysis of the stem-like classifier in the treated-only group failed to show any prognostic value to the classifier (Supplementary Figure S2), suggesting that treatment of patients in the poor prognostic high stem-like group might improve their overall survival. prognosis in the treated group compared to the untreated group, although this finding was not significant (Supplementary Figure S3). Identification of stem-like and inflammatory associated subgroups in later stage CRC Following identification of molecular subtypes in early stage patients, we carried out a similar unsupervised clustering of stage III and stage IV patients in our cohort using the same classifiers for both stem-like and inflammatory markers (Figure 1c and 1d). This approach revealed the presence of all four of our stage II molecular subtypes, in each of the later stages, alongside the individual stem-like or inflammatory groups. Although the presence of both the stem-like and the inflammatory To further test this hypothesis, patients displaying a high stem-like classification appeared to show a trend towards a benefit from treatment with adjuvant chemotherapy as highlighted by an improved overall www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 12765 e 2: Prognostic value of identified stem-like molecular sub-groups in a. Stage I and b. II colorectal cancer ts. The prognostic value of the stem-like classifier was also observed in c. Stage II patients who received no adjuvant treatment. Figure 2: Prognostic value of identified stem-like molecular sub-groups in a. Stage I and b. II colorectal cancer patients. The prognostic value of the stem-like classifier was also observed in c. Stage II patients who received no adjuvant treatment. groups could be identified in stage III and IV patients in our cohort, there was no prognostic value to our classifier in these advanced stage cases. pre-invasive early disease population. We performed survival analysis comparing the stem-high against the stem-low group in the stage I cases. As these patients have a relatively good prognosis compared to stage II patients we used a 10-year follow up for this analysis (Figure 2a). We observed a significantly worse overall prognosis for patients classified in the stem-high group with an increased risk of death over 3 times that of the low-stem group (HR = 3.118, CI 1.283 – 7.578, p = 0.0121). Prognostic value of the stem-like classifier in pre-invasive stage I colorectal patients Next, we sought to examine whether the stem- like group, which has prognostic effect in stage II, may also identify patients with poor outcome in the www.impactjournals.com/oncotarget DISCUSSION It is interesting however, that when patients in the adjuvant treated group are analyzed, the stem-like classifier is no longer prognostic (Supplementary Figure S2). Further analysis revealed that there was a clear trend for improved survival in the high stem-like subgroup following the addition of adjuvant treatment, although this effect was not significant, while there was no trend for improved survival in the low-stem group (Supplementary Figure S3). This trend towards a benefit from treatment in the otherwise poor prognostic group displays similarities to the study by Sadanandam et al., where they found no association between their stem-like subtype and DFS in patients receiving adjuvant therapy. Similar to the data presented here, when the untreated patients were selected individually the stem-like group was found to have a significant association with lower survival (Figure 2c). Given that a large proportion of stage II patients who are given adjuvant therapy do not gain any benefit from this treatment, this finding warrants further investigation, as current decisions on whether to offer adjuvant chemotherapy still rely on a number of other classical prognostic factors. g p pi g [ ] The high expression of the inflammatory-related factors CD3, CD8 and FOXP3 has been shown in a number of other studies [12–14]. FOXP3 has been reviewed to be associated with either good or neutral prognostic effect in colorectal cancer [12]. Thus, it has been suggested that the favorable prognostic effect of FOXP3 may be due to the ability to suppress tumor- promoting inflammatory responses to gut microbes [13]. It has been hypothesized that increased inflammation could either provide a favorable environment for the dissemination of tumor cells or an indication of protective host responses [14]. The improved survival associated with CD3 and CD8 expression could be an effect of micrometastatic suppression induced by a systemic immunosurveillance mechanism [15]. The role of COX2 in the immune tumor microenvironment in colon cancer is well-defined and the uses of inhibitors, such as aspirin, in prevention of hereditary cancers of the colon are commonly accepted [16, 17]. Although stage I patients currently have a very favorable outcome, with a 5-year survival rate of over 90%, we found that the stem-like IHC panel could identify a subgroup with a poor overall survival rate (Figure 2a). DISCUSSION state [21, 22]. Although both CD133 and p53 appeared to cluster within the stem-like subgroup, we chose to leave these factors out of further analysis. This was due to the overall low levels of staining observed for CD133 within our TMA (Table 2) and the current uncertainty about using p53 staining as a surrogate for mutation identification. Although given the role for both CD133 and p53 in cell self-renewal these finding do justify further investigations into the relationship of these markers within a stem-like signature.i Even though numerous studies have consistently showed evidence of different subtypes outlining the heterogeneity nature of colorectal cancer [6, 10, 11], the integration of these gene expression profiles in the clinical context still require extensive validation. Using our well-characterized retrospective cohort of patients with extensive survival follow-up, we aimed to define markers of clinically relevant and diagnostically useful subgroups in colorectal cancer. As with a number of other studies, we initially focused on the stage II patients within our cohort. Using unsupervised consensus-based clustering, we found that our dataset subdivided into three general clusters based on their IHC profiles (Figure 1, Supplementary Tables S2). Although one of these clusters represented biology which was heterogeneous in nature, it was interesting that the other two clusters were underpinned by clearly distinct driving biology, namely, a subgroup showing high expression of inflammatory-related factors (CD3, CD8, COX-2 and FOXP3) and a subgroup showing high expression of stem-like factors (CD44, LGR5, SOX2 and OCT4). The identification of these two biologically diverse inflammatory-related and stem-like clusters have been reported in a number of other studies through transcriptional profiling of colorectal cancer [6].l Our finding, which shows that stem-like phenotype is associated with worse overall survival in early stage colorectal cancer, is very similar to the findings of a number of independently published transcriptional based classification studies [8–10]. While these other studies have proposed IHC markers to define their identified subtypes, using our retrospective cohort, we have shown that our panel of CD44, LGR5, SOX2 and OCT4 can similarly identify this poor prognostic subgroup. We see a clear prognostic value of our stem-like classifier in the overall stage II population (Figure 2b) and more so in the untreated patients, where the true prognostic value can be identified (Figure 2c). www.impactjournals.com/oncotarget Oncotarget 12766 MATERIALS and METHODS p ( ) For the antigen-retrieval using the Microwave Processor, the sections were subjected to steaming at 120°C for 5 min in an antigen retrieval solution (10-mM citrate buffer, pH 6.0) (Dako, Glostrup, Denmark). To avoid non-specific targeting, they were treated with serum-free protein blocking solution (Dako, Oslo, Norway) before incubation with the marker of interest. The dilution and incubation conditions were described in Table 2. After incubation, the slides were rinsed twice in saline buffer solution before secondary antibody incubation at room temperature for an hour. For antibody detection, 3, 30-diaminobenzidine (DAB) based detection system (LSAB2; Dako, Norway) was used according to manufacturer’s specifications. The stained sections were subsequently counterstained with haematoxylin. DISCUSSION Currently the proportion of patients diagnosed at stage I is low, approximately 9% in the UK, but given the recent expansion of colorectal cancer screening programs in the UK and worldwide, patients identified at this early stage will no doubt increase from its current rate [23]. Having a routine method of testing the projected increased numbers of early stage cases for traits associated with In normal tissue homeostasis, the regulation of cellular renewal is carefully maintained by stem-cells and progenitor cells. The loss of this mechanism may be the underlying cause of deregulated cell proliferation leading to cancer development [18]. Both CD44 and LGR5 are widely recognized as key regulators of the stem cell phenotype, particularly in colorectal cancer [19, 20]. While the transcription factors SOX2 and OCT4 are increasingly reported to contribute to cancer progression through their functional control of cell self-renewal and the pluripotent www.impactjournals.com/oncotarget Oncotarget 12767 in development of colorectal cancer such as such having tumor suppressive, oncogenic, proliferative, inflammatory and stem-like properties. disease progression represents an opportunity for early intervention. Evidence for the existence of transcription profiles similar to those observed in poor prognostic groups of established colorectal cancer have been found in early serrated adenoma lesions, with suggestions that the driving biology found in the stem-like tumors may already be established in precursor lesions [7]. This latter point highlights a need for the expansion of similar studies to archived polyp collections for further investigation.l Tissue microarray construction and immunohistochemistry The details of construction of the tissue microarray has been previously described [26]. Briefly, representative areas of tumor were initially marked on hematoxylin and eosin stained slides by a pathologist. These areas were taken from the center of the colorectal tumor and were subsequently cored from the donor tissue block using a 0.6 mm diameter cylinder. The cylinder cores were then transplanted into a recipient paraffin block. These processes were carried out using an ATA-100 tissue arrayer (Chemicon International, Temecula, CA, USA). Three cores were taken from tissue blocks from each of the patients. A total of 10 tissue microarray blocks, arrayed in triplicates, were constructed for this study. Consecutive 5-μm sections were then placed on polylysine-coated slides for immunohistochemical analyses. In summary, our results clearly reflect the molecular heterogeneity of colorectal cancer and are in agreement with observations in gene expression profiling studies [6, 10, 11]. Our findings also address the relevance of inflammatory and stem-like molecular subgroups in colorectal cancer. More importantly, the difference in the survival outcome between the different subtypes suggest that colorectal cancer management could be potentially improved by the inclusion of biomarker information [24, 25]. In addition the poor-prognostic stem-like subgroup identified in this cohort appears to show a trend for improved survival rates following adjuvant chemotherapy, which may aid in selecting patients who will derive benefit from treatment. The results from our study may help shed some light on identifying this particular subtype for targeted clinical intervention, particularly at the very early stage, where there is only a small subgroup of patients who will derive any benefit from the addition of adjuvant chemotherapy following surgery. Heat-mediated antigen retrieval methods were used for all the immunohistochemical markers; these were carried out using either the MicroMED TT Microwave Processor (Milestone, Sorisole, Italy) or the Bond-Max autostainer (Leica Biosystems, Newcastle, UK). The immunohistochemistry markers used were commercially available and were validated in our previous studies (Table 2). Patient cohort and selection of immunohistochemical markers The patient cohort in this study comprises of 493 CRC cancer patients that underwent primary surgery for the disease from 1990 to 1999 in the National University Hospital of Singapore. For this study, formalin-fixed and paraffin-embedded tissue blocks were retrospectively collected from the Department of Pathology. Clinical and pathological data were extracted from the medical records for the purpose of the study. The procedure for this research was approved by the ethics committee of the National University of Singapore. Patient characteristics are summarized in Table 1. The information retrieved included gender, age, tumor size, tumor stage (AJCC), histological grade, vascular invasion, perineural invasion, and lymphatic invasion. Patients were staged according to the AJCC TNM classification and were monitored for relapse of disease and death. Survival time was measured from the date of diagnosis until the date of death. Patients alive after 60 months was considered censored. For markers that were stained using the Bond-Max autostainer, the automated immunohistochemical staining process was carried out according to the manufacturer’s protocol. Briefly, the sections were subjected to heat- induced antigen retrieval with epitope retrieval ER1 solution (Leica Biosystems) at 100°C for 20 min. Subsequently, they were incubated with the primary antibody for 15 min and were washed subsequently with Bond washing buffer (Leica Biosystems). After washing, For this study, a panel of 18 immunohistochemical markers were analyzed (Table 2). These markers were broadly selected and categorized by their putative role www.impactjournals.com/oncotarget Oncotarget 12768 the slides were incubated with the secondary antibody (Bond Polymer Refine kit, Leica Biosystems) for 8 min at room temperature. Finally, chromogenic detection of the antibody was achieved by incubation with DAB for 10 min Analysis of immunohistochemistry Staining results were assessed independently by CWO and subsequently reviewed by an experienced colorectal pathologist (MST). Patient cohort and selection of immunohistochemical markers The scorings were done y p ( ) Clinical parameters Number and percentage of cohort (total n = 493) Age < 65 247 (50%) ≥ 65 246 (50%) Gender Male 240 (48%) Female 257 (52%) AJCC stage 1 and 2 262 (53%) 3 and 4 231 (47%) Ethnicity Chinese 429 (87%) Non-Chinese 68 (13%) Tumor site Colon 109 (22%) Rectal 388 (78%) Tumor size < 5 cm 194 (56%) ≥ 5 cm 303 (44%) Tumor differentiation Poor 59 (12%) Well and moderate 438 (88%) Metastasis status Positive 121 (25%) Negative 378 (75%) Vascular invasion Present 50 (10%) Absent 447 (90%) Perineural invasion Present 41 (8%) Absent 456 (92%) Lymphatic invasion Present 19 (4%) Absent 478 (96%) Number and percentage of cohort (total n = 493) Analysis of immunohistochemistry the slides were incubated with the secondary antibody (Bond Polymer Refine kit, Leica Biosystems) for 8 min at room temperature. Finally, chromogenic detection of the antibody was achieved by incubation with DAB for 10 min. the slides were incubated with the secondary antibody (Bond Polymer Refine kit, Leica Biosystems) for 8 min at room temperature. Finally, chromogenic detection of the antibody was achieved by incubation with DAB for 10 min. Staining results were assessed independently by CWO and subsequently reviewed by an experienced colorectal pathologist (MST). The scorings were done www.impactjournals.com/oncotarget Marker Clone Manufacturer Dilution Expression frequency (N = 493) Localisation Previous publication p53 D-07 Dako, Denmark 1:250 245 (49%) Nuclear CW Ong et al., Modern Path (2010) [26] SOX2 57CT23.3.4 Abcam, MA, USA 1:1000 235 (47%) Cytoplasmic CW Ong et al., Modern Path (2010) [26] STMN1 3352 Cell Signaling, MA, USA 1:250 235 (48%) Cytoplasmic HT Tan et al., J Proteome Res. (2012) [29] Number of actual cases used in previous studies may differ from the total number of cases examined due to availability of clinical information, loss of cores or lack of tumor materials available for pathological scoring purposes during the time of study. Number of actual cases used in previous studies may differ from the total number of cases examined due to availability of clinical information, loss of cores or lack of tumor materials available for pathological scoring purposes during the time of study. Figure 3: Representative immunohistochemistry images for the markers a. CD133, b. SOX2, c. OCT4, d. P27, e. P53 and f. COX-2. immunohistochemistry images for the markers a. CD133, b. SOX2, c. OCT4, d. P27, Figure 3: Representative immunohistochemistry images for the markers a. CD133, b. SOX2, c. OCT4, d. P27, e. P53 and f. COX-2. blinded to the knowledge of patient outcomes and other clinical features. For the markers that expressed immunoreactivity in the nucleus (CD3, CD8, FOXP3, Ki67, p27 and p53), a pathological score was given semi-quantitatively as 0, +1, +2 and +3, corresponding to the percentage of positively stained nucleic (Figure 3). For the cytoplasmic and membrane expressing markers (AXL, CD133, CD44, CK7, CK20, COX2, LGR5, NHE-1, NrCAM, OCT4, SOX2 and STMN1), a four- level semi-quantitative classification corresponding to the staining intensity of the cytoplasmic and membrane immunoreactivity was used (Figure 3). univariate Cox proportional hazards regression was carried out. Subsequently, a multivariate Cox model was used to test for independent prognostic value. The data were summarized with hazard ratios (HR) with their 95% confidence intervals (CI). Survival curves were plotted using GraphPad Prism Version 5. The NMF package [30] for the R statistical software [31] was used for the cluster analysis for the immunohistochemical scores. The NMF method allowed identification of clusters in an unsupervised manner based on the Euclidean distance and average linkage. All other statistical analyses were performed using the SPSS package (version 20.0 for Mac, SPSS, USA) with significance set at the 5% level. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 12769 Table 2: Summary of characteristics and expression frequency of markers, conditions used for immunohistochemistry and previous publications using the same patient cohort Marker Clone Manufacturer Dilution Expression frequency (N = 493)* Localisation Previous publication AXL HPA037422 Sigma Aldrich, MO, USA 1:1000 219 (65%) Cytoplasmic P Dunne et al., Clin Can Res (2014) [27] CD3 LN10 Novocastra, Newcastle, UK 1:1000 246 (50%) Nuclear Unpublished CD8 11F1 Novocastra, Newcastle, UK 1:40 256 (52%) Nuclear Unpublished CD133 AC133 Miltenyi Biotech, CA, USA 1:10 60 (12%) Membrane CW Ong et al., Modern Path (2010) [26] CD44 SFF-304 Bender MedSystems, CA, USA 1:250 230 (47%) Cytoplasmic Unpublished CK7 OV-TL-12/30 Santa Cruz, CA, USA 1:500 159 (32%) Cytoplasmic CW Ong et al., Modern Path (2010) [26] CK20 KS20–8 Dako, Denmark 1:500 226 (42%) Cytoplasmic CW Ong et al., Modern Path (2010) [26] COX2 M-19 Santa Cruz, CA, USA 1:250 351 (71%) Cytoplasmic CW Ong et al., Modern Path (2010) [26] FOXP3 236A/E7 Abcam, MA, USA 1:250 234 (47%) Nuclear Unpublished Ki67 SP6 Abcam, MA, USA 1:200 183 (37%) Nuclear CW Ong et al., Modern Path (2010) [26] LGR5 EPR3065Y Novus Biologicals, CO, USA 1:300 245 (70%) Cytoplasmic Unpublished NHE1 PRS4377 Sigma Aldrich, MO, USA 1:100 335 (68%) Membrane Unpublished NRCAM ab24344 Abcam, MA, USA 1:500 272 (55%) Cytoplasmic JY Chan et al., Cancer Sci. (2011) [28] OCT4 H-143 Santa Cruz, CA, USA 1:1000 279 (56%) Cytoplasmic CW Ong et al., Modern Path (2010) [26] p27 SX53G8 Abcam, MA, USA 1:500 299 (61%) Nuclear CW Ong et al., Modern Path (2010) [26] (Continued ) www.impactjournals.com/oncotarget Oncotarget 12770 Marker Clone Manufacturer Dilution Expression frequency (N = 493)* Localisation Previous publication p53 D-07 Dako, Denmark 1:250 245 (49%) Nuclear CW Ong et al., Modern Path (2010) [26] SOX2 57CT23.3.4 Abcam, MA, USA 1:1000 235 (47%) Cytoplasmic CW Ong et al., Modern Path (2010) [26] STMN1 3352 Cell Signaling, MA, USA 1:250 235 (48%) Cytoplasmic HT Tan et al., J Proteome Res. (2012) [29] *Number of actual cases used in previous studies may differ from the total number of cases examined due to availability of clinical information, loss of cores or lack of tumor materials available for pathological scoring purposes during the time of study. www.impactjournals.com/oncotarget References 1. 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https://openalex.org/W1975993486	http://journals.aiac.org.au/index.php/IJALEL/article/download/748/678/	English	null	Perceptual Convergence as an Index of the Intelligibility and Acceptability of Three Nigerian English Accents	International journal of applied linguistics and English literature	2,012	cc-by	9,047	1. Introduction The language situation in Nigeria is a result of languages in contact. This paved the way for the evolution of bilingualism / multilingualism.. However, languages can hardly come in contact without cultures coming together. The coming together of different cultures and languages aided the evolution of Nigerian English (NE). The evolution of NE emphasizes the fact that the appropriate use of the English language (EL) in Nigeria must reflect the society as well as serve her citizens and not overwhelm them. Jowitt (1991) observed that sympathetic ‘sorry’ is the conventional translation of a one word lexeme in the mother tongues (MTs). For example, Hausa, ‘sannu,’ Igbo ‘ndo’, Yoruba: ‘pele’, Tiv: ‘nsugh’, Edo: ‘koyo’, etc. These are local words for ‘sorry’ in EL. This and others are peculiarly Nigerian and cannot be said to break general rules. They are rather justified on semantic grounds showing an adaptation of the vocabulary of the EL to fill a semantic gap that exists because of the difference between British and Nigerian cultures/languages. This too could be justifiable for NE accents. That is, accents to be used by Nigerians should be Nigerian so as to fill the phonological gaps that exist because of differences between British and Nigerian phonological set ups and articulatory settings. Another problem which NE would hopefully be a cure for is in language teaching and testing. The continued dominance of the EL and the ambiguous language policy are mostly at the root of the teaching and learning problems. This is compounded by the projection of standard British English (SBE) as being superior to all varieties of English in Nigeria. According to Odumuh cited in Ohia (1997), one of the problems of teaching English in Nigeria is in the confusion as to what to accept as permissible regional varieties and what to reject as ‘sub-standard’. An acceptable Nigerian variety of the EL would be of help in finding a lasting solution to the above problem. This is necessary at this point because the issue of the increasing functions of the EL as the language of instruction, commerce, law, etc., without a change in status as second language (L2) has a direct relationship with students’ poor performance in public examinations. The immediate concern should be to develop a norm that would be generally acceptable as standard Nigerian spoken English. Perceptual Convergence as an Index of the Intelligibility and Acceptability of Three Nigerian English Accents Fatimayin Foluke (Ph.D) National Open University Of Nigeria, Lagos. Nigeria Tel: 234 803 377 3268 E-mail: folukefatimehin@yahoo.com. Received: 27-05- 2012 doi:10.7575/ijalel.v.1n.5p.100 Accepted: 26-07- 2012 Published: 03-09- 2012 URL: http://dx.doi.org/10.7575/ijalel.v.1n.5p.100 Accepted: 26-07- 2012 Pub URL: http://dx.doi.org/10.7575/ijalel.v.1n.5p.100 Published: 03-09- 2012 Abstract This study investigated perceptual convergence as a measure of the intelligibility and acceptability of three Nigerian English (NE) accents with a view to arriving at a possible norm of usage for teaching and communication purposes. The subjects were one hundred and eighty Nigerians of varied socio-economic, educational and ethno-linguistic backgrounds drawn from various offices, institutions in Kaduna, Enugu, Ibadan. Two researcher-designed instruments were used. First is the Oral Reading Test for Accent identification made up of phonological difficulties usually exhibited by NE language users. Next is a questionnaire in the form of an intelligibility and acceptability rating scale. Based on the findings, the educated NE accent was the most intelligible and acceptable, followed by the mother-tongue based NE accent and the Regional NE accents. It was recommended among others that language policy makers confront the problem of the NE corpus to be used in teaching and the Nigerian Educational Research and Development Council be commissioned to produce texts on the phonology and phonetics of the Educated NE accent. Keywords: Perceptual convergence, index, Nigerian English, Accents, Intelligibility, Acceptability 1 Introduction Keywords: Perceptual convergence, index, Nigerian English, Accents, Intelligibility, Acceptability International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] This paper is licensed under a Creative Commons Attribution 3.0 License. 1.1 Background literature on Nigerian English (NE) The emergence of NE is no longer in doubt. Odumuh (1984) asserted that NE has emerged and stabilized. It is a term used in reference to the variety of English used in Nigeria which is neither pidgin nor Standard British. It is English with a distinctive Nigerian flavour, resulting from contact between the EL and Nigerian languages. It is an indigenized variety of English as an international language. Therefore the evolution of NE can be associated with the strive to achieve a linguistic identity. The development of NE followed the global patterns described as the result of contact between languages and cultures. Describing the development and inevitability of NE, Soyinka (1993), submitted that English was forced to play “unaccustomed roles” which turned it into ‘a new medium of communication in a new organic series of mores, social goals, relationships, all of which go into the creation of a new culture’ and by extension a new variety of the language i.e. NE. While it is true that NE has emerged and has distinct linguistic features, it is also true that it has not fully developed into a standard comparable to other world standard Englishes. This is because there are still some fundamental issues to be resolved. But out of the varieties that exist, a norm can be identified as being intelligible and acceptable by all for the linguistic purpose of the Nigerian nation. This will in turn enable the Nigerian variety assume the role of a national language as well as contribute to the varieties of World Standard Englishes (WSE: Kachru, 1992). World Englishes refers to the emergence of indigenized varieties of the English language. It consists of varieties of English used in diverse sociolinguistic contexts globally. It is an outcome of how sociolinguistic histories, other cultures and languages influence the use of English in different climes where it was transported. Crystal (2007) asserts that establishing the total number of Englishes around the world is difficult as new varieties of English are constantly being developed and discovered. The English language has enjoyed tremendous linguistic patronage in the last 300 years. This according to Kachru (1992) is because of its range and depth. Factors that aided the global spread of English are economic, political, scientific and technological. While economic considerations aided its spread across the globe, political considerations helped in stabilizing such influences. Official policies also helped. 1. Introduction This may upon approval be taken as a basis for assessing language competence in the educational system as well as for communication purposes. Efforts should be geared towards integrating the EL and the indigenous languages for effective communication and as a solution Page \| 100 Page \| 100 This paper is licensed under a Creative Commons Attribution 3.0 License. to the country’s language problems. This is really necessary as attempts at promoting the indigenous languages through the language in education policy (National Policy on Education, revised draft 2007) have not been fruitful. The most likely option now as a forward is an acceptable variety of NE. This is premised on the fact that since the local languages are not used in schools as stipulated in the policy, using and adopting a homegrown Nigerian variety of the EL is likely to be more intelligible and acceptable to users and may be a means of achieving objectives of teaching/learning. This paper is licensed under a Creative Commons Attribution 3.0 License. International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] Countries using English as Further Tongue (e.g. countries of the arabophone, Francophone and lusophone world such as Tunisia, Togo, Mexico, etc. FIG.1. Lawal’s (2006) Slightly Modified Version of Kachru’s (1992) Trichotomy Showing L1, L2 And Fl Users Of English Countries using English as Mother Tongue (e.g. England, USA and Australia Countries using English as Other Tongue (i.e. the Anglophone world e.g. Nigeria, Ghana, the Philippines) International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] Countries using English as Further Tongue (e.g. countries of the arabophone, Francophone and lusophone world such as Tunisia, Togo, Mexico, etc. FIG.1. Lawal’s (2006) Slightly Modified Version of Kachru’s (1992) Trichotomy Showing L1, L2 And Fl Users Of English Countries using English as Mother Tongue (e.g. England, USA and Australia Countries using English as Other Tongue (i.e. the Anglophone world e.g. Nigeria, Ghana, the Philippines) Countries using English as Other Tongue (i.e. the Anglophone world e.g. Nigeria, Ghana, the Philippines) Countries using English as Further Tongue (e.g. countries of the arabophone, Francophone and lusophone world such as Tunisia, Togo, Mexico, etc. FIG.1. Lawal’s (2006) Slightly Modified Version of Kachru’s (1992) Trichotomy Showing L1, L2 And Fl Users Of English Countries using English as Mother Tongue (e.g. England, USA and Australia Countries using English as Other Tongue (i.e. the Anglophone world e.g. Nigeria, Ghana, the Philippines) Countries using English as Further Tongue (e.g. countries of the arabophone, Francophone and lusophone world such as Tunisia, Togo, Mexico, etc. Countries using English as Mother Tongue (e.g. England, USA and Australia FIG.1. Lawal’s (2006) Slightly Modified Version of Kachru’s (1992) Trichotomy Showing L1, L2 And Fl Users Of English Kachru’s inner circle is of native English speaking countries. These are ‘norm providing, his ‘outer-circle’ consist of ESL countries and are ‘norm-developing, while his ‘expanding-circle’ comprises EFL countries. These are ‘norm-dependent’ in the sense that the criteria for judging usage are imported from U.K and U.S.A. NE has many varieties. Empirical work in the area of the phonology of NE can be examined in two parts. The first relates to research on varieties differentiation. Brosnaham (1958) suggested a typology of four varieties based on educational attainment. Banjo (1979) identified four varieties based on linguistic deviations from the standard. Another typology is the regional parameter separating Hausa English (northern) from Igbo English (eastern) from Yoruba English (western). The second part of the empirical research on the phonology of NE relates to tests of intelligibility and acceptability. These include Tiffen’s (1974) study, Ekong (1980), Jibril (1986). The findings suggest that while social acceptability is subject to the democratic process, international intelligibility is more elite inclined (Banjo, 1979). However, Adetugbo (1987) argued that international intelligibility is an unnecessary luxury. International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] Countries using English as Further Tongue (e.g. countries of the arabophone, Francophone and lusophone world such as Tunisia, Togo, Mexico, etc. FIG.1. Lawal’s (2006) Slightly Modified Version of Kachru’s (1992) Trichotomy Showing L1, L2 And Fl Users Of English Countries using English as Mother Tongue (e.g. England, USA and Australia Countries using English as Other Tongue (i.e. the Anglophone world e.g. Nigeria, Ghana, the Philippines) To him, there is no reason for this especially as Americans, Australians, Scots have their own varieties of the EL and nobody bothers about international intelligibility especially as these are mutually intelligible regional varieties. In support of the above, it is the opinion of this writer that language should serve users by enabling them use it to express their own identity as well as relate with others around them. It should also be a reflection of their total culture. Based on this, what is needed is national intelligibility and acceptability as a first step in the right direction for Nigerian spoken English. 1.1 Background literature on Nigerian English (NE) These are the introduction of the American Peace Corps program, and the posting of American and British military personnel to different parts of the world. These helped in the spread of the English language. In addition, the official language policy which promoted the B.B.C. and V.O.A. as means of information dissemination and sometimes propaganda aided the spread. Political associations like the E.U., the Commonwealth have helped stabilize the use of English. The advancement of science and technology has also helped. This is because scientific breakthroughs recorded in the West as well as numerous technological feats have been documented in the language. Therefore a world that is becoming dependent on technology has come to rely on the use of the English language (Awonusi, 2004). An inevitable consequence of this global spread is that the language will become open to the winds of linguistic change in totally unpredictable ways. This has already happened as evidenced in the emergence of new varieties of English in the different territories where the language has taken root. These new Englishes are somewhat like dialects we all recognize within our own country except that they are on an international scale applying to whole countries or regions. They are the inevitable consequence of the spread of the English language on a global scale. Speaking on new, national and international Englishes in an attempt to portray the rapid growth of the English Language, Kachru (1992) divided its speakers into three. The ‘inner circle’ is made up of native English speaking countries such as the U.K., U.S.A., Canada, Australia, and New Zealand. The ‘outer circle’ consists of countries in the African territories, the Indian sub-continent, and the Pacific. These are countries like Nigeria, Ghana, Kenya, India, Pakistan; Singapore, etc. English here co-exists with many indigenous languages and functions as L2. In these countries, there exist several nativized varieties of the English language. The ‘expanding circle’ of Kachru’s division is made up of countries in the far East (China, Indonesia, Japan, Taiwan, etc), Middle East (Israel, Egypt, S. Arabia), and Zimbabwe where English is a foreign language. (See fig. 1). This paper is licensed under a Creative Commons Attribution 3.0 License. Page \| 101 1.1.2 Purpose of the Study The purpose of the study is to find out whether perceptual convergence can be used as a measure of the intelligibility and acceptability of three NE accents. 1.1.3 Research Questions The following questions were addressed in the study: 1. What is the users’ self perception, other users’ perception and user’s metaperception of the intelligibility of each of NE accent 1 (i.e. ENEA), NE accent 2 (RNEA), NE accent 3 (MTBNEA). 2. What is the users’ self perception, other users’ perception and user’s metaperception of the acceptability of each of NE accent 1 (i.e. ENEA), NE accent 2 (RNEA), NE accent 3 (MTBNEA)? .1.4 Research Hypotheses The following hypotheses were formulated as a guide to the study. The following hypotheses were formulated as a guide to the study. Ho1: There is no significant difference in the users’ perception, other accents users’ perception and the users’ metaperception of the intelligibility of Accent 1 (i.e. ENEA) Ho2: There is no significant difference in the users’ perception, other accents users’ perception and the users’ metaperception of the intelligibility of the Accent 2(i.e. RNEA) Ho3: There is no significant difference in the user’ perception, other accents users’ perception and the users’ metaperception of the intelligibility of accent 3 (MTBNEA) Ho4: There is no significant difference in the user’ perception, other accents users’ perception and the users’ metaperception of the acceptability of accent 1 (ENEA) o5: There is no significant difference in the user’ perception, other accents users’ perception and the users’ metaperception of the acceptability of accent 2 (RNEA) Ho6: There is no significant difference in the user’ perception, other accents users’ perception and th users’ metaperception of the acceptability of accent 3(MTBNA) 1.1.5 Conceptual Framework This study is conceptualized around some sociolinguistic, psycholinguistic and linguistic issues and concepts. The theory of languages in contact is of importance to these concepts and issues, that is the evolution of a new speech form from the communion of two or more languages. Closely related to the contact theory is the concept of varieties. The varieties of English are a result of languages in contact. They can also be regarded as the production of psycholinguistic interaction between two or more linguistic systems, those of the mother tongues (MTs) and the second language (L2). Also important is the social contact arising from the interaction between members of these groups which results in cultural and linguistic diffusion producing different varieties. 1.1.1 Statement of the Problem 1.1.1 Statement of the Problem After almost two centuries of its advent in the country, EL usage is characterized by some Nigerians using SBE, others, educated Nigerian spoken English and for a large majority, MT interference is common. This is a result of the English language being badly taught. In addition, the influence of the native languages has made performance in the EL peculiar. The focus of this study is on the intelligibility and acceptability of NE accents in relation to the perception and metaperception of the users with a view to identifying the convergence of agreement. The convergence can serve as a pointer towards the norm and also determine the reference point for standardization. It therefore used the degree of convergence among the users’ perception, other accents users’ perception and users’ metaperception to measure the intelligibility and acceptability of NE accents. Self perception is how the user of an accent perceives the accent, other users’ perception is how other users perceive an accent not their own, and metaperception is the perception of the user of an accent about how other users perceive her/his accent. The convergence point is the point of overlap among the three perceptual types relative to each of the three NE accents. Page \| 102 Page \| 102 This paper is licensed under a Creative Commons Attribution 3.0 License. International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] ISSN 2200 3592 (Print), ISSN 2200 3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] 1.1.2 Purpose of the Study 1.1.2 Purpose of the Study These varieties are of interest in this study. Crucial to the understanding of these varieties and the present study is the term accent. The Nigerian user of the English language learns it as a second language long after his first language mainly through formal instruction in school. In addition, the phonological and articulatory settings of the first and second languages are different. These !!! Fig 2.Adapted from Lawal (2000). Assessing Acceptability/Intelligibility of NE Accents. SELF- PERCEPTION OTHERS’ PERCEPTION METAPERCEPTION SELF- PERCEPTION OTHERS’ PERCEPTION !!! Fig 2.Adapted from Lawal (2000). Assessing Acceptability/Intelligibility of NE Accents. Page \| 103 Page \| 103 result in NE accents different from the SBE accents. How intelligible and acceptable these NE accents would be to Nigerian EL users is of interest to this study. Since NE is a product of this complex sociolinguistic environment, it is obvious that its growth, understanding and acceptability will depend on the perception of those in the society. Perception and metaperception are very crucial to language understanding especially in L2 situation. This is because all language understanding begins with perception. Jowitt (1991) posits that in language understanding, perception precedes production and cognition precedes perception. Related to this is the need to know how others perceive us. Knowing how others perceive us is an important aspect of one’s social life. It will enable individuals to understand their language use as well as that of others. It follows then that users’ self perception of their own accent, other accents users’ perception and user’s metaperception would be an indicator of which accent is generally intelligible and acceptable. This is illustrated in figure 2. This paper is licensed under a Creative Commons Attribution 3.0 License. 2. Methodology This is a descriptive survey type. The population for this study comprised all Nigerian users of the EL. The target populations are Nigerians with varying levels of formal education: graduates, Nigerian Certificate in Education holder (NCE), undergraduates, primary school graduates, newscasters, professionals etc. The country was divided into three language regions i.e. North (Kaduna, Hausa dominated), East (Enugu, Igbo dominated), West (Ibadan, Yoruba dominated), using stratified sampling. Incidental sampling was used to get the organizations and establishments where the samples were reached. Random sampling was carried out to get the required number of 60 respondents per region. The distribution of the sample is represented in the table. Table1. Sampling Grid Regions Total Accent North West East Total 1 20 20 20 60 2 20 20 20 60 3 20 20 20 60 Total 60 60 60 180 Two researcher-designed instruments were used. First is the Oral Reading Test for Accent Identification. Here, an admixture of NE users read a short passage and sentences. These not only have areas of phonological difficulties usually exhibited by NE language users but all the sound segments of the language. A table of specification of items on the test was used to streamline respondents into the appropriate accents used by them. Next, the intelligibility and acceptability rating scale in form of a questionnaire with speech recording of the accents used by three speakers, one from each of the identified language regions was used to obtain respondents’ views. The questionnaire was drawn in such a way that respondents were also judges of how intelligible and acceptable the accents they listened to were. The reliability index of the instruments was determined through test-retest technique three weeks after the first administration. Using Pearson Product Moment Correlation Statistics, a reliability coefficient of 0.72 was obtained. The writer personally monitored the speech recordings, listened to respondent read and administered the questionnaire. Data was analyzed using mean, and Standard Deviation (SD) to answer the research questions, while the six hypotheses were analyzed with the statistical tool of Analysis of Variance (ANOVA). 2.1 Analysis of Results The analysis of data collected and the results of the investigation carried out are reported. The analysis is based on the research questions and hypotheses stated in the write up. The mean, SD, and Analysis of Variance (ANOVA) results are presented in tables followed by a summary of the findings. Research Question 1: What was users’ self-perception, other users’ perception and user’s metaperception of the intelligibility of each of the three NE accents? Page \| 104 This paper is licensed under a Creative Commons Attribution 3.0 License. International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] Table 2. Mean and SD results of user’s self perception, other users’ perception and user’s metaperception of the intelligibility of each of the three NE accents. ENEA (Accent1 RNEA (Accent2) MTbNEA (Accent3) User’s self perception Mean 2.54 2.46 2.71 SD .986 .943 1.02 Other accent users’ perception Mean 2.67 2.60 2.63 SD .947 .999 .870 User’s metaperception Mean 2.70 2.54 2.46 SD .975 .859 .888 metaperception of the intelligibility of each of the three NE accents. ENEA (Accent1 RNEA (Accent2) MTbNEA (Accent3) User’s self perception Mean 2.54 2.46 2.71 SD .986 .943 1.02 Other accent users’ perception Mean 2.67 2.60 2.63 SD .947 .999 .870 User’s metaperception Mean 2.70 2.54 2.46 SD .975 .859 .888 Table 2 indicates that Accent 3 was the most intelligible, followed by accent 1. Accent 2 was the least intelligible. This is based on respective user’s self perception. The table also indicates that Accent 1 was the most intelligible, Accent 2 was intelligible and Accent 3 was the least intelligible according to respective users’ metaperception of the accents. Research Question 2: What was the user’s self-perception, other users’ perception and user’s metaperception of the acceptability of each of the three NE accents? Research Question 2: What was the user’s self-perception, other users’ perception and user’s metaperception of the acceptability of each of the three NE accents? Table 3. Mean and SD results of users’self-preception , other users’ perception and metaperception of the acceptability of each of the three NE accents ENEA (Accent1 RNEA (Accent2) MTbNEA (Accent3) Users’self-perception Mean 2.84 2.68 2.48 SD .917 1.02 .944 Other Users’ perception Mean 2.76 2.72 2.73 SD .935 .937 .905 User’s metaperception Mean 2.68 2.58 2.44 SD .970 1.01 .933 Table 3. This paper is licensed under a Creative Commons Attribution 3.0 License. This paper is licensed under a Creative Commons Attribution 3.0 License. 2.1 Analysis of Results Mean and SD results of users’self-preception , other users’ perception and user’s Table 3 shows that Accent 1 was the most acceptable, Accent 2 was acceptable and Accent 3 was the least acceptable according to users’ self-perception of the acceptability of the accents. The table also indicates that Accent 1 was the most acceptable and Accent 2 was the least acceptable according to other users’ perception. Table 3 shows that Accent 1 was the most acceptable, Accent 2 was acceptable and Accent 3 was the least acceptable according to users’ self-perception of the acceptability of the accents. The table also indicates that Accent 1 was the most acceptable and Accent 2 was the least acceptable according to other users’ perception. Overall, the table shows that Accent 1 was the most acceptable while accent 3 was the least acceptable according to users’ meatperception. Hypotheses 1: There is no significant difference in users’ self–perception, other accents users’ perception and users’ metapercerption of the of the intelligibility of the intelligibility of Accent1. users’ metapercerption of the of the intelligibility of the intelligibility of Accent1. Table 4. ANOVA result of each users’ self-perception, other accents users’ perception and users’ Page \| 105 Page \| 105 International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] metaperception of the intelligibility of Accent1 Sum of square Df Mean squar e F Sig Decision Between groups Within groups Total 19.83 389.30 409.13 2 177 119 9.91 3.33 2.98 .050 Significant Duncan post hoc Test Result. Intelligibility of Accent Subset for alpha = .05 Users’self perception Other accent User’s perception User’s metaperception Significance N 49 58 73 I 5.12 5.29 5.42 .369 a a b Means of the same letter are not significantly different Hypothesis 2: There is no significant difference in users’ metaperception of the intelligibility of Accent 2. International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] metaperception of the intelligibility of Accent1 Sum of square Df Mean squar e F Sig Decision Between groups Within groups Total 19.83 389.30 409.13 2 177 119 9.91 3.33 2.98 .050 Significant Duncan post hoc Test Result. Page \| 106 This paper is licensed under a Creative Commons Attribution 3.0 License. This paper is licensed under a Creative Commons Attribution 3.0 License. users’ metaperception of the intelligibility of Accent2. 2.1 Analysis of Results Intelligibility of Accent Subset for alpha = .05 Users’self perception Other accent User’s perception User’s metaperception Significance N 49 58 73 I 5.12 5.29 5.42 .369 a a b Means of the same letter are not significantly different Hypothesis 2: There is no significant difference in users’ metaperception of the intelligibility of Accent Table 5. ANOVA result of each users’ self-perception, other accents users’ perception and users’ metaperception of the intelligibility of Accent 2 Sum of squares Df Means square F Decision Between groups Within groups Total .624 216.576 217.200 2 177 119 .312 1.851 .168 Not Significant Hypothesis 3: There is no significant difference in users’self-perception, other Accents users’ perception and users’ metaperception of the intelligibility of Accent3. Table 5. ANOVA result of each users’ self-perception, other accents users’ perception and Hypothesis 3: There is no significant difference in users’self-perception, other Accents users’ perception and users’ metaperception of the intelligibility of Accent3. Hypothesis 3: There is no significant difference in users’self-perception, other Accents users’ perception and users’ metaperception of the intelligibility of Accent3. Table 6. ANOVA result of each users’ self-perception, other accents users’ perception and users’ metaperception of the intelligibility of Accent3 Sum of square df Mean square F Sig Decision Between groups Within groups Total .624 397.64 437.13 2 17 7 19.74 3.40 119 5.8 1 .00 4 Significan t Table 6. ANOVA result of each users’ self-perception, other accents users’ perception and users’ metaperception of the intelligibility of Accent3 Table 6. ANOVA result of each users’ self-perception, other accents users’ perception and users’ i f h i lli ibili f A 3 Page \| 106 Page \| 106 International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] Duncan post hoc Test Result. Intelligibility of Accents Subset for alpha = .05 Users’self perception Other accent User’s perception User’s metaperception Significance N 58 73 49 I 5.18 5.46 5.79 .35 a b c * Means of the same letter are not significantly different Hypothesis 4: There is no significant difference in users’self-perception, other Accents users’ perception and users’ metaperception of the intelligibility of Accent1. Hypothesis 4: There is no significant difference in users’self-perception, other Accents users’ perception and users’ metaperception of the intelligibility of Accent1. Table 7. This paper is licensed under a Creative Commons Attribution 3.0 License. ypothesis 5: There is no significant difference in users’self-perception, other Accents users’ perception an This paper is licensed under a Creative Commons Attribution 3.0 License. Page \| 108 This paper is licensed under a Creative Commons Attribution 3.0 License. 2.1 Analysis of Results ANOVA result of each user’s self-perception, other accents users’ perception and users’ metaperception of the intelligibility of Accent1. Between groups Within groups Total Sum of square 12.23 280.76 292.99 Df 2 177 119 Mean square 6.12 240 F 2.55 Sig 0.43 Decision Significa nt Table 7. ANOVA result of each user’s self-perception, other accents users’ perception and users’ metaperception of the intelligibility of Accent1. Table 7. ANOVA result of each user’s self-perception, other accents users’ perception and users’ metaperception of the intelligibility of Accent1. Between groups Within groups Total Sum of square 12.23 280.76 292.99 Df 2 177 119 Mean square 6.12 240 F 2.55 Sig 0.43 Decision Significa nt Duncan post hoc Test Result. Subset for alpha = .05 Acceptability of accent 1 Users’ self perception Other accent user’ Perception. Users’ metaperception Sig N 73 58 49 I 4.90 5.18 5.50 .055 b a a * Means of the same letter are not significantly different. Duncan post hoc Test Result. Subset for alpha = .05 Acceptability of accent 1 Users’ self perception Other accent user’ Perception. Users’ metaperception Sig N 73 58 49 I 4.90 5.18 5.50 .055 b a a * Means of the same letter are not significantly different. Hypothesis 5: There is no significant difference in users’self-perception, other Accents users’ perception and users’ metaperception of the intelligibility of Accent2. users’ metaperception of the intelligibility of Accent2. Page \| 107 Page \| 107 International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] Table 8. ANOVA result of each user’s self-perception, other accents users’ perception, users’ metaperception of the intelligibility of Accent2. Sum of square df Mean squar e F Sig Decision Between groups Within groups Total 20.22 397.64 437.13 2 17 7 10.11 3.40 119 4.36 .015 Significant Duncan post hoc Test Result. Subset for alpha = .05 Acceptability of accent 1 Users’ self perception Other accent user’ Perception. Users’ metaperception Sig N 58 49 73 I 5.2105 5.3824 .055 a b c Hypothesis 6: There is no significant difference in users’self-perception, other accents users’ perception and users’ metaperception of the intelligibility of Accent3. Hypothesis 6: There is no significant difference in users’self-perception, other accents users’ perception and users’ metaperception of the intelligibility of Accent3. This paper is licensed under a Creative Commons Attribution 3.0 License. 2.1 Analysis of Results Hypothesis 6: There is no significant difference in users’self-perception, other accents users’ perception and users’ metaperception of the intelligibility of Accent3. Table 9. ANOVA result of each users’ self-perception, other accents users’ perception, users’ metaperception of the intelligibility of Accent3 Sum of square Df Mean square F Sig Decision Between groups Within groups Total 8.88 321.72 330.59 2 177 4.44 2.75 119 1.6 1 .003 Significant Duncan post hoc Test Result. Subset for alpha = .05 Acceptability of accent 1 Users’ self perception Other accent user’ Perception. Users’ metaperception Sig N 73 58 49 I 5.21 5.29 5.35 .66 a a b * Means of the same letter are not significantly different Table 9. ANOVA result of each users’ self-perception, other accents users’ perception, users’ metaperception of the intelligibility of Accent3 Table 9. ANOVA result of each users’ self-perception, other accents users’ perception, users’ metaperception of the intelligibility of Accent3 Sum of square Df Mean square F Sig Decision Between groups Within groups Total 8.88 321.72 330.59 2 177 4.44 2.75 119 1.6 1 .003 Significant Duncan post hoc Test Result. Subset for alpha = .05 Acceptability of accent 1 Users’ self perception Other accent user’ Perception. Users’ metaperception Sig N 73 58 49 I 5.21 5.29 5.35 .66 a a b * Means of the same letter are not significantly different Page \| 108 Page \| 108 International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] 2.2 Summary of Findings The following is a summary of major findings. S/N INDEX OF MEASURE MOST INTELLIGIBLE LEAST INTELLIGIBLE 1 Users’ Self-perception of accents Accent 3 (MTBNEA) Accent 2 (RNEA) 2 Other User’s Perception of accents Accent 1 (ENEA) Accent2 9RNEA0 3 User’s Metaperception of accents Accent 1 (ENEA) Accent 3 (MTBNEA) MOST ACCEPTABLE LEAST ACCEPTABLE 1 Users’ Self-perception of accents Accent 1 (ENEA) Accent 3 (MTBNEA) 2 Other Use’s Perception of accents Accent 1 (ENEA) Accent 2 (RNEA) 3 User’s Metaperception of accents Accent 1 (ENEA) Accent 3 MTBNEA) International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] The following is a summary of major findings. 2.1 Analysis of Results g y j g S/N INDEX OF MEASURE MOST INTELLIGIBLE LEAST INTELLIGIBLE 1 Users’ Self-perception of accents Accent 3 (MTBNEA) Accent 2 (RNEA) 2 Other User’s Perception of accents Accent 1 (ENEA) Accent2 9RNEA0 3 User’s Metaperception of accents Accent 1 (ENEA) Accent 3 (MTBNEA) MOST ACCEPTABLE LEAST ACCEPTABLE 1 Users’ Self-perception of accents Accent 1 (ENEA) Accent 3 (MTBNEA) 2 Other Use’s Perception of accents Accent 1 (ENEA) Accent 2 (RNEA) 3 User’s Metaperception of accents Accent 1 (ENEA) Accent 3 MTBNEA) There was no significant difference in users’ self-perception, other accents users’ perception, and users’ metaperception of the intelligibility of Accent 1. Based on the results, two of the perceptual forms (other accent users’ perception and user’s metaperception) showed that Accent 1 was the most acceptable, while all three perceptual forms indicated that Accent 1 was the most intelligible. 2.2.1 Conclusions and Recommendations Conclusions and Recommendations The following conclusions and recommendations are based on the summary of finding with close reference to research questions and hypotheses already stated. 2.2 Conclusions 2.2.2 Conclusions 2.2.2 Conclusions ased on the results and findings, the following conclusions can be reached. Based on the results and findings, the following conclusions can be reached. ducated NE accent was the most intelligible and acceptable, followed by the MTbNE accent and the RNE Educated NE accent was the most intelligible and acceptable, followed by the MTbNE accent and the RNE Accent. The least intelligible and acceptable accent was the Regional NE accent. This presents an interesting angle: could it be that NE users favour narrow linguistic ethnicism over broad linguistic regionalism and what are the reasons for this? There was no significant difference on the intelligibility of the Regional NE accent among the three perceptual forms. There is a near perfect convergence on accent 2 (regional NE accent) as the least intelligible. There was also significant difference on the acceptability of each of the three NE accents. In addition, the degree of perceptual convergence for both intelligibility and acceptability among the three perceptual forms learned towards the Educated NE accent making it the most generally intelligible and acceptable among Nigerian users of the English language. Again, this might be because the Educated NE is closest to Standard British English. References Awonusi, V.O (2004). The English language in global context. In A.B.K Dadzie, and S. Awonusi. (eds). Nigerian English, influences and characteristics. Pp. 33-45. Lagos: Concept Pub. Awonusi, V.O (2004). The English language in global context. In A.B.K Dadzie, and S. Awonusi. (eds). Nigerian English, influences and characteristics. Pp. 33-45. Lagos: Concept Pub. Adetugbo, Abiodun. (1987). Nigerian English phonology: Is there any standard? Lagos Review of English Studies IX, 64 -84. Adetugbo, Abiodun. (1987). Nigerian English and Communicative Competence. In E. Ubahakwe (ed) Varieties and Functions of English in Nigeria. pp. 167-183. Ibadan: African University Press. Babatunde, Shola T. (2002a). World Englishes and the Paradox of English Language Teaching in Nigeria. In S.T. Babatunde and D.S. Adeyanju (eds) Language, Meaning and society in honour of E.E. Adegbija at 50. pp. 69-95. Ilorin: Haytee press. Babatunde, Shola T. (2002b). The State of English language in Nigeria. In R.A. Lawal, I. Isiugo Abanihe, 1.N.Ohia (eds) Perspectives in Applied Linguistics in Language and Literature. pp. 129-140.Ibadan: Stirling – Horden. Bamgbose, Ayo. (1995). English in the Nigerian Environment. In A. Bamgbose, A. Banjo, A. Thomas (eds) New Englishes : A West African perspective. pp. 9-26. Ibadan: Mosoro. Banjo, Ayo. (1979). Beyond Intelligibility: A presidential address. In E. Ubahakwe, E (ed) Varieties and Functions of English in Nigeria. pp. 7-13. Ibadan: African University press. Banjo, Ayo. (1995). On Codifying Nigerian English: Research So Far. In A. Bamgbose, A. Banjo, A. Thomas (eds) New Englishes: AWest African Perspective. pp. 203-231.Ibadan: Mosoro. Brosnahan, Leonard F. (1958). English in Southern Nigeria. English Studies 39 (3), pp. 97-110. Crystal, D. (2007). English as a Global Language. Cambridge: Cambridge University Press. Ekong, P.A. (1980). Investigating into the Intelligibility of a Possible Standard Model for Nigerian Spoken English. In S.O. Umoh. (ed) Jounal of language Arts and Communication. 1(1), pp. 1 – 11. Federal Republic of Nigeria (2004, revised 2007). National Policy on Education. Lagos: NERDC Press. Jowitt, David. (1991). Nigeria English Usage: an introduction. Lagos: Longman. Jibril, Munzali. (1986). Sociolinguistic Variation in Nigerian English. English Worldwide 7, pp. 47-75. Kachru, Braj. (1992).World Englishes: Approaches, issues and resources: Language Teaching 25 (1). Cambridge University Press. pp.1-14. ru, B.B., Kachru, Y. and Nelson, C. (2009). The Handbook of World Englishes. Wiley-Blackwell Lawal, Adebayo. (2000). Perceptual Convergence as a measure of Acceptability and Intelligibility of Nigerian Englishes. Unpublished paper, Faculty of Education, University of Ilorin. Odumuh, Adama. (1984). This paper is licensed under a Creative Commons Attribution 3.0 License. 2.2.3 Recommendations Based on the findings, it is recommended that language policy makers realistically confront the problem of the NE corpus to be used in teaching and other educational purposes. The Nigerian Educational Research and Development Council and other allied bodies could be commissioned to produce texts on the phonology and phonetics of the educated NE accent found to be most intelligible and acceptable. This would help the process of standardization and codification. In addition, textbook writers should take note of codified variations and write textbooks based on ENE which is closest to Sandard English for the use in educational institutions. Teacher educators should be trained in the rudiments of the Educated NE accent. This would enable students learn the correct and acceptable NE accent. Page \| 109 Page \| 109 International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] ( ), ( ) Vol. 1 No. 5; September 2012 [Special Issue on General Lingu APPENDIX I APPENDIX I Dear Respondent, This questionnaire is designed to obtain valid information on which of the three accents of Nigerian English will be most intelligible and acceptance to users. I wish to assure you that all information supplied is for academic purposes and would have significant effect on the outcome of this study. Thanks Yours faithfully, Thanks Yours faithfully, This paper is licensed under a Creative Commons Attribution 3.0 License. References Some Methodological Considerations in the Identification and Description of Nigerian English. In R. Freeman & M. Jubril (eds). Papers of Nigerian English Studies Association / British Council Conference, Bayero University Kano, 25 – 31. dumuh, Adama. (1987). Nigerian English: Selected essays. Pp. iv, 67.Zaria: Ahmadu Bello University Pre Ohia Isaac N. (1997). The Lexicon of Standard Nigerian English As An Acceptability Paradigm Among the Educated Elite. An Unpublished Ph. D. Thesis. University of Ibadan. Soyinka, Wole. (1993). Art, Dialogue and Outrage. Ibadan: New Horn Press. Tiffen, Brian. (1974). The Intelligibility of Nigerian English. Unpublished Ph.D.Thesis. University of London, London. bahakwe, Ebo. (1979) (ed). Varieties and Functions of English in Nigeria. Ibadan: Ibadan University Pres Ubahakwe, Ebo. (1979) (ed). Varieties and Functions of English in Nigeria. Ibadan: Ibadan University Press. Page \| 110 Page \| 110 International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] F.F. O. Fatimayin (Mrs.) INSTRUCTION: Please indicate your response by putting a tick (ü) in the appropriate space provided. 1 PART A PERSONAL / DEMOGRAPHIC INFORMATION INSTRUCTION: Please indicate your response by putting a tick (ü) in the appropriate space provided. 1. PART A: PERSONAL / DEMOGRAPHIC INFORMATION. I. State of Origin.---------------------------------------------------------- II. Place of work.----------------------------------------------------------- III. Status / Rank / Designation.------------------------------------------- IV. Sex : Male ( ) Female ( ) V. Age Range : a. below 30 ( ) b. 30 - 40 ( ) c. 41 - 50 ( ) d. above 50 ( ) VI. What is your mother tongue? ------------------------------------------------ VII. Which region does your mother tongue belong? ------------------------- (a) North ( ) (b) East ( ) (c) West ( ) VIII. What other Nigerian language(s) are you proficient in? a ----------------------- b ----------------------- c ----------------------- IX. What is your level of Education? a Tertiary (Higher degree) b Tertiary (First degree) I. State of Origin.---------------------------------------------------------- II. Place of work.----------------------------------------------------------- III. Status / Rank / Designation.------------------------------------------- IV. Sex : Male ( ) Female ( ) V. Age Range : a. below 30 ( ) b. 30 - 40 ( ) c. 41 - 50 ( ) d. above 50 ( ) I. State of Origin.---------------------------------------------------------- II. Place of work.----------------------------------------------------------- III. Status / Rank / Designation.------------------------------------------- IV. Sex : Male ( ) Female ( ) V. Age Range : a. below 30 ( ) b 30 40 ( ) VI. What is your mother tongue? ------------------------------------------------ VII. Which region does your mother tongue belong? ------------------------- (a) North ( ) (b) East ( ) (c) West ( ) (a) North ( ) (b) East VIII. What other Nigerian language(s) are you proficient in? IX. What is your level of Education? Page \| 111 Page \| 111 International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] X Occupation. Please tick the most specific. a. Lecturer / Teacher ( ) b. Professional ( doctor, lawyer, clergyman) ( ) c. Professional writer ( ) d. Journalist ( newspapers / magazine) ( ) e. Broadcaster (radio / TV) ( ) f. Politician ( ) g. Civil servant ( ) h. Others ( Specify) ( ) PART B Dear Respondent, Vol. 1 No. This paper is licensed under a Creative Commons Attribution 3.0 License. F.F. O. Fatimayin (Mrs.) 5; September 2012 [Special Issue on General Linguistics] Page \| 112 This paper is licensed under a Creative Commons Attribution 3.0 License. X Occupation. Please tick the most specific. a. Lecturer / Teacher ( ) b. Professional ( doctor, lawyer, clergyman) ( ) c. Professional writer ( ) d. Journalist ( newspapers / magazine) ( ) e. Broadcaster (radio / TV) ( ) f. Politician ( ) g. Civil servant ( ) h. Others ( Specify) ( ) PART B Dear Respondent, Please listen attentively to the recorded accents (three of them) that would be played to your hearing. Then answer the questions below by ticking ( ü) 1. Rate each accent on the basis of its intelligibility (i.e. how easily you can understand the message of the speaker). ACCENT VERY INTELLIGIBLE 4 INTELLIGIBLE 3 FAIRLY INTELLIGIBLE 2 MINIMALLY INTELLIGIBLE 1 NOT INTELLIGIBLE AT ALL 0 1 2 3 2. Rate each accent on the basis of its acceptability (i.e. the extent to which you like or prefer the accent). ACCENT VERY ACCETABLE 4 ACCEPTABLE 3 FAIRLY ACCEPTABLE 2 MINIMALLY ACCEPTABLE 1 NOT ACCEPTABLE AT ALL 0 1 2 3 3. Rate each accent on the basis of how easily understandable the accent is. ACCENT VERY INTELLI GIBLE 4 INTELLI GIBLE 3 FAIRLY INTELLI GIBLE 2 MINIMA LLY INTELLI GIBLE 1 NOT INTELLI GIBLE AT ALL 0 1 2 3 Please listen attentively to the recorded accents (three of them) that would be played to your hearing. Then answer the questions below by ticking ( ü) 1. Rate each accent on the basis of its intelligibility (i.e. how easily you can understand the message of the speaker). ACCENT VERY INTELLIGIBLE 4 INTELLIGIBLE 3 FAIRLY INTELLIGIBLE 2 MINIMALLY INTELLIGIBLE 1 NOT INTELLIGIBLE AT ALL 0 1 2 3 Page \| 112 This paper is licensed under a Creative Commons Attribution 3.0 License. 2. Rate each accent on the basis of its acceptability (i.e. the extent to which you like or prefer the accent). ACCENT VERY ACCETABLE 4 ACCEPTABLE 3 FAIRLY ACCEPTABLE 2 MINIMALLY ACCEPTABLE 1 NOT ACCEPTABLE AT ALL 0 1 2 3 3. Rate each accent on the basis of how easily understandable the accent is. This paper is licensed under a Creative Commons Attribution 3.0 License. F.F. O. Fatimayin (Mrs.) ACCENT VERY INTELLI GIBLE 4 INTELLI GIBLE 3 FAIRLY INTELLI GIBLE 2 MINIMA LLY INTELLI GIBLE 1 NOT INTELLI GIBLE AT ALL 0 1 2 3 International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] 4.Rate each accent on the basis of how easily acceptable the accent is (i.e. how readily you feel they will accept it). ACCENT VERY ACCETABLE 4 ACCEPTABLE 3 FAIRLY ACCEPTABLE 2 MINIMALLY ACCEPTABLE 1 NOT ACCEPTABLE AT ALL 0 1 2 3 5a. Which of the accent do you prefer most? Accent A ( ) Accent B ( ) Accent C ( ) 5b Why? ------------------------------------------------------------------------ 6a Which of the accents do you most easily understand? Accent A ( ) Accent B ( ) Accent C ( ) 6b. Why? ------------------------------------------------------------------- 7. How would you rate your accent of English? Very Intelligible Intelligible Fairly Intelligible Minimally Intelligible Not Intelligible at all 8. How do you feel other accent users rate your accent of English? Very Intelligible Intelligible Fairly Intelligible Minimally Intelligible Not Intelligible at all 9. How acceptable do you think your accent is? Very Intelligible Intelligible Fairly Intelligible Minimally Intelligible Not Intelligible at all 10. How acceptable do you think your accent is to other Nigerian users of English? Very Intelligible Intelligible Fairly Intelligible Minimally Intelligible Not Intelligible at all APPENDIX II ORAL READING TESTS FOR ACCENT IDENTIFICATION Section A Jimoh: Welcome to my farm, Madam. Agric Officer: Good morning, it’s a pleasure to be here. How is the planting going? Jimoh: Madam, we’ve been waiting for you. Agric Officer: You’ve been waiting for me? It’s only half past seven. Jimoh: That’s true but we’ve been careful not to plant without your instruction. Page \| 113 This paper is licensed under a Creative Commons Attribution 3.0 License. APPENDIX II ORAL READING TESTS FOR ACCENT IDENTIFICATION Section A Jimoh: Welcome to my farm, Madam. Agric Officer: Good morning, it’s a pleasure to be here. How is the planting going? Jimoh: Madam, we’ve been waiting for you. Agric Officer: You’ve been waiting for me? It’s only half past seven. Jimoh: That’s true but we’ve been careful not to plant without your instruction. International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. Section C {SENTENCES} Page \| 114 This paper is licensed under a Creative Commons A Section C {SENTENCES} a. Attention please! Passengers for flight 557 should assemble at gate 3. b. At the end of the story, they all shouted with joy. c. The headmaster ordered the boy to go home for six days. d. They are as thick as thieves. e. They could find only a kite near the cushion chair on which the judge sat. f. Watching church programmes on television gives me great pleasure and comfor g. The seats in the zinc structure are for sitting on. h. He must endure your fury. i. There were lots of red lorries, yellow lorries, lilac lorries being loaded on the ro yams. j. The nation is taking measures to provide plenty of permanent jobs for her gradu them computer literate. k. The man wearing a purplish pint cape appeared twice on television. Section D INTONATION I am going home What is your name? Prices have fallen sharply. Are you going home? I don’t like long journeys, do you? Will Busayo accept the gift? James isn’t too young to enter University, but Angela is. You may be right. Would you rather take beans or rice? When the truth is known my stand would be clear. Section E STRESS (DI & POLY SYLLABUS) The car behind stopped at the corner. A man of words and not of deeds Take the beggar away to the foreman He likes to work everyday I have just signed a contract with the Record company. Di syllabic Poly-syllabic window personification Annoy aristocracy Behind photographic Alone separate Machine objectivity { } a. Attention please! Passengers for flight 557 should assemble at gate 3. b. At the end of the story, they all shouted with joy. c. The headmaster ordered the boy to go home for six days. d. They are as thick as thieves. e. They could find only a kite near the cushion chair on which the judge sat. f. Watching church programmes on television gives me great pleasure and comfort. g. The seats in the zinc structure are for sitting on. h. He must endure your fury. i. There were lots of red lorries, yellow lorries, lilac lorries being loaded on the road with tubers of yams. j. F.F. O. Fatimayin (Mrs.) 5; September 2012 [Special Issue on General Linguistics] Agric Officer: Now, I’m here so let’s go straight to business Jimoh: That’ll be very good Madam. Agric Officer: But where are all your assistants? Jimoh: They have all resigned. Agric Officer: They have? Section B International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] On her wedding day, the groom’s people announced their coming only with the aggressive blare of car horns. We were not very impressed by the show. Although we could not see over the hedge, we could tell from the sound that it was a small procession of eight cars. In addition, there was neither video coverage nor cameraman to air it on television. At the end of it all, the occasion was below standard and not pleasurable and one of the worst I have witnessed. Page \| 114 This paper is licensed under a Creative Commons Attribution 3.0 Lic Section C {SENTENCES} a. Attention please! Passengers for flight 557 should assemble at gate 3. b. At the end of the story, they all shouted with joy. c. The headmaster ordered the boy to go home for six days. d. They are as thick as thieves. e. They could find only a kite near the cushion chair on which the judge sat. f. Watching church programmes on television gives me great pleasure and comfort. g. The seats in the zinc structure are for sitting on. h. He must endure your fury. i. There were lots of red lorries, yellow lorries, lilac lorries being loaded on the road with tubers yams. j. The nation is taking measures to provide plenty of permanent jobs for her graduates as well as them computer literate. k. The man wearing a purplish pint cape appeared twice on television. Section D INTONATION I am going home What is your name? Prices have fallen sharply. Are you going home? I don’t like long journeys, do you? Will Busayo accept the gift? James isn’t too young to enter University, but Angela is. You may be right. Would you rather take beans or rice? When the truth is known my stand would be clear. Section E STRESS (DI & POLY SYLLABUS) The car behind stopped at the corner. This paper is licensed under a Creative Commons Attribution 3.0 License. F.F. O. Fatimayin (Mrs.) A man of words and not of deeds Take the beggar away to the foreman He likes to work everyday I have just signed a contract with the Record company. Di syllabic Poly-syllabic window personification Annoy aristocracy Behind photographic Alone separate Machine objectivity This paper is licensed under a Creative Commons Attribution 3.0 License. Section C {SENTENCES} The nation is taking measures to provide plenty of permanent jobs for her graduates as well as make them computer literate. k. The man wearing a purplish pint cape appeared twice on television. This paper is licensed under a Creative Commons Attribution 3.0 License. International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] International Journal of Applied Linguistics & English Literature ISSN 2200-3592 (Print), ISSN 2200-3452 (Online) Vol. 1 No. 5; September 2012 [Special Issue on General Linguistics] Page \| 115
https://openalex.org/W2565794606	https://europepmc.org/articles/pmc5203897?pdf=render	English	null	Effectiveness of Laser Therapy in the Management of Recurrent Aphthous Stomatitis: A Systematic Review	Scientifica	2,016	cc-by	9,701	Hindawi Publishing Corporation Scientiﬁca Volume 2016, Article ID 9062430, 12 pages http://dx.doi.org/10.1155/2016/9062430 Hindawi Publishing Corporation Scientiﬁca Volume 2016, Article ID 9062430, 12 pages http://dx.doi.org/10.1155/2016/9062430 Hindawi Publishing Corporation Scientiﬁca Volume 2016, Article ID 9062430, 12 pages http://dx.doi.org/10.1155/2016/9062430 Hindawi Publishing Corporation Scientiﬁca Volume 2016, Article ID 9062430, 12 pages http://dx.doi.org/10.1155/2016/9062430 Review Article Effectiveness of Laser Therapy in the Management of Recurrent Aphthous Stomatitis: A Systematic Review Min Han,1 Hui Fang,1 Quan-Li Li,1 Ying Cao,1 Rong Xia,2 and Zhi-Hong Zhang3 1College & Hospital of Stomatology, Key Lab of Oral Diseases Research of Anhui Province, Anhui Medical University, Hefei 230032, China 2The 2nd Hospital Affiliated to Anhui Medical University, Hefei 230601, China 3The Hospital of Anhui Province, Hefei 230001, China 2The 2nd Hospital Affiliated to Anhui Medical University, Hefei 230601, China 3The Hospital of Anhui Province, Hefei 230001, China Correspondence should be addressed to Quan-Li Li; ql-li@126.com Received 10 July 2016; Accepted 15 November 2016 Academic Editor: Umit Tursen Copyright © 2016 Min Han et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objectives. Laser therapy is a promising new treatment for patients with recurrent aphthous stomatitis (RAS). However, the clinical effect and security issue of laser therapy remain controversial. This systematic review was conducted to evaluate the clinical effectiveness and security of laser treatment in RAS patients. Methods. Five electronic databases were searched (MEDLINE (PubMed), EMBASE, ScienceDirect, the Cochrane Library, and Web of Science) to identify all studies that were about randomized controlled clinical trials, involving the effect of laser therapy in RAS patients. Conclusion. Twenty-three studies were retained for full-text analysis after screening the titles and abstracts of potential articles, but only 10 studies satisfied the inclusion criteria after the full texts were reviewed. The included studies reported a comparison of the effectiveness between the laser treatment and placebo laser therapy (or conventional drug therapy) when managing the RAS patients. It can be concluded that laser therapy has the superiority in relieving ulcer pain and shortening healing time when compared with placebo group or medical treatment group. Although laser therapy is a promising effective treatment for RAS, high-quality clinical studies with large sample size must be further performed to confirm the effectiveness of this therapy. 1. Introduction Recently, laser therapy is clinically accepted in medical fields and practices as part of physical therapy for many diseases such as hair regrowth, infantile hemangioma, incontinent great saphenous vein, and diabetic foot ulcer [21–24]. Besides, it has been applied in dental diseases such as periodontics and peri-implantitis, dentinal hypersensitivity, and dental movement [25–30]. Due to the energy output of laser therapy in a low energy density, it is also named as low-level laser therapy (LLLT) or low-power laser therapy. Thus, the possibility of clinical complications, like thermal effects and soft tissue damage, is reduced. Laser therapy is applied to treat RAS because of its potential beneficial effects, including immediate pain relief, accelerating wound healing, reducing healing period, and being anti-inflammatory [31]. Currently, clinical case reports and randomized controlled clinical trials about several different types of lasers (Nd:YAG laser, Er:YAG laser, InGaAlP laser, GaAlAs laser, etc.) are reported in the use for treatment of RAS. (1) Only randomized control trials (RCTs) were consid- ered for the systematic review. (2) Patients with reliable history of RAS, duration of 3 days or less, and presence of one or more painful ulcers were selected for the study. (3) The trial groups received positive laser therapy; the placebo group was conducted with the same laser but with an inactive probe, or the negative control was supplied with some traditional drugs. Exclusion criteria were as follows: (1) Reviews, case reports, letters, editorials, and confer- ence abstracts were excluded. (2) Patients with oral mucosal ulcers other than RAS, such as ulcerative colitis, Crohn’s disease, Behc¸et’s syndrome, or serious anemia were excluded. (3) Studies with insufficient data or without negative group were excluded. (3) Studies with insufficient data or without negative group were excluded. (4) Studies published in non-English language were excluded. (4) Studies published in non-English language were excluded. 2.3. Data Extraction and Quality Assessment of Selected Studies. Figure 1 summarizes the details of the study selec- tion process and the reasons for exclusion. Only studies published in English language and utilized RCT design were selected in the systematic review. Study inclusions and quality assessments were independently conducted by two reviewers between March 2016 and April 2016. Any disagreements were discussed by the two reviewers until consensus was reached. 1. Introduction Missing, unclear, or unpublished data were further obtained by emailing the corresponding authors.t Although earlier systematic reviews [32, 33] about laser therapy for RAS have been reported, the randomized con- trolled clinical trials they included were limited. One sys- tematic review [32] identified the randomized controlled clinical trials until 1 June 2014 and only two eligible studies [34, 35] were selected. The other systematic review [33] made an electronic search in databases until 31 December 2013 and four original articles [36–39] were included in this review. Moreover, some important issues of laser therapy on treating RAS, such as clinical security, remain controversial. Therefore, a new systematic review should be conducted. In this work, we would try our best to identify all relevant randomized controlled clinical trials to evaluate the clinical effect and security of laser treatment on RAS. Microsoft Excel was used to extract the characteristics of each study included for final analysis. The details of the selected studies included the following: study name (the name of the first author and the year of publication), country of origin, number of ulcer sites, number of patients, age of patients, types of laser, and follow-up period. The outcome evaluation (pain score, healing time, adverse events, etc.) and parameters of various types of lasers were also summarized.h 2. Materials and Methods 2.1. Search Strategy. The following electronic databases were searched from February 2016 to April 2016: MEDLINE (via PubMed), EMBASE, ScienceDirect, the Cochrane Library, and Web of Science. To identify relevant literature, we searched the following terms “(recurrent oral ulcer OR recur- rent oral ulcers OR recurrent aphthous ulcer OR recurrent aphthous ulcers OR recurrent aphthous stomatitis OR recur- rent oral stomatitis) AND (laser OR lasers)”. Manual search was also conducted in the following relevant journals pub- lished from 2000 to 2016: Lasers in Medical Sciences, Lasers in Surgery and Medicine, Photomedicine and Laser Surgery, Oral Diseases, Photodiagnosis and Photodynamic Therapy, Journal of Oral Rehabilitation, Clinical Oral Investigations, Journal of Dentistry, Journal of Oral Laser Applications, and Journal of Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology. The criteria for the evaluation of RCTs (randomization, allocation concealment, blinding, reporting loss to follow- up/withdrawal, and comparability of baseline) were com- bined with the Cochrane Handbook for Systematic Reviews of Interventions for the quality assessment of all selected trials (Table 1). The high quality of evidence was estimated when all criteria were met, moderate quality was estimated when one or more criteria were unclear or partly met, and low quality was estimated when one or more criteria were not met [44]. 1. Introduction are characterized by 1–3 mm, multiple, and clustered lesions, which may coalesce into larger ulcers and heal up to 15 days [6]. Although the exact cause of RAS is not completely understood, mucosal lesions are reported to be related to several factors, such as immune system dysfunction, genetic factors, allergic agents, stress, nutrition deficiency, hormonal changes, and infective viruses [7]. Curative therapy is cur- rently unavailable to prevent the recurrence of ulcer [8]. Conventional treatment for RAS involves the use of topical or systemic drugs only to relieve the severity of painful symptoms and prevent secondary infection. Topical agents, such as corticosteroids and other anti-inflammatory agents, including benzydamine, amlexanox, aphtheal, and triclosan, are usually provided for patients with mild symptoms in forms of mouth rinse, adhesive paste, or anesthetic gel [9–12]. However, for those patients with particularly fre- quent or severe RAS, systemic immunosuppressive treatment (corticosteroids, pentoxifylline, thalidomide, etc.) may be Recurrent aphthous stomatitis (RAS), also known as recur- rent aphthous ulcer or recurrent oral ulcer, is the most common recurrent oral mucosal lesions. The prevalence of RAS in the general population is between 2% and 50%; most estimates fall between 5% and 25% [1–3]. RAS clinically manifests as small, round or ovoid, painful, self-healing, and recurrent ulcers with circumscribed margins, erythematous haloes, and yellow or grey floors. RAS may occur during childhood or adolescence, and mucosal lesions may dis- turb patients’ daily activities, such as drinking, eating, and speaking [4]. RAS can be classified into minor (MiRAS), major (MjRAS), and herpetiformis ulcers (HUs) [5]. MiRAS, which comprises more than 80%–90% of RAS cases, presents lesions of less than 1 cm in diameter and heals within 7– 14 days without scar formation. MjRAS lesions exceed 1 cm in diameter and heal within 20–30 days with scarring. HUs 2 Scientifica 2.2. Study Inclusion and Exclusion. Inclusion criteria were as follows: 2.2. Study Inclusion and Exclusion. Inclusion criteria were as follows: necessary [13–16]. Corticosteroids, antibiotics, and analgesics play the role of mainstay in the treatment of patients with RAS, especially in improving healing of severe RAS [17, 18]. However, long-term or repeated use of these medications should be avoided as fungal infection or drug resistance or even life-threatening complications may be caused [19]. Therefore, many doctors are exploring new treatments for RAS. The history of the investigation and clinical use of laser therapy in medicine goes back to the late 1960s [20]. 3. Results and Discussion 3.1. Study Selection. A total of 676 potential relevant titles, abstracts, and articles were found through electronic and manual search. 48 duplicate articles were excluded, and 628 3 Scientifica Table 1: Grade scale for quality assessment of randomized controlled trials. Criterion Grade A B C Randomization Adequate Unclear (reported randomization but method not described) Inadequate (quasirandom method of allocation, such as alternation, date of birth, case record number) Allocation concealment Adequate Unclear (not mentioned) Clearly inadequate concealment/not used Blinding Adequate (double-blind or blinding the outcomes evaluators) Partly blinded/unclear (single-blind or not mentioned) Not used (“open-label” or “unmask”) Loss to follow-up/withdrawal Adequate reporting (including numbers and causes) Partly reported Not reported Comparability of baseline Comparable (at least including age) Unclear Not comparable Table 1: Grade scale for quality assessment of randomized controlled trials. Table 1: Grade scale for quality assessment of randomized controlled trials. Criterion Grade A B C Randomization Adequate Unclear (reported randomization but method not described) Inadequate (quasirandom method of allocation, such as alternation, date of birth, case record number) Allocation concealment Adequate Unclear (not mentioned) Clearly inadequate concealment/not used Blinding Adequate (double-blind or blinding the outcomes evaluators) Partly blinded/unclear (single-blind or not mentioned) Not used (“open-label” or “unmask”) Loss to follow-up/withdrawal Adequate reporting (including numbers and causes) Partly reported Not reported Comparability of baseline Comparable (at least including age) Unclear Not comparable Records excluded according to inclusion and exclusion criteria (n = 316) titles and abstracts Records screened through (n = 339) Records after removing duplicates (n = 628) Databases searched: MEDLINE (via PubMed), EMBASE, ScienceDirect, the Cochrane Library, and Web of Science (n = 676) Studies included for review (n = 10) Full-text articles excluded, with reasons: non-RCTs and non-English (n = 13) Full-text articles assessed for eligibility (n = 23) Figure 1: Flow chart showing details of the study selection process. articles were further excluded according to inclusion andt finally included in the systematic review [34–43]. (i) Laser Therapy versus Inactive “Laser” Therapy (i) Laser Therapy versus Inactive “Laser” Therapy The visual analog scale (VAS) scoring system or a numer- ical rating scale scoring system, as well as healing time, the size of ulcers, the degree of erythema and exudation (range 0–3), erythema dynamics, and epithelization time, were used to evaluate the outcomes in the included trials. The scoring systems of the VAS and numerical rating scale in assessing pain are usually 0–10 cm or 0–100 mm (the ends of the scale are defined as “no pain” and “severe pain”). (1) Healing Time. Zand et al. [37] evaluated the healing time of a CO2 laser (10600 nm). The healing time after treatment in the laser group (4.8 ± 2.4 days) was dramatically shorter than that in the placebo group (7.6 ± 2.5 days), with 𝑝value of 0.02. (1) Healing Time. Zand et al. [37] evaluated the healing time of a CO2 laser (10600 nm). The healing time after treatment in the laser group (4.8 ± 2.4 days) was dramatically shorter than that in the placebo group (7.6 ± 2.5 days), with 𝑝value of 0.02. Prasad and Pai [38] also applied a CO2 laser (wavelength not reported) with a significant reduction in the mean healing time, 4.08 ± 0.81 days for the laser treatment group and 7.84 ± 0.90 days for the placebo group (𝑝< 0.001). 3.3. Quality Assessment of Selected Studies. 10 studies were finally included in the systematic review [34–43]. As shown in Table 2, five trials [34–36, 39, 42] were defined as moderate- quality evidence and the remaining five trials [37, 38, 40, 41, 43] were rated as low-quality evidence. The randomized method was clearly described in one study [35], in which patients were randomly allocated by tossing a coin. Blinding was conducted in most of the included trials, but only one study [40] was double-blinded and one study [43] was reported to be nonblinded. Only one study [40] reported allocation concealment, whereas the allocation concealment in the remaining nine trials was unclear. Three studies [34– 36] reported that these trials were without loss to follow- up/withdrawals. The baselines were comparable in six studies [34–38, 40] and the last 4 trials [39, 41–43] were unclear. y g 𝑝 Aggarwal et al. 3. Results and Discussion The inclu- y of Comparable (at least including age) Unclear Not comparable Records excluded according to inclusion and exclusion criteria (n = 316) titles and abstracts Records screened through (n = 339) Records after removing duplicates (n = 628) Databases searched: MEDLINE (via PubMed), EMBASE, ScienceDirect, the Cochrane Library, and Web of Science (n = 676) Studies included for review (n = 10) Full-text articles excluded, with reasons: non-RCTs and non-English (n = 13) Full-text articles assessed for eligibility (n = 23) Figure 1: Flow chart showing details of the study selection process. Records excluded according to inclusion and exclusion criteria (n = 316) titles and abstracts Records screened through (n = 339) Records after removing duplicates (n = 628) Databases searched: MEDLINE (via PubMed), EMBASE, ScienceDirect, the Cochrane Library, and Web of Science (n = 676) Studies included for review (n = 10) Full-text articles excluded, with reasons: non-RCTs and non-English (n = 13) Full-text articles assessed for eligibility (n = 23) Figure 1: Flow chart showing details of the study selection process. Databases searched: MEDLINE (via PubMed), EMBASE, ScienceDirect, the Cochrane Library, and Web of Science (n = 676) Records excluded according to inclusion and exclusion criteria (n = 316) titles and abstracts Records screened through (n = 339) Full-text articles excluded, with reasons: non-RCTs and non-English (n = 13) Full-text articles assessed for eligibility (n = 23) Studies included for review (n = 10) Figure 1: Flow chart showing details of the study selection process. finally included in the systematic review [34–43]. The inclu- sion process is shown in Figure 1. finally included in the systematic review [34–43]. The inclu- sion process is shown in Figure 1. articles were further excluded according to inclusion and exclusion criteria after screening the titles and abstracts. We reviewed the full texts of the remaining 23 articles. Of the 23 articles, twelve full-text articles reported nonrandomized controlled clinical trials and one non-English randomized controlled clinical trial were excluded. Ten studies were 3.2. Study Characteristics. The systematic review included 384 patients. All studies compared the effectiveness of one kind of laser with a negative treatment (an inactive laser 4 Scientifica Table 2: Quality of the included trials. 3. Results and Discussion Criterion Study Zand et al., 2009 [36] Zand et al., 2012 [37] Prasad and Pai, 2013 [38] Sattayut et al., 2013 [40] Aggarwal et al., 2014 [34] Albrektson et al., 2014 [35] Tezel et al., 2009 [41] De Souza et al., 2010 [39] Lalabonova and Daskalov, 2014 [42] Jijin et al., 2016 [43] Randomization B B B B B A B B B B Allocation concealment B B B A B B B B B B Blinding B B B A B B C B B C Loss to follow- up/withdrawal A C C C B A A A B C Comparability of baseline A A A A A A A B B B Quality assessment Moderate quality Low quality Low quality Low quality Moderate quality Moderate quality Low quality Moderate quality Moderate quality Low quality Study therapy or conventional medical therapy). Among these reports, four studies [36–38, 40] made a comparison between the CO2 laser treatment and an inactive laser treatment (placebo group). In those studies applied with CO2 laser, before irradiation, a layer of transparent, high-water, and nonanesthetic gel was placed on the lesion in patients of the laser and placebo groups. CO2 laser is one type of special laser when compared to the conventional low-level laser therapy (Nd:YAG laser, InGaAlP laser, GaAlAs laser, etc.) as its wavelength and energy density are obviously longer and stronger than other lasers. Although it seemed that it is not a low-level laser, the studies of CO2 laser therapy in the management of RAS were still included in our systematic review. The importance of the nonanesthetic gel and the reason why we included studies applied with CO2 laser therapy will be mentioned in Discussion.h 3.4. Meta-Analysis Results. This systematic review attempted to conduct a meta-analysis by using RevMan 5.3 software, which could summarize the extracted data from included tri- als. However, owing to the fact that a quite high heterogeneity was found among the included studies, a meta-analysis was considered to be inappropriate. Therefore, we only provided a descriptive assessment of the included data in the systematic review. 3.5. Review 3.5.1. Description of Studies. The characteristics of the included studies, the outcome evaluation of the included studies, and the parameters of various types of lasers are shown in Tables 3, 4, and 5, respectively. (i) Laser Therapy versus Inactive “Laser” Therapy (i) Laser Therapy versus Inactive “Laser” Therapy [34] reported that the complete healing time in the AMD laser group (810 nm) was observed to be 3.05 ± 1.10 days and 8.90 ± 2.45 days in the sham placebo group with 𝑝value of <0.001. (2) Changes in Pain Level. Zand et al. [36] compared the effectiveness of CO2 laser (10600 nm) against that of control group. VAS scores system of 10 cm was used for the evaluation of pain level. Immediately after laser treatment, the mean scores for noncontact and contact pain in the laser therapy group decreased significantly (𝑝 < 0.001). These mean scores of immediate pain relief in the placebo group were not changed. At 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h after Scientifica 5 Table 3: Details of the included studies. (i) Laser Therapy versus Inactive “Laser” Therapy Study Zand et al., 2009 [36] Zand et al., 2012 [37] Prasad and Pai, 2013 [38] Sattayut et al., 2013 [40] Aggarwal et al., 2014 [34] Albrektson et al., 2014 [35] Tezel et al., 2009 [41] De Souza et al., 2010 [39] Lalabonova and Daskalov, 2014 [42] Jijin et al., 2016 [43] Country Iran Iran India Thailand India Sweden Turkey Brazil Bulgaria India Number of ulcers (test group/control group) 15/15 10/10 25/25 7/7 30/30 20/20 10/10 15/5 90/90 25/25 Number of patients (male/female) 15 (2/13) 10 (1/9) 25 14 30 (18/12) 20 20 (7/13) 20 180 (31/149) 50 (29/21) Mean ages or age range 37.9 ± 10.9 (24–56) 35.6 (18–40) (18–39) Not clear 25 32 ± 7.7 (1–70) 43.01 ± 1.25 (15–55) Laser group Nonanesthetic gel + CO2 laser Nonanesthetic gel + CO2 laser Nonanesthetic gel + CO2 laser Nonanesthetic gel + CO2 laser AMD laser GaAlAs laser Anesthetic gel + Nd:YAG laser InGaA1P diode laser SIX Laser TS diode laser system AMD laser Control group Nonanesthetic gel + placebo “laser” Nonanesthetic gel + placebo “laser” Nonanesthetic gel + placebo “laser” Nonanesthetic gel + placebo “laser” Placebo “laser” Placebo “laser” Medication (triamci- nolone acetonide given three times daily) Medication (triamci- nolone acetonide given four times daily) Medication (Granofurin and solcoseryl given twice daily) Medication (5% amlexanox given four time daily) Follow-up time Immediately, 4 h, 8 h, 12 h, day 1, day 2, day 3, day 4 Not reported Immediately, day 1 Immediately, day 1, day 2, day 3, day 5, day 7 Immediately, day 1, day 2, day 3 Immediately, day 1, day 2, day 3 Day 1, day 4, day 7 Day 1 to day 7 Day 1, day 2, day 3, day 5, Day 1, day 3, da 7 Scientifica 6 Table 4: Details of the effects of different intervention measures for RAS patients in the included studies. Table 4: Details of the effects of different intervention measures for RAS patients in the included studies. (i) Laser Therapy versus Inactive “Laser” Therapy Study Intervention measures Pain scoring systems Outcomes Side effects (Laser group/control group) Zand et al., 2009 [36] CO2 laser/placebo “laser” VAS (10 cm) system Healing time: NR Changes in pain level: immediately after treatment, these differences were statistically significant between study groups (𝑝< 0.001) Laser group: noncontact pain: 6.2 ± 1.3 (baseline) → 0.07 ± 0.3 (immediately); contact pain: 8.4 ± 1.3 (baseline) →0.7 ± 0.8 (immediately) Placebo group: not changed At 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h after laser treatment, these differences of both noncontact and contact pain were significant compared to control group (𝑝< 0.001) Size of ulcers: NR No Zand et al., 2012 [37] CO2 laser/placebo “laser” NR Healing time: 4.8 ± 2.4 days in the laser group and 7.6 ± 2.5 days in the placebo group (𝑝= 0.02) Changes in pain level: NR Size of ulcers: NR No Prasad and Pai, 2013 [38] CO2 laser/placebo “laser” A numerical rating scale of 0–10 Healing time: 4.08 ± 0.81 days in the laser group and 7.84 ± 0.90 days in the placebo group (𝑝< 0.001) Changes in pain level: Immediately after treatment, these differences were statistically significant between study groups (𝑝< 0.001). Laser group: 8.48 ± 0.71 (baseline) →0.68 ± 0.63 (immediately); Placebo group: 8.08 ± 0.70 (baseline) → 7.96 ± 0.84 (immediately) At day 1, laser group also showed a significant reduction in pain compared to placebo group (𝑝< 0.001) Size of ulcers: NR No Sattayut et al., 2013 [40] CO2 laser/placebo “laser” VAS (100 mm) system Healing time: NR Changes in pain level: Although the pain scores after treatment and daily activity-disturbance scores of the laser group were lower than the placebo group in every point of assessment, a statistically significant difference of the pain score between the groups was found only on day 3 (𝑝< 0.001). The immediate pain reliefs between the groups were not achieved. (i) Laser Therapy versus Inactive “Laser” Therapy Pain scores after treatment (Laser group; Placebo group): 40.99 (baseline 1) →42.67 (baseline 2) →25.39 (immediately) →38.92 (day 1) →33.25 (day 2) →21.45 (day 3) →3.03 (day 5) →0 (day 7); 45.17 (baseline 1) →56.85 (baseline 2) →32.98 (immediately) →42.43 (day 1) →34.98 (day 2) →33.22 (day 3) →7.36 (day 5) →0.44 (day 7) Daily activity-disturbance scores (Laser group; Placebo group): 39.38 (baseline 1) →43.54 (baseline 2) →24.25 (immediately) →34.31 (day 1) →28.21 (day 2) →17.98 (day 3) →0 (day 5) →0 (day 7); 52.40 (baseline 1) →65.65 (baseline 2) →39.70 (immediately) →52.61 (day 1) →40.12 (day 2) →39.82 (day 3) →7.22 (day 5) →0 (day 7) Size of ulcers (mm2): There was no statistically significant difference in the size of ulcers between the laser groups and the placebo groups. No Pain scores after treatment (Laser group; Placebo group): 40.99 (baseline 1) →42.67 (baseline 2) →25.39 (immediately) →38.92 (day 1) →33.25 (day 2) →21.45 (day 3) →3.03 (day 5) →0 (day 7); 45.17 (baseline 1) →56.85 (baseline 2) →32.98 (immediately) →42.43 (day 1) →34.98 (day 2) →33.22 (day 3) →7.36 (day 5) →0.44 (day 7) Daily activity-disturbance scores (Laser group; Placebo Sattayut et al., 2013 [40] CO2 laser/placebo “laser” VAS (100 mm) system Sattayut et al., 2013 [40] CO2 laser/placebo “laser” VAS (100 mm) system Daily activity-disturbance scores (Laser group; Placebo group): 39.38 (baseline 1) →43.54 (baseline 2) →24.25 (immediately) →34.31 (day 1) →28.21 (day 2) →17.98 (day 3) →0 (day 5) →0 (day 7); 52.40 (baseline 1) →65.65 (baseline 2) →39.70 (immediately) →52.61 (day 1) →40.12 (day 2) →39.82 (day 3) →7.22 (day 5) →0 (day 7) 2h Size of ulcers (mm ): There was no statistically significant difference in the size of ulcers between the laser groups and the placebo groups. g p p g p Laser group: 4.25 (baseline 1) →4.00 (baseline 2) → 4.75 (day 1) →5.25 (day 2) →7.00 (day 3) →4.50 (day 5) →2.00 (day 7); Placebo group: 3.00 (baseline 1) →4.75 (baseline 2) → 6.25 (day 1) →6.00 (day 2) →4.50 (day 3) →2.50 (day Placebo group: 3.00 (baseline 1) →4.75 (baseline 2) → 6.25 (day 1) →6.00 (day 2) →4.50 (day 3) →2.50 (day 7 Scientifica Table 4: Continued. (i) Laser Therapy versus Inactive “Laser” Therapy Study Intervention measures Pain scoring systems Outcomes Side effects (Laser group/control group) Aggarwal et al., 2014 [34] AMD laser/placebo “laser” VAS (10 cm) system Healing time: 3.05 ± 1.10 days in the laser group and 8.90 ± 2.45 days in the placebo group (𝑝< 0.001) Changes in pain level: The laser group showed a statistically significant reduction in pain scores from baseline values as compared to the sham controlled group at immediately, day 1, day 2, day 3 after laser treatment. Laser group: 4.79 ± 0.86 (immediately); 4.58 ± 1.2 (day 1); 5.41 ± 2.04 (day 2); 4.72 ± 1.22 (day 3) Placebo group: 0.13 ± 0.35 (immediately); 0.17 ± 0.38 (day 1); 0.48 ± 1.57 (day 2); 0.79 ± 0.62 (day 3) Size of ulcers (mm): The laser group showed a statistically significant reduction in lesion size from baseline values as compared to the controlled group at immediately, day 1, day 2, day 3 after laser treatment. Laser group: no change (immediately); 0.65 ± 0.6 (day 1); 1.79 ± 0.94 (day 2); 3.17 ± 1.03 (day 3) Placebo group: no change (immediately); 0.10 ± 0.3 (day 1); 0.17 ± 0.38 (day 2); 0.48 ± 0.57 (day 3) No Albrektson et al., 2014 [35] GaAlAs laser/placebo “laser” VAS (100 mm) system Healing time: NR Changes in pain level: the laser group showed a statistically significant reduction in pain scores as compared to the placebo group (𝑝< 0.0001) Laser group: 81.7 (baseline) →56.2 (day 1) →31.5 (day 2); Placebo group: 84.7 (baseline) →80.7 (day 1) →76.1 (day 2) The difference was also significant when comparing the percentage for participants who reported moderate or severe difficulty with daily activities between the laser and the placebo group (𝑝< 0.0001) Size of ulcers: NR No Tezel et al., 2009 [41] Nd:YAG laser/medication (triamcinolone acetonide) VAS (10 cm) system Healing time: NR Changes in pain level: laser treatment always presented with a significantly greater efficacy in ulcer pain relieving than medical therapy on days 1, 4, and 7 (𝑝< 0.05). (i) Laser Therapy versus Inactive “Laser” Therapy Laser group: 7.87 ± 0.78 (before treatment) →1.34 ± 0.76 (day 1) →0.18 ± 0.23 (day 4) →0 (day 7) Medication group: 7.72 ± 0.67 (before treatment) →6.19 ± 0.76 (day 1) →3.71 ± 0.69 (day 4) →0.54 ± 0.60 (day 7) The difference was also significant when comparing the pains of daily activity-disturbance such as chewing and speaking between the laser and the medication group (𝑝< 0.05) Change in erythema and exudation level (signs of healing): there were no statistically significant differences at any time point during the study between groups in erythema. Laser group had a significantly lower exudation (𝑝< 0.05) than medication group at the final patient visit at the end of the study Erythema (laser group; medication group): 1.91 ± 0.43 (baseline) →0.09 ± 0.29 (posttreatment); 2.04 ± 0.48 (baseline) →0.17 ± 0.39 (posttreatment) Exudation (laser group; medication group): 2.23 ± 0.61 (baseline) →0.14 ± 0.57 (posttreatment); 2.34 ± 0.77 (baseline) →0.43 ± 0.51 (posttreatment) Size of ulcers: NR No Table 4: Continued. Laser group: 7.87 ± 0.78 (before treatment) →1.34 ± 0.76 (day 1) →0.18 ± 0.23 (day 4) →0 (day 7) Medication group: 7.72 ± 0.67 (before treatment) →6.19 ± 0.76 (day 1) →3.71 ± 0.69 (day 4) →0.54 ± 0.60 (day 7)hfi Scientifica 8 Table 4: Continued. Table 4: Continued. Study Intervention measures Pain scoring systems Outcomes Side effects (Laser group/control group) De Souza et al., 2010 [39] InGaA1P diode laser/medication (triamcinolone acetonide) A numerical rating scale of 0–3 Healing time: there was no statistically significant difference in the healing times between the laser group and the medication group (𝑝= 0.4345) Changes in pain level: NR Size of ulcers: NR No Lalabonova and Daskalov, 2014 [42] SIX Laser TS diode laser system/medication (Granofurin and solcoseryl) A 10-point visual analog scale system The authors reported the percentage of patients in pain levels and erythema and epithelization levels at different time point of assessment (before treatment and on days 1, 2, 3, and 5) Changes in pain levels: 0 points (no pain); 1 to 5 points (mild pain); 6 to 10 points (severe pain) Laser group showed a statistically significant reduction in pain levels as compared to medication group in every point of assessment (excepting on day 1, mild pain) with a 𝑝value < 0.01. A 10-point visual analog scale system Lalabonova and Daskalov, 2014 [42] SIX Laser TS diode laser system/medication (Granofurin and solcoseryl) NR: not reported. (i) Laser Therapy versus Inactive “Laser” Therapy Healing time/change in erythema and epithelization levels (signs of healing) Erythema levels: erythema, erythema decreases, no erythema; epithelization levels: no epithelization, initial epithelization, epithelization completed At day 3 in laser group, all of the patients were pain-free and had their ulcers successfully treated, while the patients in medication group still felt some pain and their ulcers were failed to be successfully treated even at day 5 Erythema and epithelization processes were evolving through several levels. In nearly every point of assessment (days 1, 4, and 7), laser treatment presented with a significantly greater efficacy in patients than medical therapy (𝑝< 0.05) Size of ulcers: NR No Jijin et al., 2016 [43] AMD laser/medication (5% amlexanox) A numerical rating scale of 0–10 Healing time: NR Changes in pain level: the difference in pain levels between the amlexanox group and the laser group was significant on the third day (𝑝= 0.006); however, the difference was not significant on the seventh day (𝑝= 0.171) Laser group: 6.80 (day 1) →5.20 (day 3) →2.64 (day 7); Medication group: 6.36 (day 1) →4.16 (day 3) →1.8 (day 7) Size of ulcers (mm): both therapies resulted in a significant reduction in the sizes of ulcers on day 3 and day 7 compared with the first day of treatment. However, the difference was not statistically significant between these two therapies both on day 3 (𝑝= 0.54) and on day 7 (𝑝= 0 78) No Study Intervention measures Pain scoring systems Outcomes Side effects (Laser group/control group) De Souza et al., 2010 [39] InGaA1P diode laser/medication (triamcinolone acetonide) A numerical rating scale of 0–3 Healing time: there was no statistically significant difference in the healing times between the laser group and the medication group (𝑝= 0.4345) Changes in pain level: NR Size of ulcers: NR No Lalabonova and Daskalov, 2014 [42] SIX Laser TS diode laser system/medication (Granofurin and solcoseryl) A 10-point visual analog scale system The authors reported the percentage of patients in pain levels and erythema and epithelization levels at different time point of assessment (before treatment and on days 1, 2, 3, and 5) Changes in pain levels: 0 points (no pain); 1 to 5 points (mild pain); 6 to 10 points (severe pain) Laser group showed a statistically significant reduction in pain levels as compared to medication group in every point of assessment (excepting on day 1, mild pain) with a 𝑝value < 0.01. (i) Laser Therapy versus Inactive “Laser” Therapy Healing time/change in erythema and epithelization levels (signs of healing) Erythema levels: erythema, erythema decreases, no erythema; epithelization levels: no epithelization, initial epithelization, epithelization completed At day 3 in laser group, all of the patients were pain-free and had their ulcers successfully treated, while the patients in medication group still felt some pain and their ulcers were failed to be successfully treated even at day 5 Erythema and epithelization processes were evolving h h l l l I l f No Erythema and epithelization processes were evolving through several levels. In nearly every point of assessment (days 1, 4, and 7), laser treatment presented with a significantly greater efficacy in patients than medical therapy (𝑝< 0.05) Changes in pain level: the difference in pain levels between the amlexanox group and the laser group was significant on the third day (𝑝= 0.006); however, the difference was not significant on the seventh day (𝑝= 0.171) Jijin et al., 2016 [43] AMD laser/medication (5% amlexanox) A numerical rating scale of 0–10 Jijin et al., 2016 [43] AMD laser/medication (5% amlexanox) A numerical rating scale of 0–10 NR: not reported. laser treatment, these differences of both noncontact and contact pain were significant compared to control groups (𝑝< 0.001). placebo group: 8.08 ± 0.70 (baseline) to 7.96 ± 0.84 (immedi- ately). At day 1, the difference was also significant compared to control group. 𝑝 Prasad and Pai [38] compared CO2 laser treatment (wave- length not reported) with a placebo laser. In the group treated by laser, the pain level (a numerical analog scale of 0–10) was from 8.48 ± 0.71 (baseline) to 0.68 ± 0.63 (immediately) with a significant reduction (𝑝< 0.001) when compared to the g p Sattayut et al. [40] reported the change of pain scores (a 100 mm VAS system) of a CO2 laser (10605 nm) group com- pared to the placebo group. At immediately, day 1, day 2, and day 3 after laser treatment, a statistically significant difference of the pain score between the groups was found only on day 3 9 9 Scientifica Table 5: Parameters of various types of lasers. (i) Laser Therapy versus Inactive “Laser” Therapy Study Type of laser Wavelength Output power Energy Frequency of treatment Irradiation time Energy density Zand et al., 2009 [36] CO2 laser 10600 nm, CW 1 W NR NR 5–10 s NR Zand et al., 2012 [37] CO2 laser 10600 nm, CW 1 W NR NR 5–10 s NR Prasad and Pai, 2013 [38] CO2 laser NR, CW 0.7 W NR NR 5–8 s NR Sattayut et al., 2013 [40] CO2 laser 10605 nm, CW 2 W NR NR 5 s 110.67 J/cm2 Aggarwal et al., 2014 [34] AMD laser 810 nm, CW 0.5 W NR NR 45 s; 4 times daily NR Albrektson et al., 2014 [35] GaAlAs laser 809 nm, CW 60 mW NR 1800 Hz 80 s; once daily 6.3 J/cm2 Tezel et al., 2009 [41] Nd:YAG laser 1064 nm, P 2 W 100 mJ 20 Hz 2-3 min NR De Souza et al., 2010 [39] InGaA1P diode laser 670 nm, P 50 mW 300 mJ NR 1 min; once daily 3 J/cm2 Lalabonova and Daskalov, 2014 [42] SIX Laser TS diode laser system 658 nm, CW 27 mW NR 5.8 Hz 1.14 min; once daily 27 J/cm2 Jijin et al., 2016 [43] AMD laser 810 nm 0.1 W NR NR 30 s (3 times daily with 2-minute interval) 6 J/cm2 P: pulsed wave; NR: not reported; CW: continuous wave. were no significant difference in the size of ulcers between the laser groups and the placebo groups. It seemed that laser group did not accelerate the wound healing. (𝑝< 0.001). The immediate pain reliefs between the groups were not achieved. Besides, the statistically significant differ- ences were also not observed in the daily activity-disturbance scores between the groups (𝑝> 0.05). It seems that there were no statistically significant differences in pain relief between the two groups. (𝑝< 0.001). The immediate pain reliefs between the groups were not achieved. Besides, the statistically significant differ- ences were also not observed in the daily activity-disturbance scores between the groups (𝑝> 0.05). It seems that there were no statistically significant differences in pain relief between the two groups. (ii) Laser Treatment versus Medical Treatment. Tezel et al. [41] compared the results by using Nd:YAG laser treatment (1.064 nm) with conventional medication, a topical corti- costeroid (0.1% triamcinolone acetonide given three times daily). (i) Laser Therapy versus Inactive “Laser” Therapy The pain degree (evaluated by a 10 cm VAS scale) and erythema and exudation level (the sign of healing) were used to assess the treatment effect. Laser treatment group always presented with a significantly greater efficacy in ulcer pain relieving than medical therapy on days 1, 4, and 7 (𝑝< 0.05). As for the pretreatment of topical anesthetic gel to the ulcers area in the laser treatment group, the study did not report the immediate pain relieving after the treatment. Although there was no statistically significant difference in erythema level at any time point during the study (𝑝< 0.05), laser therapy group had significantly lower exudation level than medical therapy group at the final patient visit at the end of the study (𝑝< 0.05). Aggarwal et al. [34] reported a statistically significant reduction in pain by using AMD diode laser unit (810 nm) for RAS patients. The laser group showed a statistically significant reduction of VAS scores (a horizontal 10 cm VAS system) in the immediate pain relief as compared to the sham controlled group. At day 1, day 2, and day 3, the differences were also significant compared to control group. i Albrektson et al. [35] compared the results by using GaAlAs (gallium aluminum-arsenic) semiconductor laser treatment (810 nm) with that placebo laser. Pain scores (a horizontal 100 mm VAS system) and patients’ subjective experience of eating, drinking, and brushing were registered. The pain scores changed dramatically with statistically signif- icant difference (𝑝< 0.0001) in the laser group compared to the placebo group. The difference was also significant when comparing moderate or severe difficulty with eating, drinking, and brushing between the laser and the placebo group (𝑝< 0.0001). De Souza et al. [39] evaluated the effectiveness of InGaA1P diode laser treatment (670 nm) with medication treatment (a topical triamcinolone acetonide given four times daily). The results revealed that no significant difference was noted in RAS regression time between the patients treated with corticoid agent and those treated with laser (𝑝= 0.4345). De Souza et al. [39] evaluated the effectiveness of InGaA1P diode laser treatment (670 nm) with medication treatment (a topical triamcinolone acetonide given four times daily). The results revealed that no significant difference was noted in RAS regression time between the patients treated with corticoid agent and those treated with laser (𝑝= 0.4345). (i) Laser Therapy versus Inactive “Laser” Therapy Lalabonova and Daskalov [42] reported a statistically significant reduction in pain level and erythema and epithe- lization dynamics (the sign of healing) by using SIX Laser TS diode laser system (658 nm) compared to medication treatment (Granofurin and solcoseryl given twice daily) for RAS patients. (3) Size of Ulcers. Aggarwal et al. [34] found that the AMD diode laser unit (810 nm) group showed a statistically significant reduction in lesion sizes at each follow-up time, especially on day 3 (𝑝< 0.001). Lalabonova and Daskalov [42] reported a statistically significant reduction in pain level and erythema and epithe- lization dynamics (the sign of healing) by using SIX Laser TS diode laser system (658 nm) compared to medication treatment (Granofurin and solcoseryl given twice daily) for RAS patients. Sattayut et al. [40] also reported the change of ulcer sizes after CO2 laser (10605 nm) therapy. Clinical data were recorded at baseline, day 1, day 2, day 3, day 5, and day 7 after treatment between the laser group and the control group. However, the authors were disappointed to find that there 10 Scientifica Jijin et al. [43] made a comparison between the laser therapy group (AMD lasers, 810 nm) and the medication treatment group (5% amlexanox oral paste given to the ulcers 4 times daily). Both the participants in 5% amlexanox group and AMD laser group showed a significant reduction in their pain scores and ulcer size on the third day and the seventh day compared with the first day. Also, the intergroup difference in pain levels between the laser group and amlexanox group was significant on the third day (𝑝= 0.006); however, the difference in pain levels was not significant on the seventh day (𝑝= 0.171). In the meantime, the intergroup difference in the reduction of ulcer sizes was not statistically significant both on the third day (𝑝= 0.54) and on the seventh day (𝑝= 0.78) after treatment. traditional medical treatment. However, Jijin et al. [43] stated that the difference in pain levels between the laser group and amlexanox group was significant on the third day but not significant on the seventh day. When converted to the effectiveness in ulcer healing, Tezel et al. [41] and De Souza et al. (i) Laser Therapy versus Inactive “Laser” Therapy [39] conducted that no significant difference was noted between the laser group and medical therapy group, though Lalabonova and Daskalov [42] reported that there was a statistically significant reduction in erythema dynamics and epithelization time (the sign of healing) in the laser therapy group. So it was difficult to make sound conclusions if laser group showed a significantly greater efficacy than medical therapy in the management of RAS. This review also aimed to explore the security of laser treatment. No visible side effects were reported in the trials included. Some factors may be contributed to the absence of side effects: firstly, the characteristic of the low-power density of the laser beams and, secondly, the use of gel in the application of CO2 laser. The laser beams in LLLT, with a narrow spectral width (600 nm–1000 nm) and power den- sities (1 mw–5 W/cm2), are so low that the resulting biological effects are not associated with macroscopic thermal effects [47]. Additionally, it should be noted that, in contrast to conventional low-level laser therapy (Nd:YAG laser, Er:YAG laser, InGaAlP laser, and GaAlAs laser), CO2 laser has been used as a very useful high-power, thermal laser in surgery for cutting, ablation, and coagulation of the tissues for many years [31]. However, this high-power laser can also be used as a low-power therapeutic laser in RAS. In the studies about the CO2 laser treatment in RAS, before laser irradiation, a thick layer of a transparent, nonanesthetic gel with high-water content is placed on the lesion to reduce the beam absorption by the tissue. The final laser power output is reduced to 2– 5 mW which is in the range of low-power lasers, after passing through the gel [31]. As a result, the conventional high-power laser is reduced to a low-power, nonthermal, and noninvasive laser after passing through the gel. This technique is called nonthermal, nonablative CO2 laser therapy (NACLT) [37]. Due to the low-level therapeutic nature of NACLT, studies involving the application of low-power nature CO2 laser therapy in RAS were included in our systematic review. Although we have observed no visual side effects after the application of laser phototherapy, it should be noted that we should not let down our vigilance against the complications such as the aggravation of the lesions or the thermal damage (tissue ablation, ulceration, and erythema) to the normal oral mucosa. (iii) Clinical Complications. No adverse reactions or clinical complications were reported in all included ten trials. 3.6. Discussion. Although many positive findings were reported in animal models, clinical case reports, and random- ized controlled clinical trials, the clinical use of LLLT therapy for oral stomatitis remains controversial. This possibly is due to two main reasons: firstly, the mechanisms of the treatment effects are incompletely understood now; and, secondly, the complexity of treatment protocols choosing such as wavelength, frequency, power density, pulse structure, and treatment timing has led to the difficulty in classification and evaluation of the negative studies as well as positive ones. The exact mechanisms of LLLT are not completely understood. There are several theories that explain the mechanisms of LLLT at present. The adenosine triphosphate (ATP) hypothesis [45] believes that the laser light is absorbed by cytochromes in the mitochondria and then converted into energy—ATP. The cell thus enters a photobioactivated state, during which the target photo activating cellular can use the energy to stimulate its membrane or organelles. Besides, the enhancing level of ATP causes hyperpolarization of the neurons and obstruction of pain stimuli, resulting in an obvious decrease in the induction of pain stimuli, leading to a result of symptomatic pain relief. In addition to the ATP hypothesis, other mechanisms such as the singlet oxygen hypothesis, the redox properties alteration hypothesis, and nitrous oxide (NO) hypothesis may be operating in LLLT [46]. Generally, the mechanisms of low-level light therapy should be continually explored in the future. Th d h l ff f This systematic review assessed the actual effectiveness of laser treatment on RAS by analyzing 10 RCTs. As you see, despite the application of various types of lasers with different laser parameters, the results indicated that the laser therapy group showed significant changes on healing time, pain level (especially the immediate pain relief), and the size of ulcers at different follow-up times compared with the placebo group (an inactive laser probe). However, when we tried to draw comparisons between the laser groups and the medical treatment groups, things were going to be different. In those four studies, when talking about the effectiveness in pain relieving, Tezel et al. (i) Laser Therapy versus Inactive “Laser” Therapy Additionally, some negative experiments reported that DNA damage and reversible cell damage can be observed directly after the laser irradiation in vitro [48]. The amount of DNA damage and cytotoxicity may be related to duration of the laser irradiation, which is dependent on the power density (mW/cm2) of each laser. (iii) Clinical Complications. No adverse reactions or clinical complications were reported in all included ten trials. 4. Conclusions In conclusions, although most of studies included in this review reported that laser treatment could significantly alle- viate pain (especially the immediate pain relief) and facilitate healing compared with placebo “laser,” it was difficult to make sound conclusions if laser group showed a significantly greater efficacy than medical therapy in the management of RAS. Furthermore, no adverse reactions or clinical complica- tions after the application of laser phototherapy were reported in all included trials. Of course, the evidence of the retrieved studies is weak. Therefore, rigorously designed, long-term, randomized, controlled, and large sample-sized clinical trials must be conducted to confirm the effectiveness of laser on RAS therapy. [11] B. Murray, N. McGuinness, P. Biagioni, P. Hyland, and P.-J. 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No adverse reactions or clinical complications were reported in all included ten trials. [41] and Lalabonova and Daskalov [42] reported that laser group showed a statistically significant reduction in pain at different follow-up times, compared to The results showed a relatively high statistical hetero- geneity, maybe owing to various laser types (Nd:YAG laser, Er:YAG laser, CO2 laser, etc.) and laser treatment protocols, such as different wave length, output and energy density, frequency, and follow-up period. Furthermore, several lim- itations must be addressed in this systematic review. Firstly, 11 Scientifica [7] R. G. Shashy and M. B. 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https://openalex.org/W2979912804	https://eprints.soton.ac.uk/435071/1/journal.pone.0223614.pdf	English	null	Increased aggression and reduced aversive learning in honey bees exposed to extremely low frequency electromagnetic fields	PloS one	2,019	cc-by	7,454	RESEARCH ARTICLE Editor: Adam G Dolezal, University of Illinois at Urbana-Champaign, UNITED STATES Editor: Adam G Dolezal, University of Illinois at Urbana-Champaign, UNITED STATES Copyright: © 2019 Shepherd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Abstract Honey bees, Apis mellifera, are a globally significant pollinator species and are currently in decline, with losses attributed to an array of interacting environmental stressors. Extremely low frequency electromagnetic fields (ELF EMFs) are a lesser-known abiotic environmental factor that are emitted from a variety of anthropogenic sources, including power lines, and have recently been shown to have a significant impact on the cognitive abilities and behav- iour of honey bees. Here we have investigated the effects of field-realistic levels of ELF EMFs on aversive learning and aggression levels, which are critical factors for bees to main- tain colony strength. Bees were exposed for 17 h to 100 μT or 1000 μT ELF EMFs, or a sham control. A sting extension response (SER) assay was conducted to determine the effects of ELF EMFs on aversive learning, while an intruder assay was conducted to deter- mine the effects of ELF EMFs on aggression levels. Exposure to both 100 μT and 1000 μT ELF EMF reduced aversive learning performance by over 20%. Exposure to 100 μT ELF EMFs also increased aggression scores by 60%, in response to intruder bees from foreign hives. These results indicate that short-term exposure to ELF EMFs, at levels that could be encountered in bee hives placed under power lines, reduced aversive learning and increased aggression levels. These behavioural changes could have wider ecological impli- cations in terms of the ability of bees to interact with, and respond appropriately to, threats and negative environmental stimuli. Sebastian ShepherdID1,2, Georgina Hollands1, Victoria C. Godley1, Suleiman M. Sharkh3, Chris W. Jackson1, Philip L. Newland1 Sebastian ShepherdID1,2, Georgina Hollands1, Victoria C. Godley1, Suleiman M. Sharkh Chris W. Jackson1, Philip L. Newland1 1 Biological Sciences, University of Southampton, Highfield Campus, Southampton, United Kingdom, 2 Department of Entomology, Purdue University, West Lafayette, Indiana, United States of America, 3 Mechatronics, Mechanical Engineering, University of Southampton, Highfield Campus, Southampton, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * shephe24@purdue.edu * shephe24@purdue.edu OPEN ACCESS Citation: Shepherd S, Hollands G, Godley VC, Sharkh SM, Jackson CW, Newland PL (2019) Increased aggression and reduced aversive learning in honey bees exposed to extremely low frequency electromagnetic fields. PLoS ONE 14 (10): e0223614. https://doi.org/10.1371/journal. pone.0223614 Editor: Adam G Dolezal, University of Illinois at Urbana-Champaign, UNITED STATES Received: June 7, 2019 Accepted: September 24, 2019 Published: October 10, 2019 Copyright: © 2019 Shepherd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Adam G Dolezal, University of Illinois at Urbana-Champaign, UNITED STATES Received: June 7, 2019 Accepted: September 24, 2019 Published: October 10, 2019 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees Competing interests: The authors have declared that no competing interests exist. known abiotic environmental factor that has the potential to affect insect biology and thus may contribute to the environmental stress load that insects currently experience in global eco- systems [5, 6]. Competing interests: The authors have declared that no competing interests exist. Extremely low frequency electromagnetic fields (ELF EMFs) are a specific type of non-ion- ising electromagnetic radiation in the frequency range 3–300 Hz that are emitted from anthro- pogenic devices. Pollution of the environment with ELF EMFs has increased dramatically in the last century, with a major source for ELF EMFs being power transmission lines [7]. ELF EMF exposure has recently been associated with a variety of different effects on insects includ- ing changes in developmental biology [8, 9], locomotor behaviour [6, 10], molecular biology [11, 12], and immune response [13]. Honey bees may be particularly at risk to ELF EMF pollution in the environment. At ground level, ELF EMF intensity under power transmission lines can reach 100 μT, while fly- ing insects can be exposed to much higher levels close to conductors where ELF EMF levels can be over 1,000 μT [5]. Some studies suggest exposure to ELF EMFs from power lines may be stressful for honey bees [14, 15] whilst it has also been reported [16] that bees hived under power lines will readily abscond. Moreover, Greenberg et al. [17] found that bee hives exposed to power lines had increased motor activity, abnormal propolisation, reduced weight gain of hives, queen loss, impaired production of queen cells, decreased sealed brood and poor winter survival, leading to a federal US precaution to not store hives under power lines [18]. While these studies show no direct experimental evidence for ELF EMF effects on bees, they at least suggest that ELF EMF exposure may be a factor that contributed to, or caused, the stress responses of the bees observed in these studies. In their environment bees are exposed to a variety of negative environmental stimuli and cues, which are also critical for bees to perceive and respond to, such as weather, toxins [19], or biotic threats such as colony diseases and parasites [20, 21], invading robber bees from other colonies [20] and predators [21–23]. Introduction Funding: SS was funded by a Mayflower Studentship from the University of Southampton. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publication of this article was funded in part by Purdue University Libraries Open Access Publishing Fund. Over the last 30 years there has been a decline in the numbers of the economically and ecolog- ically important honey bee [1, 2]. Honey bee declines are part of a much larger global problem of pollinator declines [3] with major causes attributed to a combination of interacting, and mainly anthropogenically driven, environmental stressors including, habitat loss, pesticide exposure, pathogens and parasites [4]. Electromagnetic pollution is emerging as a lesser- PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 1 / 13 Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 Animals Honey bees were kept at the University of Southampton Highfield Campus apiary (50˚ 56’ 10’’N, 1˚ 23’ 39’’W) and experiments conducted from June-August, 2017. Foragers were iden- tified by the pollen in their corbiculae and transported to an insectary in the Institute for Life Sciences at the University of Southampton, where they were immobilized on wet ice and trans- ferred into appropriate containers for SER and Intruder Assay experiments. Magnetic fields Electromagnetic fields were generated with a custom-made Helmholtz coil [5] which produced homogenous 50 Hz sinusoidal AC electromagnetic fields with a range of field strength from ~10 μT—10,000 μT. Field strength (magnetic flux density) was measured with a Model GM2 Magnetometer (Alphalab Inc., USA). For control exposures no current was passed through the coil system. For SER experiments control, 100 μT and 1000 μT 50 Hz EMF treatments were applied, while for intruder assay experiments control and 100 μT ELF EMF treatments were used. How colonies respond to these environmental stresses is critical to their long-term fitness. Bees must be able to detect these negative stimuli [20], learn that they are associated with a negative effect [19], enact an appropriate aggressive response [22], and even communicate this information to other individuals [23]. For example, guard bees when confronted with a threat (e.g. predator or intruder) may enter the hive to release alarm pheromone by extruding their sting, raising their abdomen and fanning their wings [24, 25]. Surprisingly little is known about aversive learning, and how it is affected by environmental stimuli, despite its importance in maintaining colony fitness. A sting extension response (SER) assay [26, 27] has been developed to study aversive learning in bees in which a conditioned stimulus (CS) (often olfactory) is applied and associated with an unconditioned stimulus (US) of a weak electric shock. Over repeated conditioning trials bees learn to associate the negative US with the CS. The SER assay can therefore provide valuable information in a controlled experimental environment of how potential stressors such as ELF EMFs can affect bees [28]. For example, SER has been used to investigate the impacts of the neonicotinoid insecticide imidacloprid on honey bee aversive learning [29]. In addition, intruder assays have been used to assess aggressive responses of honey bees, including to conspecifics [30–33]. Environmental stresses which could affect the ability of bees to learn about negative environmental cues, or respond appropriately to environmental cues, could therefore be detrimental to honey bee col- ony health. Here we have used both the SER and intruder assays to determine whether short term expo- sure to ELF EMFs, at levels equivalent to those found at ground level under high-voltage trans- mission power lines, can affect aversive learning and aggression in honey bees. We have utilised these well-established assays in the laboratory where the levels of EMF exposure of PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 2 / 13 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees individual bees can be precisely controlled, and under consistent conditions free from stray fields and other confounding stimuli. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 Sting extension response assay Bees were collected individually from 3 hives and harnessed in custom made SER cradles cut from Perspex, with a similar design to Vergoz et al. [27]. Bees were placed ventral side upwards in a metal fork of the cradle, such that the fork held the bee by the thorax, with prongs in place around the petiole and neck of the bee (Fig 1A). This fork also served as an electrode for an Fig 1. Sting extension response protocol. A) Harnessing of a bee in an SER cradle for EMF exposure. Tesa© tape was applied around the thorax to hold the bee between the fork prongs. B) Aversive sting extension response to the CS in SER conditioning trials. The inset shows the extended stinger in more detail. C) SER Timetable showing a representation of an individual conditioning trial. The bee was acclimatised to the arena for 20 s, before CS (linalool) application. After 6 s of CS, CS and US (12 V shock) were paired for 2 s, after which both CS and US were switched off. A further 32 s of clear airflow was allowed for odour to be removed from the arena. https://doi.org/10.1371/journal.pone.0223614.g001 Fig 1. Sting extension response protocol. A) Harnessing of a bee in an SER cradle for EMF exposure. Tesa© tape was applied around the thorax to hold the bee between the fork prongs. B) Aversive sting extension response to the CS in SER conditioning trials. The inset shows the extended stinger in more detail. C) SER Timetable showing a representation of an individual conditioning trial. The bee was acclimatised to the arena for 20 s, before CS (linalool) application. After 6 s of CS, CS and US (12 V shock) were paired for 2 s, after which both CS and US were switched off. A further 32 s of clear airflow was allowed for odour to be removed from the arena. https://doi.org/10.1371/journal.pone.0223614.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 3 / 13 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees aversive shock stimulus during the SER assay (Fig 1B). Tesa© tape was then placed laterally across the cradle and between the prongs of the fork across the thorax to restrain the bee in the cradle. Bees were then fed to satiation with a 50% w/v sucrose solution and were then ready for overnight treatment (17 h). Sting extension response assay An experimental arena (W × D × H = 60 × 45 × 55 cm) was used with an odour delivery system at one end and an extraction fan at the other to remove any odours from the arena. The odour delivery system allowed a constant airflow to be supplied to the arena. A clear airflow, and the CS, were delivered in separate channels in the multichannel system which joined via Teflon tubing before it discharged into the arena at a single release point. Electronic valves allowed the airflow to switch between CS and clear airflow channels. The CS used was 8 μl of 97% linalool (Sigma-Aldrich, UK) which was pipetted onto filter paper to be placed in the CS delivery channel. The channel with clear air was always open when no odour was delivered. To deliver the CS, airflow was switched from the clear air channel to the odour delivery channel such that bees were supplied with a constant airflow, and would associate any stimulus with the odour and not a change in airflow. For SER experiments bees were exposed to control, 100 μT or 1000 μT EMFs for 17 h and following exposure SER trials began immediately. This treatment was chosen to represent a field-realistic scenario where bee hives are placed under transmission and where bees have been reported to show negative responses [17]. 357 bees completed the SER assay. An SER cra- dle containing a harnessed bee was placed into the experimental arena of the odour delivery system. Bees were exposed to a clear airflow for 20 s (Fig 1C). During this time the SER cradle was attached to a DC power-supply with a 12 V output. The airflow was then switched from clean air to linalool airflow, representing the CS. The CS lasted 8 s. For the final 2 s of the CS the bee was shocked at 12 V from the DC power supply, representing the unconditioned stim- ulus (US) thus pairing US and CS for 2 s. The US and CS finished at the same time (28 s into the trial). The clear airflow was then left on for 32 s with the bee in the arena to reinforce the association of the CS with the US and to allow the extractor to remove linalool from the arena. The length of one complete conditioning trial for a bee was 60 s (Fig 1C). Sting extension response assay Conditioning trials were repeated 5 times for each individual bee with an inter-trial interval of 10 min. If a bee did not respond during linalool delivery or electric shock then a ‘failed response’ was recorded. Bees that failed to respond more than once in conditioning trials (n = 16, 4.5% of 357) were excluded from analyses. No bees exhibited a pre-learned aversive response to linalool in the first conditioning trial, and therefore no bees had to be excluded from analysis for this reason. After all exclusions were made, 341 bees remained that com- pleted the SER assay for inclusion in statistical analyses (S1 Table). If a bee responded only after the shock stimulus then a non-conditioned sting extension response was recorded (i.e. the bee responded to US but not CS). As in previous aversive learn- ing studies responses to the conditioned stimulus have been described only when a bee extends its sting during the CS application, and are defined as a ‘sting extension response’ (Fig 1A and 1B). The proportions of conditioned sting extension responses over 5 trials were analysed to assess the effects of short-term ELF EMF exposure on aversive learning in honey bees. This aversive learning approach therefore measures acquisition and short-term retention of information, and thus has comparability with the results of the intruder assay where bees encounter a new individual from a foreign hive. Intruder assay Bees were collected from 5 different hives in groups of 20 bees from the same hive of origin. Each group of 20 was split into 2 paired cohorts of 10 (S2 Table), and stored in separate petri 4 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees Table 1. Aggressive severity behavioural index used in the intruder assay adapted from Richard et al. [31]. Behaviour Definition Aggressive Severity Index Aggressive antennation Antennation directed towards the intruder or touching the intruder with antennae 1 Stalking Follows and moves towards intruder for more than 5 seconds 1 Crawl over Moves directly on top of the intruder 1 Antennation with mandibles open Antennation directly towards the intruder with mandibles open 2 Biting Uses mandibles to grasp the intruder 3 Abdomen flexion The abdomen is flexed but the stinger is not extruded 4 Stinging attempts The stinger is visibly extruded towards the intruder 5 https://doi.org/10.1371/journal.pone.0223614.t001 https://doi.org/10.1371/journal.pone.0223614.t001 dishes fitted with 50% w/v sucrose feeders. For each pair of 10-bee cohorts (from the same hive of origin) 1 cohort was exposed to a 100 μT ELF EMF and the other exposed to control conditions (both at 22 ± 1˚C) for 17 h overnight. The intruder assay was conducted the next day. The sample period for the intruder assay began when a forager bee from a 6th (and differ- ent) hive was introduced into each petri dish. Focal sampling of the ‘intruder’ bee was con- ducted continuously for 10 min to assess the behaviour of recipient bees towards the intruder. Behaviours were categorized on an aggressive severity index adapted from Richard et al. [31] (Table 1) and the aggressive severity indices summed for a full 10 min sample period to give an overall aggression score for that sample. In total 60 intruder assay samples were conducted (n = 30 per treatment, with 6 assays/treatment/hive). Statistical analysis Data were analysed in SPSS (v.24, IBM SPSS Inc.) and Graphpad Prism (v.7, Graph Pad Soft- ware Inc.). Where appropriate, homogeneity of variance and normality assumptions were tested. For all models assessing the effects of treatments on binomial SER data, binomial error structure and logit link function were used, and where appropriate pairwise contrasts with Bonferroni adjusted significance were used in post-hoc analyses. To determine whether ELF EMF exposure or ‘hive or origin’ affected the initial aversive responsiveness of bees a generalized linear model (GLM) with ‘EMF treatment’ and ‘hive of origin’ as interacting factors was used. To analyse the effect of ELF EMF exposure on sting extension responses, a generalized mixed effect model (GLMM) was used with ‘EMF treat- ment’, ‘hive of origin’, and ‘conditioning trial’ as interacting factors. For GLMMs trial 1 was not included in analyses (i.e. trials 2–5 were used), as learning cannot occur in the first trial. For intruder assay analysis, aggression scores were totalled from each trial and data log10- transformed to satisfy normality assumptions for parametric statistical analyses. A two-way Repeated Measures ANOVA was conducted to determine the effects of ‘EMF’, and ‘Hive of Origin’ on log-transformed aggression score data, with data paired by their collection cohort. Data plotted in aggression score graphs is back-transformed. Sting extension response ELF EMFs do not reduce the ability of bees to respond to aversive stimuli. To deter- mine whether short-term exposure to EMF (control, 100 μT, or 1000 μT) affected the ability of bees to respond with an aversive extension of the sting, the proportions of bees which did not 5 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees Fig 2. Aversive responses of honey bees in the SER assay. The effect of ELF EMF treatment on the proportion of aversive responsiveness to 12 V electric shock aversive stimuli. Exact proportions are plotted. Results show that ELF EMFs had no effect on the aversive responses of bees to electrical stimulation. https://doi.org/10.1371/journal.pone.0223614.g002 Fig 2. Aversive responses of honey bees in the SER assay. The effect of ELF EMF treatment on the proportion of aversive responsiveness to 12 V electric shock aversive stimuli. Exact proportions are plotted. Results show that ELF EMFs had no effect on the aversive responses of bees to electrical stimulation. https://doi.org/10.1371/journal.pone.0223614.g002 fail to respond to the US (i.e. non-learned sting extension to an aversive stimulus) between each treatment were compared. After 17 h control exposure 95.0% of bees (n = 119) exhibited aversive responses (Fig 2), whereas 96.6% (n = 118) responded following exposure to 100 μT and 95.0% (n = 120) responded following exposure to 1000 μT EMFs. Thus, the initial aversive responsiveness of honey bees was not affected by any interaction between the ELF EMF ‘treat- ment’ or the honey bee ‘hive of origin’ (GLM, χ2<0.001, d.f. = 4, P > 0.99), nor were there any main effects of ‘treatment’ (GLM, χ2<0.001, d.f. = 2, P > 0.99) or ‘hive of origin’ (GLM, χ2<0.001, d.f. = 2, P > 0.99). fail to respond to the US (i.e. non-learned sting extension to an aversive stimulus) between each treatment were compared. After 17 h control exposure 95.0% of bees (n = 119) exhibited aversive responses (Fig 2), whereas 96.6% (n = 118) responded following exposure to 100 μT and 95.0% (n = 120) responded following exposure to 1000 μT EMFs. Thus, the initial aversive responsiveness of honey bees was not affected by any interaction between the ELF EMF ‘treat- ment’ or the honey bee ‘hive of origin’ (GLM, χ2<0.001, d.f. Sting extension response = 4, P > 0.99), nor were there any main effects of ‘treatment’ (GLM, χ2<0.001, d.f. = 2, P > 0.99) or ‘hive of origin’ (GLM, χ2<0.001, d.f. = 2, P > 0.99). ELF EMFs reduce learning performance of the sting extension response. For control bees, and those exposed to 100 μT and 1000 μT ELF EMFs, the proportion of bees exhibiting a sting extension response increased with each conditioning trial (GLMM, F3,1352 = 26.08, P < 0.0001). For bees maintained under control conditions 29% showed SER after trial 3 while 50% showed SER after conditioning trial 5 (Fig 3). By contrast, after bees were exposed to 100 μT ELF EMFs only 12% of bees showed SER after trial 3 and 32% after trial 5. Following exposure to 1000 μT ELF EMFs 19% showed an SER after trial 3 and 27% after trial 5. EMF treatments were found to significantly reduce the proportions of SER in honey bees (GLMM, F2,1352 = 15.01, P < 0.0001). A greater proportion of control exposed bees exhibited SER than PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 6 / 13 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees Fig 3. Effects of ELF EMFs on aversive learning in honey bees. Effect of short-term ELF EMF exposure on the proportion of aversive responses to the conditioned stimulus (linalool) for each of the trials. For each treatment the Fig 3. Effects of ELF EMFs on aversive learning in honey bees. Effect of short-term ELF EMF exposure on the proportion of aversive responses to the conditioned stimulus (linalool) for each of the trials. For each treatment the proportion of bees showing a learned response increased. The exact proportion of responses is plotted. https://doi.org/10.1371/journal.pone.0223614.g003 Fig 3. Effects of ELF EMFs on aversive learning in honey bees. Effect of short-term ELF EMF exposure on the proportion of aversive responses to the conditioned stimulus (linalool) for each of the trials. For each treatment the proportion of bees showing a learned response increased. The exact proportion of responses is plotted. https://doi.org/10.1371/journal.pone.0223614.g003 https://doi.org/10.1371/journal.pone.0223614.g003 both 1000 μT (Pairwise comparison, Bonferroni adjusted P < 0.001) and 100 μT (Pairwise comparison, Bonferroni adjusted P = 0.001) exposed bees. There was no ‘treatment’ ‘trial’ interaction (GLMM, F1,1352 = 0.82, P = 0.56). Sting extension response In this analysis of the effects of ELF EMF exposure on sting extension responses, hive of origin was removed as a factor to improve model fit as it was found to have no effect on the proportion of SER to the CS (GLMM, F2,1328 = 0.17, P = 0.84), nor any interaction with ‘treat- ment’ (GLMM, F4,1328 = 1.38, P = 0.24) ‘conditioning trial’ (GLMM, F6,1328 = 0.24, P = 0.96) or three-way interaction (GLMM, F12,1328 = 0.33, P = 0.99). Intruder assay Bees exposed to 100 μT ELF EMF exhibited greater aggressive behaviour to introduced bees, than bees not exposed to ELF EMFs (Fig 4). Bee cohorts which received a control treatment PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 7 / 13 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees displayed an aggression score of 12.87 ± 1.69 (mean ± SEM) whereas bee cohorts exposed to 100 μT EMF exhibited a mean aggression score of 20.70 ± 2.14 (mean ± SEM, Standard Error of the Mean). EMF exposure significantly increased the average aggression scores across bees from all hives (F1,25 = 11.42, P = 0.0024). There was no impact of Hive (F4,25 = 0.65, P = 0.63) or any HiveEMF interaction effect (F4,25 = 0.75, P = 0.56) on aggression score. This indicates Fig 4. The effect of ELF EMFs on honey bee aggression levels. Exposure to a 100 μT ELF EMF significantly increased the Aggression Score. Mean ± SEM are shown. Statistical analyses were conducted on log-transformed data. Data plotted are reverse log-transformed from data used in statistical analysis. https://doi.org/10.1371/journal.pone.0223614.g004 Fig 4. The effect of ELF EMFs on honey bee aggression levels. Exposure to a 100 μT ELF EMF significantly increased the Aggression Score. Mean ± SEM are shown. Statistical analyses were conducted on log-transformed data. Data plotted are reverse log-transformed from data used in statistical analysis. https://doi.org/10.1371/journal.pone.0223614.g004 Fig 4. The effect of ELF EMFs on honey bee aggression levels. Exposure to a 100 μT ELF EMF significantly increased the Aggression Score. Mean ± SEM are shown. Statistical analyses were conducted on log-transformed data. Data plotted are reverse log-transformed from data used in statistical analysis. Fig 4. The effect of ELF EMFs on honey bee aggression levels. Exposure to a 100 μT ELF EMF significantly increa the Aggression Score. Mean ± SEM are shown. Statistical analyses were conducted on log-transformed data. Data plotted are reverse log-transformed from data used in statistical analysis. https://doi.org/10.1371/journal.pone.0223614.g004 Fig 4. The effect of ELF EMFs on honey bee aggression levels. Exposure to a 100 μT ELF EMF significantly increased the Aggression Score. Mean ± SEM are shown. Statistical analyses were conducted on log-transformed data. Data plotted are reverse log-transformed from data used in statistical analysis. Discussion Short-term exposure to 50 Hz ELF EMFs reduced aversive learning performance and increased aggression at levels as low as 100 μT. This directly shows, for the first time, that short-term ELF EMF exposure at levels which can be encountered at ground level under high- voltage transmission power lines can affect honey bees, in terms of both their conditioning to negative stimuli, and the intensity of their aggressive behaviour. In locusts ELF EMFs have been shown to affect neural circuits controlling limb movement and muscular force [6]. During the stinging response in honey bees the protraction of the tip of the abdomen, and the alternate sliding of barbed lancets of the stinging apparatus, are coor- dinated by four large abdominal muscles [34–36] whose activity are regulated by neural cir- cuits in the terminal abdominal ganglion [22]. Given that a sting extension response was evoked by the US in over 95% of trials, it is unlikely that the effects on aversive learning were due to the effects of EMF at the neuromuscular level. Similarly, the effects of EMF were not due to changes in the sting extension motor pattern as bees could still extend their abdomens to electric shocks. Instead ELF-EMF induced reductions in SER performance are solely down to a reduced ability to learn the aversive stimuli, and not the motor pattern involved in responding to the stimuli. p g The mechanisms underlying the effects of ELF EMFs on honey bee aversive learning and aggression may be diverse. While the neural pathways underlying appetitive learning in the honey bee brain are well characterised [37, 38], less is known of the neural architecture under- lying aversive learning. The biogenic amines dopamine and octopamine have critical roles in associative learning in honey bees [39]. Vergoz et al. [27] for example, found that aversive learning is impaired after the injection of dopaminergic antagonists, and Jarriault et al. [40] found that dopamine was released in mushroom bodies in the honey bee brain after electric shock stimulation of the abdomen. These findings suggest that dopamine may have a key role in memory formation in honey bee aversive learning. Furthermore, the honey bee alarm pher- omone has been shown to increase levels of the biogenic amines serotonin and dopamine, with increases in these amine levels being associated with increased likelihood of a bee to sting [41]. Intruder assay https://doi.org/10.1371/journal.pone.0223614.g004 https://doi.org/10.1371/journal.pone.0223614.g004 displayed an aggression score of 12.87 ± 1.69 (mean ± SEM) whereas bee cohorts exposed to 100 μT EMF exhibited a mean aggression score of 20.70 ± 2.14 (mean ± SEM, Standard Error of the Mean). EMF exposure significantly increased the average aggression scores across bees from all hives (F1,25 = 11.42, P = 0.0024). There was no impact of Hive (F4,25 = 0.65, P = 0.63) or any HiveEMF interaction effect (F4,25 = 0.75, P = 0.56) on aggression score. This indicates PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 8 / 13 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees that short-term ELF EMF exposure, at levels that can be encountered at ground level or in proximity to a high voltage transmission power lines, led to an increase in aggressive behaviour of bees directed towards conspecifics. Discussion Some studies investigating the effects of EMF on invertebrates have suggested that increased biogenic amine levels lead to increases in behavioural activity [42, 43]. While no studies have yet analysed changes in dopamine levels following ELF EMF exposure, these pre- vious studies suggest that biogenic amine levels may be a potential area to investigate to eluci- date the underlying mechanisms of ELF EMF induced changes in insect behaviour. Moreover, ELF EMFs have been shown to have effects on neuronal signalling in insects [6], and therefore there is the potential for ELF EMF induced effects on dopaminergic neurons or other neural circuits which are involved in aversive learning pathways. ELF EMF induced changes in behav- iour could also be underpinned by molecular changes such as gene expression. For example short-term ELF EMF exposure has been shown to increase heat-shock protein expression in locusts [6] and Drosophila [12]. The ecological implications of these effects are diverse. On the one hand the reduced ability to learn new negative stimuli could lead to an increased latency of honey bee colonies to respond to novel threats. Maliszewska et al. [10] found that short-term exposure of American cockroaches to 7,000 μT ELF EMFs increased the latency of responses to a negative heat stimu- lus. The increase in latency could clearly be detrimental to individuals in the ability to avoid PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 9 / 13 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees harmful environmental stimuli. On the other hand, we found that bees exposed to ELF EMFs showed increased aggression levels. Rittschof et al. [33] found that increased levels of aggres- sion in honey bees are associated with greater resilience to environmental stresses and to immune challenge. However, direct short-term ELF EMF exposure at 2,000 μT in Lepidop- teran larvae has been associated with changes in immune response parameters such as increased apoptotic-like hemocytes, reduced hemolyph total protein and reduced hemocyte cell count, which could suggest short-term ELF EMF exposures might lead to reduced resil- ience to immune challenge [13]. It is not known if ELF EMFs affect immune response in honey bees at field-realistic ELF EMF intensities, lower than those that have been studied with Lepidoptera, and thus it is not known if ELF EMF exposure would confer greater resilience to immune challenge alongside increased aggression levels in bees. Discussion In addition, in the environ- ment if a bee perceives a negative stimulus a sting response often results in sting autonomy, with a rupture of the abdomen that causes the eventual death of the bee [44, 45]. Less aggres- sive responses to negative stimuli such as aggressive buzzing and flight bombardment can be successful methods of warding off threats in a manner that is less detrimental to a colony in terms of bee loss [25, 45]. The effects of environmental stressors and the consequences of increased aggression on this aversive decision making processes (other than increased sting autonomy) are not-known. While it is unclear what the ecological consequences of increased aggression may be for bees exposed to ELF EMFs, the implications of reduced aversive learning performance are more distinct. It is imperative that honeybees are able to perceive, learn, and avoid threats in the environment [28, 39]. Reductions in the ability to learn about negative stimuli could have implications for the abilities of bees to deal with predatory/invader threats [20, 22], detecting/ avoiding deleterious stimuli [19] and responding to negative stimuli that require action e.g. attacking/removing diseased individuals from the hive [20], all of which could have detrimen- tal effects on bee colonies. Although it is not yet known how bees will actually respond in the field, it is clear that the reduction in aversive learning seen here with short-term 100 μT expo- sures could be detrimental to honeybees on an ecological level. A number of studies have described bee colonies failing that are hived under high-voltage transmission power lines, where EMF levels can reach 100 μT [14–17]. There is the possibility that with hives located under power lines, the long-term chronic exposure to ELF EMFs could continually reduce cognitive abilities both with regards to aversive and appetitive learning, potentially leading to some of the negative effects found in these studies. Reductions in learning could be detrimental to individual and colony survivability. There are large potential ecological consequences for reduced ability to learn about aversive and appetitive stimuli for bees. Future studies should focus on whether there are ecological effects of ELF EMF exposure, with direct measurements of chronic EMF exposure under power lines, as well as determining what physiological/molecular processes may be affected by this kind of exposure. Discussion These effects may not be confined to managed honey bees as there may be much wider implications for wild bees and even other pollinators that require power line strips for critical habitat refuge [46–50]. The underlying mechanisms, as well as the potential ecological implications of ELF EMF pollution in the field must be further investigated to determine the effects of ELF EMF pollution on insect biology and ecology, including crucial pollination eco- system services. S1 Table. The number of bees in SER analyses (after exclusions) for each hive and treatment. (DOCX) Formal analysis: Sebastian Shepherd. Investigation: Sebastian Shepherd, Georgina Hollands, Victoria C. Godley. Methodology: Sebastian Shepherd, Georgina Hollands, Victoria C. Godley, Chris W. Jackson, Philip L. Newland. Resources: Suleiman M. Sharkh. Supervision: Chris W. Jackson, Philip L. Newland. Supervision: Chris W. Jackson, Philip L. Newland. Writing – original draft: Sebastian Shepherd, Philip L. Newland. Writing – review & editing: Sebastian Shepherd, Georgina Hollands, Victoria C. Godley, Suleiman M. Sharkh, Philip L. Newland. Supporting information S1 Table. The number of bees in SER analyses (after exclusions) for each hive and treatment. (DOCX) 10 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees Author Contributions Conceptualization: Sebastian Shepherd, Suleiman M. Sharkh, Chris W. Jackson, Philip L. Newland. Formal analysis: Sebastian Shepherd. References https://doi.org/10.1002/bem.20709 PMID: 21953292 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223614 October 10, 2019 11 / 13 Extremely low frequency electromagnetic fields increase aggression and reduce aversive learning in honey bees 12. Li SS, Zhang ZY, Yang CJ, Lian HY, Cai P. 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https://openalex.org/W2800052201	https://europepmc.org/articles/pmc5797373?pdf=render	English	null	Quasispecies variant of pre-S/S gene in HBV-related hepatocellular carcinoma with HBs antigen positive and occult infection	Infectious agents and cancer	2,018	cc-by	6,929	Hatazawa et al. Infectious Agents and Cancer (2018) 13:7 DOI 10.1186/s13027-018-0179-4 Hatazawa et al. Infectious Agents and Cancer (2018) 13:7 DOI 10.1186/s13027-018-0179-4 * Correspondence: yanoyo@med.kobe-u.ac.jp 1Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan 2Division of Molecular Medicine & Medical Genetics, Department of Pathology, Kobe University Graduate School of Medicine, Kobe, Japan Full list of author information is available at the end of the article Quasispecies variant of pre-S/S gene in HBV-related hepatocellular carcinoma with HBs antigen positive and occult infection Yuri Hatazawa1, Yoshihiko Yano1,2, Rina Okada1, Toshihito Tanahashi3, Hiroki Hayashi1, Hirotaka Hirano1, Akihiro Minami1, Yuki Kawano1, Motofumi Tanaka4, Takumi Fukumoto4, Yoshiki Murakami5, Masaru Yoshida1 and Yoshitake Hayashi2 © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Hepatocellular carcinoma (HCC) can develop in patients who are negative for the hepatitis B surface antigen (HBsAg) in serum but positive for hepatitis B virus (HBV) DNA in the liver, referred to as occult HBV infection (OBI). Previous reports showed that HBV variants in OBI-related HCC are different from those in HBsAg-positive HCC. In the present study, HBV quasispecies based on the pre-S/S gene in OBI-related HCC patients were examined by high throughput sequencing and compared with those in HBsAg-positive HCC. Methods: Nineteen tissue samples (9 OBI-related and 10 HBsAg-positive non-cancerous tissues) were collected at the time of surgery at Kobe University Hospital. The quasispecies with more than 1% variation in the pre-S/S region were isolated and analysed by ultra-deep sequencing. Results: There were no significant differences in the major HBV populations, which exhibit more than 20% variation within the entire pre-S/S region, between OBI-related HCC and HBsAg-positive HCC. However, the prevalences of major populations with pre-S2 region mutations and of minor populations with polymerized human serum albumin-binding domain mutations were significantly higher in OBI-related HCC than in HBsAg-positive HCC. Moreover, the major variant populations associated with the B-cell epitope, located within the pre-S1 region, and the a determinant domain, located in the S region, were detected frequently in HBsAg-positive HCC. The minor populations of variants harbouring the W4R, L30S, Q118R/Stop, N123D and S124F/P mutations in the pre-S region and the L21F/S and L42F/S mutations in the S region were detected more frequently in OBI-related HCC than in HBsAg-positive HCC. Conclusions: Ultra-deep sequencing revealed that the B-cell epitope domain in the pre-S1 region and alpha determinant domain in the S region were variable in HBsAg-positive HCC, although the quasispecies associated with the pre-S2 region were highly prevalent in OBI-related HCC. l registration: Ref: R000034382/UMIN000030113; Retrospectively registered 25 November 2017. Trial registration: Ref: R000034382/UMIN000030113; Retrospectively registered 25 November 2017. Keywords: HBV, Pre-S/S, Quasispecies, Occult, HCC, Ultra-deep sequencing HBV extraction and ultra-deep sequencing analysis HBV extraction and ultra-deep sequencing analysis Viral DNA was extracted from 200 μg of non-cancer tis- sue using the QIAGEN DNA Mini Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. The pre-S/S region was amplified by nested PCR using spe- cific primers [18]. The presence of an amplified product was confirmed by electrophoresis on 2% agarose gels, which were stained with ethidium bromide and visual- ized on a UV transilluminator. All of the necessary precautions were taken to prevent cross-contamination. A negative control was included in each assay. A serologically negative HBsAg but positive HBV DNA status in serum or the liver is referred to as occult HBV infection (OBI) [9, 10]. OBI is a potential risk fac- tor for cirrhosis and HCC. Although it has been re- ported that HBV variants of the pre-S/S region are related to HCC generation and HBsAg seroclearance [11–13], the cause of carcinogenesis in OBI patients re- mains unclear. Ultra-deep sequencing was used to detect the varia- tions in HBV quasispecies. The concentrations of the PCR products were measured using the Qubit high- sensitivity double-stranded DNA assay (Invitrogen, Carlsbad, CA, USA). Next, a library of the viral genomic PCR products (< 500 bp; 50 or 0.2 ng) was prepared using the Nextera DNA sample prep kit (Illumina, San Diego, CA, USA) or Nextera XT DNA sample prep kit (Illumina), according to the manufacturer’s instructions. The PCR products were uniformly sheared to 500 bp fragments using these kits, and the PCR products were mixed with 1% 8 pM PhiX as a control and run on the MiSeq sequencer (Genome Analyzer; Illumina) for paired-end 151 bp sequencing. Finally, image-based de- tection of the fluorescent signals was performed using MiSeq control software (Illumina), and the images were used to obtain the sequence data in FASTQ format. Illu- mina paired-end sequencing was performed to generate overlapping read pairs among relatively short sequences combined with relatively long sequences. A quality check and data trimming were necessary before assem- bling the sequences using Genomics Workbench soft- ware version 6.0.1 (CLC bio, Aarhus, Denmark). The sequencing results used in this analysis had a read qual- ity of > 80% of the consensus sequence estimated from a quality score of 30 according to the manufacturer’s (Illu- mina) data. HBV extraction and ultra-deep sequencing analysis Overlapping read pairs and filtering of reads based on a quality score of 30 were used to reveal se- quencing errors and eliminate any false-positive variants generated by PCR errors during sequencing, to increase The variation among viral quasispecies is convention- ally analysed by direct sequencing. However, it is difficult to detect variation levels less than 20% using this method. The analysis of drug-resistance and disease- specific mutations in viruses has recently become pos- sible following technical advances in next-generation se- quencing [14, 15]. In particular, thousands of viral quasispecies within a single host can be analysed by ultra-deep sequencing methods, and it has been sug- gested that the detection of minor quasispecies popula- tions are related to the clinical efficacy [16, 17]. This study focused on quasispecies based on the HBV pre-S/S gene in surgically resected HCC specimens. Using ultra-deep sequencing methods, the quasispecies were analysed and compared between OBI-related HCC and HBsAg-positive HCC. We investigated the specific mutations or variation patterns in OBI-related HCC tissues. Correspondence: yanoyo@med.kobe-u.ac.jp Hatazawa et al. Infectious Agents and Cancer (2018) 13:7 Page 2 of 8 Background hyaluronic acid and quantitative HBsAg levels (HBsAg- HQ: Fujirebio, Tokyo, Japan) at the time of surgery were retrieved from patients’ medical records. In addition, hepatitis B core antibody (anti-HBc; Architect AUSAB, Abbott Japan, Tokyo, Japan) and HBV DNA levels (Cobas® TaqMan HBV Test, v2.0, Roche Diagnostics, Ba- sel, Switzerland) were also examined. The cut-off levels of HBsAg-HQ and HBV DNA were estimated 5 mIU/ml and 2.06 log copies/ml, respectively. Patients negative for anti-HBc or positive for hepatitis C virus antibodies were excluded from this study. g It is estimated that approximately 248 million people worldwide are infected with the hepatitis B virus (HBV), with the highest prevalence occurring in Asia and Africa [1]. The clinical courses of chronic hepatitis B vary. Whereas some patients may progress from chronic hepatitis to cirrhosis and hepatocellular carcinoma (HCC) after HBV infection, others may naturally elimin- ate HBV from the blood and become hepatitis B surface antigen (HBsAg) negative [2, 3]. HBsAg is used as a marker to screen for HBV infection, and the HBsAg titre generally decreases with long-term immune responses in the host. The annual seroclearance rate of HBsAg is 0.7%–1.2% [4–6]. Recently, the goal of chronic hepatitis B therapy has been to achieve seroclearance of HBsAg [7]. However, once HBV infects the host, it integrates into the host genome and establishes a latent infection in the liver for life [8]. Statistical analysis Statistical analyses were performed using SPSS version 24.0 (IBM Corporation, S&I, Tokyo, Japan). Differences between groups were examined by Student’s t-test or analysis of variance. P values < 0.05 were considered sta- tistically significant. Patients and sample collection Nineteen HCC patients who underwent surgical resec- tion at Kobe University Hospital were enrolled in this study. Of the 19 patients, 9 (age 69.7 ± 5.6 years, 8 males and 1 female) were serologically negative for HBsAg, while 10 (age 53.0 ± 11.5 years, 9 males and 1 female) were positive for HBsAg. Laboratory data including platelet counts, transaminase levels, haemoglobin A1c, Hatazawa et al. Infectious Agents and Cancer (2018) 13:7 Page 3 of 8 Page 3 of 8 for more than 1% of the variation comprised 55.1% of the aa in the pre-S/S domain, with no significant difference between the OBI and HBsAg-positive groups (Table 2). The consensus sequence was determined by the NCBI Blast program (available at https://blast.ncbi.nlm.nih.- gov/Blast.cgi) using the sequences obtained in this study. Based on the phylogenetic analysis, all of our strains were classified into genotype C2. The closest reference HBV genotype was C2 (GenBank accession no. AB014394). The viral quasispecies were evaluated in terms of their prevalence in the viral population using the setting “read conflict” in Genomics Workbench. Based on the nucleotide alterations detected, the viruses with aa changes were determined to be variants. The proportion of a variant population was determined as the percentage of aa substitutions per aa coverage depth (entire number of sequence reads at each position) [19]. the confidence that the sequence reads can detect viral variants of low abundance. for more than 1% of the variation comprised 55.1% of the aa in the pre-S/S domain, with no significant difference between the OBI and HBsAg-positive groups (Table 2). This study was approved by the Ethical Review Board of Kobe University. g p g p ( ) The consensus sequence was determined by the NCBI Blast program (available at https://blast.ncbi.nlm.nih.- gov/Blast.cgi) using the sequences obtained in this study. Based on the phylogenetic analysis, all of our strains were classified into genotype C2. The closest reference HBV genotype was C2 (GenBank accession no. AB014394). The viral quasispecies were evaluated in terms of their prevalence in the viral population using the setting “read conflict” in Genomics Workbench. Based on the nucleotide alterations detected, the viruses with aa changes were determined to be variants. The proportion of a variant population was determined as the percentage of aa substitutions per aa coverage depth (entire number of sequence reads at each position) [19]. Clinical characteristics The average age of the OBI-related HCC patients was significantly higher than that of the HBsAg-positive HCC patients (67.7 ± 5.6 vs. 53.6 ± 11.5 years, respect- ively). There was no statistical significance of platelet count and transaminase levels between two groups. Among the OBI-related HCC patients, serum HBV DNA levels were undetectable, but the anti-HBc titre was 10.1 ± 1.2 (range 8.56–12.27) S/CO. Regarding the staging of liver fibrosis, no difference was found in the hyaluronic acid level and the proportion of histologically F3 (pre-cirrhosis) and F4 (cirrhosis) patients at the time of surgery between the groups (OBI 56% vs. HBsAg- positive 60%) (Table 1). The aim of this study was to reflect the quasispecies variants in relation to hepatocarcinogenesis; therefore, we analysed variants detected in > 1% of the total viral population. A similar cut-off was applied in previous studies [20, 21]. In addition, > 20% of the total viral population is possible to detect the conventional sequen- cing method. In this reason, the viral quasispecies were divided and analysed; viral quasispecies detected in > 1% of the total viral population were classified as a major population (20–80% of the total population) and a minor population was defined as that comprising > 1% to < 20% of the total population, respectively. Ultra-deep sequencing analysis The numbers of mapped reads and average coverage of the pre-S/S region were 859,778 ± 682,646 (range 270,715–2,941,784) and 106,038 ± 81,043 (range 30,487– 301,730), respectively. The amino acids (aa) accounting Amino acid variations in the pre-S region Within the 174 aa pre-S region, the number of major variant populations detected were 4.22 ± 3.31 in OBI- Table 1 Clinical and virological characteristics of the patients Table 1 Clinical and virological characteristics of the patients Table 1 Clinical and virological characteristics of the patients HBsAg-positive HCC (n = 10) OBI-related HCC (n = 9) P value Age (years) 53.6 ± 11.5 69.7 ± 5.6 0.002* Sex (male/female) 9/1 8/1 0.737 PLT (× 104/mm3) 17.9 ± 4.9 23.0 ± 8.4 0.131 AST (U/L) 59.1 ± 37.9 38.9 ± 15.0 0.145 ALT (U/L) 33.2 ± 11.0 31.4 ± 15.4 0.782 HbA1c (%) 5.29 ± 0.45 5.46 ± 0.50 0.461 HBsAg (mIU/ml) 3278 ± 3045 0 Anti-HBc antibody (C.O.I.) 12.6 ± 1.8 10.1 ± 1.2 0.002* HBV DNA (log copies/ml) 5.41 ± 1.84 < 2.06 Hyaluronic acid (ng/ml) 62.7 ± 51.0 107.1 ± 123.2 0.338 Fibrosis (F0–2/F3–4) 4/6 4/5 0.605 Total reads (mean ± SD) 4,451,542 ± 2,357,824 3,708,797 ± 2,365,839 0.503 Mapping reads (mean ± SD) 1,075,698 ± 845,602 619,866 ± 347,934 0.144 Coverage (mean ± SD) 131,921 ± 97,014 77,279 ± 49,215 0.139 P < 0.05 Hatazawa et al. Discussion Several epidemiological and molecular studies have re- ported OBI as a risk factor for cirrhosis and HCC [10, 22–25]. The detection of OBI is variable in depend on the HBsAg detection kit. In particular, several older HBsAg detection kits cannot detect escape mutations in the alpha determinant region. More recently, improve- ments in HBsAg detection kits have made it possible to detect HBsAg, including escape mutants. The sensitivity of HBsAg detection is also improving, and most of the available kits can detect very low levels of HBsAg. The Lumipulse HBsAg HQ assay, which was used in this study, can detect extremely low levels of HBsAg, with a cut-off level of > 0.0005 IU/ml [26]. In this study, all serum HBV DNA levels were below the sensitivity threshold but HBV DNA was detected in the liver tissues. OBI was clearly confirmed, even when using a highly sensitive HBsAg kit. According to the various domains of the pre-S region, major HBV populations based on aa mutations in the B- cell epitope within the pre-S1 region were detected only in HBsAg-positive HCC (P = 0.01) (Table 2). On the other hand, the number of minor populations based on mutations in the polymerized human serum albumin (pHSA) domain within the pre-S2 region was signifi- cantly higher in OBI-related HCC compared with HBsAg-positive HCC (2.44 ± 2.30 vs. 0.80 ± 1.48, P = 0.039). The different mutations detected were W4R, S5A/P/T, L30S, G35R, H51P/R, E54K, A60V, W77R/ Stop, I84T, N98 K/T, G102R, Q118R/Stop, N123D and S124F/P. Among them, the W4R, L30S and Q118R/Stop mutations were more prevalent in OBI-related HCC (Table 3). Ultra-deep sequencing analysis Infectious Agents and Cancer (2018) 13:7 Page 4 of 8 Table 2 Amino acid mutations in the pre-S/S region of the major quasispecies populations Domain AA Position (number) HBsAg Positive (%) OBI (%) P PreS1 aa1–119 (119) 2.80 ± 1.69 4.22 ± 3.31 0.12 NTCP aa2–48 (47) 0.60 ± 0.84 0.33 ± 0.50 0.21 HSP70 aa74–118 (45) 0.60 ± 0.70 0.33 ± 0.50 0.18 S promoter aa66–111 (46) 0.70 ± 0.67 0.33 ± 0.50 0.10 NBS aa103–127 (25) 0.20 ± 0.63 0.56 ± 0.88 0.16 T cell epitope aa21–30,52–67 (26) 0.50 ± 0.71 0.67 ± 0.87 0.33 B cell epitope aa12–47,72–78,94–117 (67) 0.70 ± 0.82 0.00 ± 0.00 0.01 S aa1-227 (227) 8.10 ± 8.62 7.33 ± 4.74 0.41 Pre-a determinant aa100–119 (20) 1.20 ± 0.79 1.11 ± 0.33 0.38 a determinant aa 124–148 (25) 0.70 ± 0.82 0.00 ± 0.00 0.01* Post a determinant aa 149–169 (21) 0.30 ± 0.67 0.22 ± 0.67 0.40 HLA class I aa87–98, 186–197, 215–223 (29) 1.80 ± 1.81 1.44 ± 0.73 0.29 HLA class II aa97–106, 171–179, 206–215 (29) 0.50 ± 0.85 0.44 ± 0.73 0.44 *P < 0.05 mino acid mutations in the pre-S/S region of the major quasispecies populations attributed to the L21F/S, L42F/S, W182Stop, and L213F/ I/T mutations. I126S/T, known as an escape mutation in the alpha determinant region, was found only in HBsAg- positive HCC (Table 3). related HCC and 2.80 ± 1.69 in HBsAg-positive HCC, re- spectively. Although no statistical difference in the pro- portion of the major quasispecies populations in the entire pre-S or pre-S1 regions was detected, the propor- tion in the pre-S2 region was significantly higher in OBI-related HCC than in HBsAg-positive HCC (2.89 ± 2.32% vs. 1.30 ± 0.95%, P = 0.041) (Fig. 1). Amino acid variations in the S region All patients who were negative for HBsAg but positive for anti-HBc were treated in this study. This condition is generally considered seropositive OBI, and is treated separately from anti-HBc- and anti-HBs-seronegative OBI [9, 10]. Seropositive OBI is considered to indicate the resolved phase of acute hepatitis or the recovery phase of chronic hepatitis. Chu and Liaw reported that the presence of cirrhosis is an independent predictive factor for HBsAg seroclearance [4]. In this study, all pa- tients showed high anti-HBc antibody titres and were thought to have had overt chronic active hepatitis. In In the 227 aa S domain, major HBV quasispecies popu- lations were detected in 7.33 ± 4.74 of OBI-related HCC and 8.10 ± 8.62 of HBsAg-positive HCC, but there was no significant difference between the groups (Fig. 1). Major populations based on the alpha determinant re- gion (aa 124–148, 25 aa) were detected in HBsAg- positive HCC (P = 0.01) but not in OBI-related HCC (Table 2). Differences in the major populations were at- tributed to the I126S/T, Y200F/S, and Y206C/H/S muta- tions, while those in the minor populations were Hatazawa et al. Infectious Agents and Cancer (2018) 13:7 Page 5 of 8 Fig. 1 Number of aa mutations in the pre-S/S region. Number of aa mutations in the pre-S/S region (a), and mutations were also shown in pre- S1 and pre-S2 region (b). No significant difference in the average number of aa mutations was detected between HBsAg-positive and OBI-related HCC. The proportion of the major variant populations based on the pre-S2 region was significantly higher in OBI-related HCC than in HBsAg-positive HCC Fig. 1 Number of aa mutations in the pre-S/S region. Number of aa mutations in the pre-S/S region (a), and mutations were also shown in pre- S1 and pre-S2 region (b). No significant difference in the average number of aa mutations was detected between HBsAg-positive and OBI-related HCC. The proportion of the major variant populations based on the pre-S2 region was significantly higher in OBI-related HCC than in HBsAg-positive HCC fact, there was no difference in liver fibrosis between the OBI and HBsAg-positive cases, and five of the nine OBI cases had a histologically advanced stage (F3/4). next-generation sequencing, an increased number of re- ports have revealed that minor variants are related to the clinical condition [30, 31]. In this study, several mu- tations in the minor populations were detected in OBI patients. Amino acid variations in the S region Further studies are necessary to clarify the clin- ical implications. HBV generally exists as numerous quasispecies in the host because of the lack of a proofreading function dur- ing the reproduction process. Many studies have re- ported that the small number of viral variants was related to disease progression and the therapeutic re- sponse. It is thought that the small number of viral vari- ants and the extent of variation are partly related to the clinical condition. Several reports revealed that the vari- ation in, or deletion of, the pre-S/S region was related to the development of liver cancer as well as disappearance of the HBsAg in OBI patients. Previously, the M1L, Q2K and S182Stop in the pre-S2 region and T105C in the pre-S/S region were reported as specific mutations in OBI patients [27]. In this study, we could not detect any unique mutations in the OBI patients. So far, direct se- quencing is the most common method used to detect viral mutations. However, variations present in approxi- mately less than 20% of viral quasispecies cannot be de- tected by direct sequencing [28, 29]. The recent introduction of next-generation sequencing enabled ana- lysis of viral sequence variations in more detail. Using The HBsAg is produced from the S region of HBV. The pre-S/S region contains many important domains, including the major hydrophilic region (MHR) related to the host immune response, a specific domain recogniz- ing hepatocytes in the host, and an antigen determinant region (Fig. 2). The HBsAg becomes the target of host immunoreactivity by vaccine-induced and naturally ac- quired antibodies; in particular, the domain of the aa 100–169 is known as MHR, which contains the B-cell epitope and is recognized by HBs antibody. Though the detailed border of the recognition part is unknown, it has been clarified that antigen determining region α (aa124–147) is strongly related to the binding by anti- HBs antibody. In addition, it is known that the muta- tions and the variety in MHR domain are related to the antigenic change of the HBsAg, the evasion of the im- mune response from HBs antibody, the responsiveness from immune induction therapy such as globulin, and a Hatazawa et al. Amino acid variations in the S region In addition, the pre-S2 region contains the T-cell epitope, B-cell epitope, and pHSA-binding domain domains (Fig. 2). pHSA is one of the receptors that binds the HBV Dane particle [37]. The pre-S2 region is associated with HCC. It was reported that deletion of aa 1–6 of the pre- S2 region encoding the B cell epitope is related to HCC Table 3 The frequencies of specific amino acid mutations associated with the major and minor population of HBV quasispecies Region Position HBsAg positive (n = 10) OBI (n = 9) P value Major population S I126S/T 30 0 0.040 Y200F/S 0 33.3 0.025 Y206C/H/S 30 0 0.040 Minor population Pre-S W4R 0 33.3 0.025 S5A/P/T 30 0 0.040 L30S 10 77.8 0.001 G35R 30 0 0.040 H51P/R 30 0 0.040 E54K 50 0 0.006 A60V 30 0 0.040 W77R/Stop 30 0 0.040 I84T 60 11.1 0.014 N98 K/T 30 0 0.040 G102R 30 0 0.040 Q118R/Stop 0 44.4 0.008 N123D 10 44.4 0.049 S124F/P 0 33.3 0.025 S L21F/S 70 100 0.040 L42F/S 0 33.3 0.025 W182Stop 30 0 0.040 L213F/I/T 40 0 0.017 Bold indicated the amino acid position which mutational frequencies in OBI were higher than those in HBsAg positive diagnostic failure by the conventional diagnosis tool of HBsAg [32, 33]. In this study, the prevalence of the major variant population in antigen determining region α was significantly lower among OBI-related HCC than in HBsAg-positive HCC. We suggest that this region would be conserved in OBI patients because the host immune response was smaller than that in HBsAg- positive patients. Several specific mutations, including G119R, C124Y, I126S, Q129R, S136P, C139R, T140I, K141E, D144A, and G145R, have been reported as being related to the decrease of HBsAg secretion [34]. In present study, I126S/T mutants were detected only in HBsAg-positive cases, and it was suggested that the titre of HBsAg of these cases might decrease over a long- term follow-up period. The present study revealed that the prevalence of the major variant populations associated with the B-cell epi- tope, within the pre-S1 region, was significantly higher in HBsAg-positive HCC than in OBI-related HCC. The greater variation in the pre-S/S region, especially the B- cell epitope, in HBsAg-positive HCC compared with OBI-related HCC, is possibly a mechanism to avoid im- mune pressure from the host. Amino acid variations in the S region Infectious Agents and Cancer (2018) 13:7 Page 6 of 8 Page 6 of 8 Table 3 The frequencies of specific amino acid mutations associated with the major and minor population of HBV quasispecies Region Position HBsAg positive (n = 10) OBI (n = 9) P value Major population S I126S/T 30 0 0.040 Y200F/S 0 33.3 0.025 Y206C/H/S 30 0 0.040 Minor population Pre-S W4R 0 33.3 0.025 S5A/P/T 30 0 0.040 L30S 10 77.8 0.001 G35R 30 0 0.040 H51P/R 30 0 0.040 E54K 50 0 0.006 A60V 30 0 0.040 W77R/Stop 30 0 0.040 I84T 60 11.1 0.014 N98 K/T 30 0 0.040 G102R 30 0 0.040 Q118R/Stop 0 44.4 0.008 N123D 10 44.4 0.049 S124F/P 0 33.3 0.025 S L21F/S 70 100 0.040 L42F/S 0 33.3 0.025 W182Stop 30 0 0.040 L213F/I/T 40 0 0.017 Bold indicated the amino acid position which mutational frequencies in OBI were higher than those in HBsAg positive diagnostic failure by the conventional diagnosis tool of HBsAg [32, 33]. In this study, the prevalence of the major variant population in antigen determining region α was significantly lower among OBI-related HCC than in HBsAg-positive HCC. We suggest that this region would be conserved in OBI patients because the host immune response was smaller than that in HBsAg- positive patients. Several specific mutations, including G119R, C124Y, I126S, Q129R, S136P, C139R, T140I, K141E, D144A, and G145R, have been reported as being related to the decrease of HBsAg secretion [34]. In present study, I126S/T mutants were detected only in HBsAg-positive cases, and it was suggested that the titre of HBsAg of these cases might decrease over a long- term follow-up period. The present study revealed that the prevalence of the major variant populations associated with the B-cell epi- tope, within the pre-S1 region, was significantly higher in HBsAg-positive HCC than in OBI-related HCC. The greater variation in the pre-S/S region, especially the B- cell epitope, in HBsAg-positive HCC compared with OBI-related HCC, is possibly a mechanism to avoid im- mune pressure from the host. In contrast, the prevalence of major populations in the pre-S2 region, especially the pHSA domain, was significantly higher in OBI-related HCC than in HBsAg-positive HCC. The pre-S2 region comprises 55 aa and, together with the pre-S1 and S re- gions, encodes the surface proteins of HBV [35, 36]. Competing interests Competing interests Competing interests The authors declare that they have no competing interests. Funding This study was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Grant number: 16H05826. 10. Raimondo G, Caccamo G, Filomia R, Pollicino T. Occult HBV infection. Semin Immunopathol. 2013;35:39–52. Received: 27 November 2017 Accepted: 26 January 2018 Received: 27 November 2017 Accepted: 26 January 2018 Abbreviations HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; OBI: Occult HBV infection 7. European Association for the Study of the Liver: European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol. 2017; 67: 370–98. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Availability of data and materials 11. Huang X, Qin Y, Li W, Shi Q, Xue Y, Li J, et al. Molecular analysis of the hepatitis B virus presurface and surface gene in patients from eastern China with occult hepatitis B. J Med Virol. 2013;85:979–86. The data sets supporting the conclusions of this article are included within the article. 12. Zhang Q, Cao G. Genotypes, Mutations, and viral load of hepatitis B virus and the risk of hepatocellular carcinoma: HBV properties and hepatocarcinogenesis. Hepat Mon. 2011;11:86–91. Conclusion 1. Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet. 2015;386:1546– 55. 1. Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet. 2015;386:1546– 55. Viral quasispecies based on the pre-S/S region were less diverse in OBI-related HCC than in HBsAg-positive HCC. The most significant variation was in the B cell epitope within the pre-S1 region and the MHR domain within the S region. It is suggested that the evasion mechanism from the host immune response is weaker in OBI-related HCC. Furthermore, because mutations in the pre-S2 region were significantly prevalent in OBI- related HCC, variation in this region may be related to hepatocarcinogenesis in OBI patients. 2. Lee WM. Hepatitis B virus infection. N Engl J Med 1997; 337: 1733–1745. 3 H f l JH D E Li TJ Fl i h R d L k AS M f 2. Lee WM. Hepatitis B virus infection. N Engl J Med 1997; 337: 1733–1745. 3. Hoofnagle JH, Doo E, Liang TJ, Fleischer Rand Lok AS. Management of hepatitis B: summary of a clinical research workshop. Hepatology. 2007;45: 1056–75. 4. Chu CM, Liaw YF. HBsAg seroclearance in asymptomatic carriers of high endemic areas: appreciably high rates during a long-term follow-up. Hepatology. 2007;45:1187–92. 5. Wong RJ, Nguyen MT, Trinh HN, Chan C, Huynh A, Ly MT. et al, Hepatitis B Surface Antigen Loss and Sustained Viral Suppression in Asian Chronic Hepatitis B Patients: A Community-Based Real World Study. J Viral Hepat. 2017 Jun 5; doi: 10.1111/jvh.12736. 6. Liu J, Liu J, Yang HI, Lee MH, Lu SN, Jen CL, et al. REVEAL-HBV Study Group . Incidence and determinants of spontaneous hepatitis B surface antigen seroclearance: a community-based follow-up study. Gastroenterology. 2010; 139:474–82. Acknowledgements This study was funded by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (16H05826). 8. Tuttleman JS, Pourcel C, Summers J. Formation of the pool of colalently closed circular viral DNA in hepadnavirus-infected cells. Cell. 1986;47:451–60. p 9. Raimondo G, Allain JP, Brunetto MR, Buendia MA, Chen DS, Colombo M, et al. Statements from the Taormina expert meeting on occult epatiti B virus infection. J Hepatol. 2008;49:652–7. 9. Raimondo G, Allain JP, Brunetto MR, Buendia MA, Chen DS, Colombo M, et al. Statements from the Taormina expert meeting on occult epatiti B virus infection. J Hepatol. 2008;49:652–7. Amino acid variations in the S region In contrast, the prevalence of major populations in the pre-S2 region, especially the pHSA domain, was significantly higher in OBI-related HCC than in HBsAg-positive HCC. The pre-S2 region comprises 55 aa and, together with the pre-S1 and S re- gions, encodes the surface proteins of HBV [35, 36]. In addition, the pre-S2 region contains the T-cell epitope, B-cell epitope, and pHSA-binding domain domains (Fig. 2). pHSA is one of the receptors that binds the HBV Dane particle [37]. The pre-S2 region is associated with HCC. It was reported that deletion of aa 1–6 of the pre- S2 region, encoding the B cell epitope, is related to HCC development [38]. In addition, a previous study reported Bold indicated the amino acid position which mutational frequencies in OBI were higher than those in HBsAg positive Fig. 2 Scheme of the HBV pre-S/S region including the functional domains. NCTP-binding site in the pre-S1 region plays an important role in viral recognition and entry into hepatocytes. The alpha determinant epitope domain in the pre-S2 is involved in HBsAg production Fig. 2 Scheme of the HBV pre-S/S region including the functional domains. NCTP-binding site in the pre-S1 region plays an important role in viral recognition and entry into hepatocytes. The alpha determinant epitope domain in the pre-S2 is involved in HBsAg production Hatazawa et al. Infectious Agents and Cancer (2018) 13:7 Page 7 of 8 Page 7 of 8 Page 7 of 8 that aa mutations in the pre-S2 region, especially the pHSA domain, are prevalent in HBV-related HCC [36], and HBV-infected patients with mutations in the pHSA region frequently progress to cirrhosis and HCC [39]. These previous studies suggested that the pre-S2 region does not play an important role in the synthesis or se- cretion of viral particles or in the infectiousness of the virus, but the accumulation of viral particles in hepato- cytes with pHSA mutations is related to cytotoxic accu- mulation and progression to cirrhosis and HCC. The results of the present study suggest that a high level of variation in the pre-S2 region, particularly the pHSA domain, is related to hepatocarcinogenesis, even in OBI patients with low viral tites. Author details 1 The limitations of the present study include the small number of Japanese cases evaluated. In addition, the average age of present study is different in two groups. It is usual that HBsAg positive HCC patients are younger than other cause of HCC patients. Although HBV is nat- urally mutated over time, it was none the less important that variation in the pre-S2 was detected in OBI patients. Future studies with larger cohorts including several viral genotypes and various host genetic factors are required to overcome these limitations. 1Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan. 2Division of Molecular Medicine & Medical Genetics, Department of Pathology, Kobe University Graduate School of Medicine, Kobe, Japan. 3Department of Internal Medicine, Tokushima Prefectural Naruto Hospital, Tokushima, Japan. 4Division of Hepato-Biliary-Pancreatic Surgery, Department of Surgery, Kobe University Graduate School of Medicine, Kobe, Japan. 5Department of Hepatology Osaka City University Graduate School of 5Department of Hepatology, Osaka City University Graduate School of Medicine, Osaka, Japan. Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. Amino acid variations in the S region Ethics approval and consent to participate pp p p The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Medical and Health Research Ethics Committee (MHREC), Kobe University Graduate School of Medicine (Trial registration: Ref: 1856 [Approved 22 March 2016]). All subjects gave their written informed consent before enrolment. Authors’ contributions YH, YY, and YH designed the study; MT, and TF were responsible for the recruitment of subjects; RO, TT, HH, HH, AM, YK, YM, and MY performed experiments and conducted data management; YH and YY wrote the manuscript. All authors read and approved the final manuscript. 13. Sinn DH, Choi MS, Gwak GY, Paik YH, Lee JH, Koh KC, et al. Pre-S mutation is a significant risk factor for hepatocellular carcinoma development: a long- term retrospective cohort study. Dig Dis Sci. 2013;58:751–8. Page 8 of 8 Page 8 of 8 Hatazawa et al. 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https://openalex.org/W2734495729	https://europepmc.org/articles/pmc5507395?pdf=render	English	null	Time for trauma immunology	PLoS medicine	2,017	cc-by	1,610	PERSPECTIVE Time for trauma immunology Timothy R. Billiar, Yoram Vodovotz Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America billiartr@upmc.edu While it is intuitive that the damage to bones and soft tissues causes both short- and long-term “organic” dysfunction, the “illness” caused by injury is less appreciated. This results from the systemic manifestations that follow the physiologic, immunologic, and metabolic changes induced by shock from blood loss and direct tissue destruction. The immunologic changes following injury are profound, and have been measured in the circulating leukocytes of injured humans as a massive activation of the innate immune system and a near-simultaneous im- pairment in adaptive immune responses [1]. This “immune dysfunction,” seen after even mod- erate injury, can be appreciated clinically as inflammation-associated organ dysfunction (typically peaking on day 2 to day 3 postinjury) and a sustained increase in the susceptibility to secondary infections, especially pneumonia [1,2]. The 2 processes are thought to be linked by the proximal events that activate the immune system immediately following a traumatic event. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 PAMP/DAMP paradigm The quest to understand how the immune system becomes activated by trauma lags consider- ably behind, and is ultimately informed by, discoveries regarding mechanisms of microbial recognition by immune cells. That immune recognition systems are gene-encoded in mam- mals was predicted by studies in the 1960s, showing that certain strains of mice are resistant to bacterial endotoxin [3]. By 1993, the parallels between microbial immune recognition and injury recognition were drawn closer when DeMaria and colleagues showed that the endo- toxin-resistant C3H/HeJ mouse strain was also resistant to hemorrhagic shock-induced death [4]. The discovery that the endotoxin-resistant mouse strain possessed a mutation in the Toll- like receptor (TLR)4 gene, the mammalian homologue to the pattern-recognition receptor first described in drosophila [5], subsequently confirmed the prediction that the innate recog- nition of microbial (nonself) antigens by the host would involve gene-encoded receptors [6]. Of course, we now know that several families of pattern-recognition receptors, including TLRs, Nod-like receptors (NLRs), C-type lectin receptors (CLRs), and retinoic-acid–inducible protein 1 (RIG-I)-like receptors, continuously survey the extra- and intracellular compart- ments for molecules expressed by foreign invaders. These molecules are referred to as patho- gen-associated molecular patterns (PAMP). OPEN ACCESS Citation: Billiar TR, Vodovotz Y (2017) Time for trauma immunology. PLoS Med 14(7): e1002342. https://doi.org/10.1371/journal.pmed.1002342 Published: July 11, 2017 Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Published: July 11, 2017 Copyright: © 2017 Billiar, Vodovotz. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors received no specific funding for this work. 1. Xiao W, Mindrinos MN, Seok J, Cuschieri J, Cuenca AG, Gao H, et al. A genomic storm in critically injured humans. J Exp Med. 2011 Dec 19; 208(13): 2581–2590. doi: 10.1084/jem.20111354. PMID: 22110166 2. Namas RA, Vodovotz Y, Almahmoud K, Abdul-Malak O, Zaaqoq A, Namas R, et al. Temporal patterns of circulating inflammation biomarker networks differentiate susceptibility to nosocomial infection Trauma immunology The immune response associated with trauma-induced “critical illness” is characterized by both its magnitude and unique inflammatory mediator patterns [1,2]. Other major health con- ditions associated with aberrant immune regulation have led to highly specialized areas within immunology, for example, cancer (tumor immunology), cell and organ transplantation (trans- plant immunology), and the broad number of diseases resulting from abnormal recognition of self by the immune system (autoimmunity). The creation of clearly defined fields of study within immunology has contributed in the translation of basic discoveries into new therapies. Based on the unique dynamics of the immune response to trauma, the scope of the clinical problem (third overall cause of death), and the tremendous opportunity for targeting the immune system in appropriate patients, the rationale appears to exist to define a new field within immunology: “trauma immunology.” The goal would be to bring trauma under the umbrella of mainstream immunology and build upon the recent advances that have begun to link early postinjury events to immune pathways previously thought to be activated only in chronic, autoimmune, or inflammatory processes [11]. Given the likely need to integrate pre- existing host factors such as genetic variability and microbiome composition with DAMP and PAMP signaling, this nascent field is poised to benefit from major strides in systems and computational immunology [12]. Likewise, insights derived from the study of the immunology of traumatic events (whose origin in time can be mapped precisely to the moment of injury) will aid in understanding chronic inflammation and immune disorders in the larger context. (e.g., hyaluronan). Evidence continues to accumulate to support a DAMP “surge” postinjury as the seminal event in trauma-induced immune activation [2,10]. This release may be an active process induced by tissue hypoxia resulting from hypo-perfusion, or passive as the result of cell lysis. Persistent or repeated cycles of DAMP release may heighten or maintain the overactiva- tion of the immune system during the escalation phase that characterizes the path to immune dysfunction, essentially forming a forward feedback loop of tissue damage leading to inflamma- tion that in turn leads to further tissue damage and cellular dysfunction. The immune dysfunction process following trauma is dominated by the pattern-recogni- tion receptors TLR2, TLR4, and TLR9, although roles for other pattern-recognition receptors are likely. To date, TLR4 appears to be the most promiscuous DAMP-responsive receptor, rec- ognizing a wide array of endogenous molecules independent of gram-negative bacterial endo- toxin [7]. TLR9 is also particularly interesting as a DNA receptor, which may be linked to the immune activation resulting from the release of mitochondrial DNA [8,10]. Mitochondria may be an especially important source of DAMP molecules, based not only on their proposed bacterial origins but also because mitochondrial DNA and other mitochondrial molecules are elevated in trauma patients that progress to critical illness [10]. It is tempting to refer to trauma-induced immune activation as “sterile inflammation”; how- ever, it is just as likely that the propagation of the immune response involves PAMP molecules derived from the host’s own microbiome or microbes invading traumatized tissues. This inter- play between DAMP and PAMP signaling remains a fruitful area for additional investigation. Competing interests: The authors have declared that no competing interests exist. It was soon realized that a subset of these receptors could also recognize host-derived mole- cules and that this recognition could lead to robust immune-cell activation and production of inflammatory mediators, following insults common to severely injured humans—hemor- rhagic shock and tissue damage [7,8]. This realization provided support for the concept that the immune system recognizes not just nonself but any threat to host homeostasis—the so-called “danger theory” [9]. Self-derived molecules that trigger pattern-recognition receptor signaling are referred to as damage-associated molecular pattern (DAMP) molecules and encompass both cellular (e.g., high-mobility group box 1, mitochondrial DNA) and matrix components Abbreviations: CLR, C-type lectin receptor; DAMP, damage-associated molecular pattern; NLR, Nod- like receptor; PAMP, pathogen-associated molecular pathways; RIG-I, retinoic-acid–inducible protein I; TLR, Toll-like receptor. Provenance: Commissioned; not externally peer reviewed Provenance: Commissioned; not externally peer reviewed PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1002342 July 11, 2017 1 / 3 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1002342 July 11, 2017 References 2 / 3 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1002342 July 11, 2017 following blunt trauma in humans. Ann Surg. 2016 Jan; 263(1): 191–198. doi: 10.1097/SLA. 0000000000001001. PMID: 25371118 following blunt trauma in humans. Ann Surg. 2016 Jan; 263(1): 191–198. doi: 10.1097/SLA. 0000000000001001. PMID: 25371118 3. Sultzer BM. Genetic control of leucocyte responses to endotoxin. Nature. 1968 Sep 21; 219 (5160):1253–4. PMID: 4877918 4. DeMaria EJ, Pellicane JV, Lee RB. Hemorrhagic shock in endotoxin-resistant mice: improved survival unrealted to deficient production of tumor necrosis factor. J Trauma. 1993 Nov; 35(5):720–4; discus- sion 724–5. PMID: 8230336 5. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. The dorsoventral regulatory gene cas- sette spaetzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 1996 Sep 20; 86(6):973–983. PMID: 8808632 6. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science. 1998 Dec 11; 282(5396):2085–8. PMID: 9851930 7. Mollen KP, Anand RJ, Tsung A, Prince JM, Levy RM, Billiar TR. Emerging paradigm: Toll-like receptor 4-sentinel for the detection of tissue damage. Shock. 2006 Nov; 26(5):430–437. doi: 10.1097/01.shk. 0000228797.41044.08. PMID: 17047512 8. Gill R, Ruan X, Menzel CJ, Namkoong S, Loughran PA, Hackam DJ, et al. Systemic Inflammation and Liver Injury following Hemorrhagic Shock And Peripheral Tissue Trauma Involve Functional TLR9 Sig- naling on Bone Marrow-Derived Cells and Parenchymal Cells. Shock. 2011 Feb; 35(2):164–70. doi: 10. 1097/SHK.0b013e3181eddcab. PMID: 20577143. 9. Matzinger P. The danger model: a renewed sense of self. Science. 2002 Apr 12; 296(5566): 301–305. doi: 10.1126/science.1071059. PMID: 11951032 10. Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, et al. (2010) Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature. 2010 Mar 4; 464: 104–107. doi: 10.1038/nature08780. PMID: 20203610 11. Abboud AN, Namas RA, Ramadan M, Mi Q, Almahmoud K, Abdul-Malak O, et al. Computational analy- sis supports an early, type 17 cell-associated divergence of blunt trauma survival and mortality. Crit Care Med. 2016 Nov; 44(11): e1074–e1081. doi: 10.1097/CCM.0000000000001951. PMID: 27513538 12. Brown D, Namas RA, Almahmoud K, Zaaqoq A, Sarkar J, Barclay DA, et al. Trauma in silico: individual- specific mathematical models and virtual clinical populations. Sci Transl Med. 2015 Apr 29; 7: (285):285ra261. doi: 10.1126/scitranslmed.aaa3636. PMID: 25925680 3 / 3
https://openalex.org/W3161592726	https://hal.archives-ouvertes.fr/hal-03173184/file/Vicdan%2C%20Perezts%20%26%20Firat%20%28forthcoming%202022%29%20License%20to%20heal.pdf	English	null	License to Heal: Understanding a Healthcare Platform Organization as a Multi-Level Surveillant Assemblage	M@n@gement	2,021	public-domain	13,370	To cite this version: Handan Vicdan, Mar Perezts„ A. Fuat Firat. License to heal: Understanding a healthcare Platform Organization as a Multi-level Surveillant Assemblage. M@n@gement, In press. hal-03173184 HAL Id: hal-03173184 https://hal.science/hal-03173184v1 Submitted on 18 Mar 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. Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 Abstract Platform organizations bring renewed attention to power disparities and risks in the rise of surveillance capitalism. However, such critical accounts provide a partial understanding of the complexity of surveillance phenomena in such shifting socio-technical and digital environments. Findings from a netnographic investigation of a healthcare platform organization, PatientsLikeMe, unravel how platforms become the locus where multi-level flows of surveillance converge, thereby constituting what we identify as a surveillant assemblage. We develop a comprehensive approach for understanding how platforms constitute a dynamic crossroads of micro, meso and macro surveillance phenomena within and beyond the online communities they create. Our study highlights this surveillant assemblage’s emerging practices and potentially empowering outcomes that enable multi-stakeholder involvement in big data and knowledge generation in healthcare. Broader implications of multi-level surveillance in and through platforms are discussed. License to heal: Understanding a healthcare Platform Organization as a Multi-level Surveillant Assemblage Handan VICDAN Associate Professor of Marketing Emlyon Business School & Lifestyle Research Centre 23 av. Guy de Collongue 69134 Ecully, France Mar PEREZTS (perezts@em-lyon.com) Associate Professor of Philosophy and Organization Emlyon Business School & OCE Research Centre 23 av. Guy de Collongue 69134 Ecully, France A. Fuat FIRAT Professor of Marketing, Department of Marketing Vackar College of Business & Entrepreneurship The University of Texas – Rio Grande Valley 1201 W. University Dr. 78541 Edinburg, Texas A. Fuat FIRAT Professor of Marketing, Department of Marketing Vackar College of Business & Entrepreneurship The University of Texas – Rio Grande Valley 1201 W. University Dr. 78541 Edinburg, Texas Introduction Mass datafication in the rise of platform organizations has renewed legitimate concerns regarding surveillance phenomena owing to platform’s overwhelming algorithmic data-extraction power. Beyond anything in Orwell’s (1949) dystopian imagination or Foucault’s (1979) descriptions of disciplinary institutions, surveillance in platforms seems ubiquitous (Andrejevic, 2012) and ever more complex today (Farinosi, 2011). As spatially delimited digital infrastructures bringing together a network of stakeholders, platforms are reconfiguring socio-economic ecosystems (Orlikowski & Scott, 2014), combining self-quantification with community dynamics and market relations (Chanal & Caron-Fasan, 2010) to produce new or challenge existing forms of organization, provision of services and wealth creation (Srnicek, 2016). Through their “data mining capabilities of discovering meaningful patterns and distilling them into predictive analytics” (Cohen, 2013, p. 1920), they are ‘shaping and computing the everyday’ (Alaimo & Kallinikos, 2017). Among other things, on platforms, people are massively, continuously, knowingly (or not) and willingly (or reluctantly) being surveilled for a profit (Fuchs, 2011; Zuboff, 2019). This brings renewed questions concerning how surveillance functions and what outcomes it yields in and through platforms where diverse human/nonhuman stakeholders participate in the production, distribution, monitoring and usage of information at an unprecedented scale. While scholarly attention has overly focused on critical accounts, the way surveillance actually functions in such shifting socio-technical environments as platforms remains an understudied and complex issue. One such platform captured our attention: PatientsLikeMe, a for- profit ad-free healthcare platform harboring several disease-specific online communities. Our extensive netnographic study of PatientsLikeMe suggests that it is more than extracting and selling health data from surveilling its online community. Both in and through the platform, various levels and surveillance mechanisms were at play, yielding outcomes other than coercive control and exploitation. These included a series of relational and learning mechanisms for patients to care of each other while increasing their own disease literacy and empowerment (micro); changing patient-healthcare provider relationships via personalized medicine and normalizing data sharing in and beyond the platform (meso); and shifting power hierarchies in healthcare and medical knowledge generation through connecting multiple stakeholders, tools and practices (macro). Such findings problematize the theoretical and ideological assumptions (Alvesson & Sandberg, 2011) that dominate surveillance regarding platforms, seen predominantly as a macro- level phenomenon aimed at control and profit. Keywords l surveillance, surveillant assemblage, platform organization, netnography, healthcare Multi-level surveillance, surveillant assemblage, platform organization, netnography, healthcare 1 1 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 Introduction Indeed, some scholars have noted how surveillance can be as much about controlling as about caring (e.g; Lyon, 2006; Sewell & Barker, 2006; Iedema & Rhodes, 2010) highlighting a tension at the crossroads of multiple levels and outcomes of surveillance that warrant further attention. This leads us to argue that in overly focusing on the – important, yet not sole – issue of power disparities, control and profit-making, other dynamics and outcomes of surveillance in platforms have been both neglected and easily dismissed as either naïve or as the product of the ideological apparatus sustained by platform giants. What has been particularly overlooked is to address in greater depth the multi-level complexity of surveillance 2 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 practices in and through platforms and their outcomes, which is the objective of our research. Our findings unpack the dynamics of surveillance flows constituting multi-level sociomaterial interactions involving and affecting diverse human/nonhuman stakeholders, including PatientsLikeMe community, healthcare providers, pharmaceuticals, governmental bodies, research institutions, tracking tools and algorithms, and data-tracking and aggregation processes. In studying how such multi-level surveillance flows were intricately woven together on PatientsLikeMe, we bring forward two main contributions. First, we draw attention to how surveillance functions in and through platforms, which constitute a dynamic crossroads of micro, meso and macro surveillance phenomena both within their online communities and beyond. This led us to understand platforms as digitally-mediated spaces for surveillance, i.e. as loci where multi-level surveillance phenomena converge, thereby constituting a surveillant assemblage, a term coined by Haggerty and Ericson (2000) that has revitalized studies of contemporary surveillance (Galič et al., 2017). Drawing selectively on Deleuzian (Deleuze & Guattari, 1980; Deleuze, 1990) analysis of surveillance transformations in the digital age, they apply the notion of assemblages to surveillance studies to theorize the multiple objects in flow (information, bodies, desires, institutions and processes) that make several systems of surveillance converge into a functional entity that transforms the purposes and hierarchies of surveillance. Second, we answer calls to explore surveillance’s outcomes other than discipline, control and capitalistic exploitation, seeking to explore its potentially productive (Lyon, 2007), participatory (Albrechtslund, 2008), collaborative (Pridmore, 2013) or even existential (Hafermalz, 2020) outcomes. In our case, in becoming a new pole where flows of information, actors and surveillance practices converge, i.e. Introduction in constituting a surveillant assemblage, PatientsLikeMe fosters an empowering potential of surveillance in healthcare, drawing attention to enabling and constraining power dynamics on healthcare platforms. We begin by reviewing the literature on platforms and surveillance in order to point out some limitations and neglected aspects. We then present our case and netnographic method, followed by our findings that allow us understand surveillance in platforms as a multi-level surveillant assemblage. Finally, we discuss implications and contributions, and outline some potentials and pitfalls for future research as concluding remarks. Surveillance capitalism and its risks Surveillance capitalism and its risks Platforms, as dominant organizational forms today are “data-based organizations that extract value and make profit from the social everyday they themselves engineer” (Alaimo & Kallinikos, 2017, p. 176). As put by Fuchs (2011, p. 289): “The discussion of surveillance in web 3 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 2.0 is important because such platforms collect huge amounts of personal data in order to work”, i.e. by commodifying every digital trace of those surveilled (Lyon, 2001, 2002; West, 2019). Since surveillance is “a technology-dependent concept” (Galič et al., 2017: 26), how surveillance unfolds in such specific socio-technical organizational environments is inescapably linked to the fact that they have become mastodons of wealth creation (Srnicek, 2016). Although links between surveillance and capitalism go much further back than today’s Internet giants such as Google and Facebook (Fuchs, 2011, Foster & McChesney, 2014), the commodification capacities of platforms’ infrastructure for extracting data asymmetrically, without dialogue or consent (Galič et al., 2017) have brought renewed concerns for ‘surveillance capitalism’ that relies on surveillance as its key modus operandi, which “tunes” and “herds” (Zuboff, 2019, p. 294-295) our online behaviors into commoditized assets. Such production of information becomes the source of data-driven and algorithmically commercialized instrumentarian power that Zuboff christens “‘Big Other’ (…) that effectively exile(s) persons from their own behavior while producing new markets of behavioral prediction and modification”. (2015, p. 75). Furthermore, such transformations in information accumulation rely on compatible legal structures that paradoxically enable it by considering the data extracted to be ‘raw’ data, i.e. being in the ‘public domain’, available for the taking (Cohen, 2018). This has fostered numerous preoccupations about how it secures government collusion and about the strength of its ideological and narrative justifications, including: the free and open network, instant and customized personalization, and new forms of consumer power. However, the ‘sharing’ economy has been shown to not always deliver on the promise of social connection, and instead harbors labor exploitation, discrimination and inequality as well power asymmetries, algorithmic biases and discriminations, tailored advertising and invasion of privacy (e.g. Gillespie, 2017; Schor & Attwood-Charles, 2017; Visser et al., 2019; West, 2019). Surveillance capitalism and its risks Reversely, the concept of privacy – which is defined as freedom from and resistance to surveillance (Lyon, 2001; Solove, 2002; Farinosi, 2011) – is also sustained by strong and equally contestable ideologies and often obscure power dynamics that play out in eminently complex ways in platforms. The complexity and ambivalence of surveillance Economic dominance of platforms today makes considerations of surveillance in this context to almost exclusively “emphasize how they cumulatively pose a threat to civil liberties” (Haggerty & Ericson, 2000, p. 610), leading numerous relevant issues on surveillance in platforms to be neglected, easily dismissed, or deprioritized. These concerns nevertheless reflect only a partial picture, given the complexity of how surveillance works in and through platforms. Since big data is both “a powerful tool to address various societal ills, offering the potential of new insights into areas as diverse as cancer research, terrorism, and climate change (… and) a troubling manifestation of Big Brother, enabling invasions of privacy, decreased civil freedoms, and increased state and corporate control” (boyd & Crawford, 2012, p. 664), some studies encourage us to not completely downplay the productive potentialities of surveillance in favor of its risks. 4 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 Owing to this inherent ambivalence, surveillance studies tend to neglect potential positive developments of surveillance practices (e.g. in science and medicine, relevant to our empirical case) (Galič et al., 2017). In unpacking this ambivalence, we remark that most of the dark claims operate at a macro-economic level, and that more in-depth studies of how specific organizational forms such as platforms, participate in surveillance (whether capitalistic or not) are insufficiently developed. This is surprising, particularly considering that surveillance studies have always been sensitive to the organizational, material and technological dimensions that make surveillance possible, at least since Bentham’s seminal work on the Panopticon and architecture-based conceptions of surveillance (Galič et al., 2017) which have over-stretched this metaphor (Haggerty, 2006; Brivot & Gendron, 2011; De la Roberterie & Lebrument, 2019; Hafermalz, 2020). But in ‘going beyond’ the Panopticon, paying attention to the organizational architecture, the infrastructure and the technological materiality of surveillance need not be forgotten. Without denying the risks, other outcomes besides control and exploitation are worth noticing, with leading scholars in surveillance studies continuously stressing the ambivalence of surveillance as being simultaneously about controlling and caring (Haggerty & Ericson, 2000; Lyon 2001, 2006, 2014). This yields a bilateral tension between surveillance’s coercive (supervision and subordination) and caring (protection from deviance) roles (Sewell & Barker, 2006). For instance, studies on self-surveillance and organizational surveillance (Vaz & Bruno, 2003; Iedema & Rhodes, 2010) have shown how power and care are actually very difficult to separate. The complexity and ambivalence of surveillance This might explain why researchers have overlooked caring surveillance in favor of more obvious issues of domination and often disguised control (Weiss, 2005). Binary conceptualizations of surveillance – e.g., coercive-caring, passive-resistant (Iedema & Rhodes, 2010; Sewell & Barker, 2006) – tend to provide a polarized and therefore limited picture, leaving much unchartered territory in emerging surveillance phenomena (Albrechtslund, 2008; Pridmore, 2013), particularly in digital environments. Scholars developed the concepts of reverse surveillance (Brin, 1998) and sousveillance (Mann et al., 2003) to draw attention to the disciplinary effect exerted by the bottom-up movement to counter the top-down scrutiny by the corporate elite. Being watched (e.g. followed on social media) is also an asset (Lyon, 2007) fulfilling exhibitionist and narcissistic desires (Koskela, 2004) while making surveillance playful and entertaining (Albrechtslund & Dubbeld, 2005). Voluntary visibilization practices through surveillance technologies can even be a matter of ‘existential recognition’ of belonging (Hafermalz, 2020). Consequently, considering surveillance as a potentially empowering (Haggerty & Ericson, 2000, Albrechtslund, 2008) and a productive practice (Lyon, 2007) warrants further attention, as it is a controversial, counter-intuitive issue. Consequently, we aim to uncover how surveillance plays out on the various levels that platforms operate within, shifting relations and directions of visibility mediated by various technologies. Platforms become a privileged site to study the transformations of the surveillant gaze since they increasingly blur the differences between agents and targets of surveillance and mitigate power relations (Galič et al., 2017). In platforms, surveillance technologies are more than an ‘electronic panopticon’ (Lyon, 1994) that imposes a centralized gaze (usually by the State or 5 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 the corporation) on passive others (citizens, employees or consumers). They simultaneously foster lateral surveillance (Brivot & Gendron, 2011), self-surveillance (Albrechtslund, 2008; Vaz & Bruno, 2003), among other variants where the increased visibility between surveyor and surveilled make them more active in this process (Farinosi, 2011). Following Hafermalz (2020, p. 7, quoting Han 2015) it is worth asking what happens when “the inhabitants of the digital panopticon (…) are engaged in ‘lively communication’ and actively produce and share personal information and ‘bare’ themselves to one another ‘of their own free will’?” Reviewing the literature on the notion of surveillant assemblages, the next section adds the theoretical missing piece for uncovering this complex and dynamic context, inclusive of some of the neglected aspects and ambivalent tensions detailed above. Surveillant assemblages: studying surveillance in the digital context In a recent review, Galič and colleagues (2017) provide a tripartite chronological-thematic overview of surveillance studies and locate Zuboff’s development of surveillance capitalism within the second phase of post-panoptic or “infrastructural theories”. However, it is the other two approaches included in this phase that interest us here: Deleuze’s (1990) analysis of control societies (Deleuze & Guattari, 1980) and its reinterpretation by Haggerty and Ericson (2000) to conceptualize the notion of surveillant assemblage. What makes this phase particularly interesting is that it signals the move from physical materiality (e.g. the prison with its central tower) to digital materiality (e.g. algorithms, connected devices) on the one side, and from a (relatively) single direction of explicit gazing to a networked, decentralized, often invisible gazing on the other. Furthermore, the goal of surveillance is no longer discipline, but control, since it is no longer institutions but corporations that orchestrate surveillance. This shifts the temporality of surveillance from a potentially omnipresent gaze on individuals to an effective omnipresent recording of what Deleuze calls dividuals, i.e. the fragmented divided individual, whose variety can be recorded and stored in many different data banks in the rise of digital technologies for consumerist purposes. Building selectively on this Deleuzian heritage that we have very succinctly summarized, Haggerty and Ericson (2000, p. 606) note that we are “witnessing a convergence of what were once discrete surveillance systems to the point that we can now speak of an emerging ‘surveillant assemblage’”. They argue that this can better account for technological transformations, the move from a human gaze to machine-based tracking in a rhizomatic and networked expansion, and a certain leveling of hierarchies (later developed and nuanced by Haggerty, 2006). An assemblage is a ‘functional entity’ grouping heterogeneous objects in flow (including people, institutions, processes, algorithms, information…) that ‘work’ together, serving a surveillance purpose of generating data and knowledge through a process of monitoring ongoing interaction between these objects (Delanda, 2016). Assemblages are a ‘potentiality’: their apparent stability conceals in fact a multiplicity of flows in temporarily more or less fixed arrangements, each in turn composed of multiple discrete assemblages. A range of desires – understood as immanent, active and positive forces, including desires for control, governance, security, profit and entertainment – hold the 6 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 assemblage together (Haggerty & Ericson, 2000, p. 609). Surveillant assemblages: studying surveillance in the digital context Surveillant assemblages “can hence be seen as ‘recording machines’, since their task is to capture flows and convert them into reproducible events” (Galič et al., 2017, p. 21), particularly now with computerized datafication, as “centres of appropriation where these flows can be captured.” (Haggerty & Ericson, 2000, p. 608). Here, humans work alongside technology in a ‘multi-agency’ constitution of assemblages, and human bodies are understood as hybrid compositions, flesh-technology-information amalgams, broken down into dividuals or ‘data doubles’ (e.g. consumer profiles), which are then re-assembled (e.g. consumption patterns). Before they can be controlled, molded or punished, bodies must be known, by ‘centres of calculation’ such as statistical institutions, the police or laboratories that open or close access to information, for the people behind such ‘data doubles’. At the interface of de-corporealized measurable bodies and technologies, surveillance reduces flesh (but also non-human bodies) to information, turning them into a renewed form of ‘surplus value’. Previously scattered records are digitally combined to serve new purposes, such as marketing, policing, and entertainment (Haggerty, 2006). Combinations are triggered and intensified “when there is some perceived ex post facto or prospective need” (Haggerty & Ericson, 2000, p. 618), and are therefore potentially infinite. In sum, the surveillant assemblage “can be seen as the first recognition that surveillance needs to be analyzed in context” (Galič et al., 2017, p. 34), as we shall do through our empirical case discussed next. Seeking to understand the complexity of how surveillance functions in and through platforms in these terms, we ask: how do multi-level flows, directions, agents and targets of surveillance interact on platforms and with which outcomes? Relevance of the healthcare sector Control over knowledge about the body but also the conditions of discourse on this knowledge (e.g. the ‘truth’ about disease) has traditionally been the object of one-way scrutiny by ‘sages’ (e.g. the medieval clergy, the rational ‘medical gaze’) as shown by Foucault’s (1975) genealogy of the clinic. As such, each system of truth prevents collaboration and contestation of the conditions as well as of the content of the discourse by actors other than its main gatekeepers (clergy, then doctors), and first and foremost by patients themselves. To date, patients usually do not have an inherent right to their data that healthcare providers hold, as medical data flows predominantly to experts. This power asymmetry rooted in control of expert information operates at various levels. At the macro-level of policies (cf. Fotaki, 2009), governments and healthcare providers set boundaries on the distribution of patient health records to third parties with the excuse of protecting patient privacy, and large pharmaceutical companies promote health ideologies structured around 7 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 their products (such as vaccinology) (Picard et al., 2017), two sensitive issues that the COVID19 pandemic has recently revamped. At the micro-level, consumerist approaches to healthcare reinforce the myth of patient choice and promote ‘prosumption’ as a means to exploit patients (by making them produce the service they need, in making them responsible for managing their diseases), thereby increasing patients’ vulnerability and inequality in terms of access to care, particularly when it depends on and is mediated by supposedly ‘neutral’ technologies that gloss over patients’ differences (Visser et al., 2019). However, albeit being intertwined, access to health- care and access to health-information are two different things. While the consumerist approach focuses on the former, our paper focuses on the latter. On the one hand, on literacy and knowledge that patients have access to, share, contribute to producing, and on the other on the conditions for this discourse to emerge, how this is technologically enabled and which actors, previously disconnected, does it integrate and with what effects. With the emergence of participatory medicine and patient-generated data (Topol, 2015), Web 2.0 technologies are transforming the ways patients seek information and manage their health by tracking and generating scientific health information in real-time (Eysenbach, 2008; Hesse et al., 2010). Relevance of the healthcare sector Hierarchies of knowledge production and dissemination have started to become more leveled with increased access to medical knowledge and services (Rier & Indyk, 2006), to “anyone who is curious, regardless of their training” (boyd & Crawford, 2012, p. 664). As argued by a parallel and complementary study on PatientsLikeMe “unconventional, Internet-based organizational forms address traditional expert problems (medical research) through the systematic involvement of non-professionals (patients)” in “stark contrast to the complex, expert-dominated, prestige-laden, and costly institutional arrangements characteristic of medical research” (Kallinikos & Tempini, 2014, p. 817; Tempini, 2015). The way this challenges the power hierarchies in healthcare is changing what ‘research’ and medical knowledge generation mean. 1 (http://www.patientslikeme.com/about accessed November 4th, 2019.) 2 According to the latest available list (dated January 2019, available here: https://patientslikeme- bibliography.s3.amazonaws.com/PLM%20Research%20Manuscripts%20Bibliography.pdf) PatientsLikeMe has participated in the production of 111 scientific publications. 1 (http://www.patientslikeme.com/about accessed November 4th, 2019.) Research context: PatientsLikeMe.com PatientsLikeMe is a Medicine 2.0 platform described as a web-based service using social media technologies for social networking, participation, and collaboration among healthcare actors (Eysenbach, 2008), connecting over 750,000 patients suffering from severe and/or chronic illnesses and collecting self-reported health data about over 2800 conditions 1. This data is compiled by PatientsLikeMe and used for scientific and commercial research2. PatientsLikeMe was founded as a privately funded for-profit organization in 2004 by the Heywood Brothers, after one of them was diagnosed with a life-changing neuro-degenerative disease, Amyotrophic Lateral Sclerosis (ALS). It crowdsources patient care (Topol, 2014), and is established as an opt-in service, 8 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 not a healthcare provider, in order to circumvent the Federal Health Insurance Portability and Accountability Act of 1996 (HIPAA) rules that control the flow of patient data in the USA. At no monetary cost, PatientsLikeMe enables patients first of all to manage their care by tracking their disease’s evolution through their profiles and a variety of self-reporting and datafication tools, which was otherwise almost impossible to do and comprehensively visualize by patients themselves3(see Kallinikos & Tempini, 2014; Tempini, 2015). PatientsLikeMe creates disease-specific communities and enables patients with the same condition to share information and experiences with other community members as well as other external actors such as healthcare providers, pharmaceuticals and the government. PatientsLikeMe aggregates this patient data (plus patient-reported measures on quality of life; forum discussions and patient surveys) into a massive goldmine on drug side effects, and patient health and lifestyles. These patient-reported data are continuously and systematically recorded, pooled and shared with partners for medical research in anonymized aggregated form, and these partners also recruit PatientsLikeMe patients for randomized clinical trials. PatientsLikeMe partners with non-profit organizations and academic research institutions for free, and sells aggregate patient data to pharmaceutical companies – its only revenue-generating source. It foregoes advertising to provide comfort for patients in sharing their data, hence relying only on word-of-mouth sustainability. 3 See for instance the profile and charts of Stephen Heywood (deceased, 2006), made openly available on: https://www.patientslikeme.com/members/40 Methodology We conducted an extended observational netnography of PatientsLikeMe (Caliandro & Gandini, 2017; Kozinets, 2010), a methodology and design that are particularly rich for studying online communities (Park et al., 2019). Since healthcare can be considered a sensitive research topic, this allowed us to precisely follow naturally occurring dynamics of members (their archival, live dialogues and interactions through field-notes) in an unobtrusive way, avoiding undesirable outsider effects (Langer & Beckman, 2005). We ensured trustworthiness in netnographic research (Kozinets, 2010) through extended immersion, ongoing observation and triangulation over a two-year period. We complied with ethical research standards, since: firstly, “the behavior being observed is commonly performed in public (…), and in a setting where the anonymity of the person being observed is assured” (Zikmund & Babin, 2006, p. 242). Secondly, we collected and analyzed the existing publicly available archival and observational data, and thirdly, the researcher’s presence was fully disclosed while not interacting with members (Kozinets, 2010). Anonymity is maintained to protect participants’ both real and pseudo identities (Park et al., 2019). Our netnographic data collection was conducted in four iterative steps. First, the decision to research PatientsLikeMe was initially inspired by a cover story in Business 2.0 magazine 9 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 (Schonfeld & Morrison, 2007) that identified 10 exemplar organizations – including PatientsLikeMe – that had the potential to rewrite the rules of existing industries and create new markets. We began by observing and learning about PatientsLikeMe and its members and why and how they interact with the tracking technologies and other actors on the platform. (Schonfeld & Morrison, 2007) that identified 10 exemplar organizations – including PatientsLikeMe – that had the potential to rewrite the rules of existing industries and create new markets. We began by observing and learning about PatientsLikeMe and its members and why and how they interact with the tracking technologies and other actors on the platform. Second, we sought to develop a deep understanding of what makes people share their sensitive private health and lifestyle data (our exploratory question) and contribute to medical knowledge production and distribution, and how this process is organized on PatientsLikeMe. To do this, we registered with a guest account, which enables various researchers to engage on this platform. --- TABLE 1 (see end of document) --- Fourth, our analysis (see next section) used the keyword method (keywords: disclosure, privacy, sharing, tracking tools, openness, transparency, community, secrecy, monitoring, tracking, big data, crowdsourcing, empowerment, Web 2.0, pharmaceutical, government) in order to go through the immense number of posts (cf. Table 1). This was informed by our exploratory question on private health data tracking and sharing (Kozinets, 2010). We then realized that in the discussions and in the practices of sharing, tracking and monitoring each other’s health, PatientsLikeMe was actually fostering what appeared to be a complex surveillance network among multiple stakeholders. In our iterative analysis we were increasingly directed towards surveillance literature, which led us to progressively inductively study PatientsLikeMe through this lens and ultimately as a surveillant assemblage, as our analysis will detail next. Methodology We began our observations in the public community blog, where there were ongoing high-traffic discussions around privacy, data sharing and tracking, patient empowerment, and big data use. We observed that the majority of the posts on these issues were from patients in Multiple Sclerosis (MS) and Mood sub-communities, which then directed us to continue our investigation in the forums of these sub-communities. Third, we triangulated data from a variety of sources in multiple rounds, involving: (1) archival data from news and articles about PatientsLikeMe in business magazines (e.g., New York Times, Business 2.0, Forbes, Economist, Wall Street Journal, BusinessWeek), the public community blog, YouTube videos, published research by PatientsLikeMe and industry partners in scientific journals, (2) field notes, and (3) textual data from numerous member posts and their interactions in different sub-communities and the community blog (see Table 1 for detailed information on data sources and use). Attention was given to all exchanges in MS and Mood sub- communities (patient-to-patient, patient-to-administrators, researchers, and founders’ interactions). Data Analysis Observations and downloading of textual data were done over two years until saturation was reached (Glaser & Strauss, 1967), with on-going initial reflections by the first author. Subsequently, together we iteratively proceeded to the initial data coding to discover the main 10 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 themes, particularly relevant for understanding the dynamic complexity of online interaction phenomena (Caliandro & Gandini, 2017). Following the inductive coding schema (Glaser & Strauss, 1967), we compared first-order codes (informant-centric terms to define digitally- mediated practices and interactions on PatientsLikeMe) as a means to clarify themes through an inductive and recursive process and create second order theory-laden constructs (Gioia et al., 2013). This process was performed individually, and then together, in order to establish the credibility of codes discovered. In the open coding phase, also known as constant comparative method, we identified two broad categories: how actors engaged on PatientsLikeMe (through 15 digitally-mediated practices), and why (16 desired or resulting outcomes of these practices), looking for emerging patterns as well as staying alert to irregularities. Once coded, we realized that these broad categories were operating at micro, meso or macro levels, which led us to group initial findings into such levels for greater clarity and analytical purposes. Our final data structure is summarized in figure 1 below. Micro level Self-surveillance. Actors reorganize their roles and identities as dividuals (in particular, patients) through interactions with tracking tools on the platform. In their profiles, patients objectify themselves by listing symptoms via disease-specific and general tracking technologies, treatments received, and different lifestyles led. For example, epilepsy patients use a weekly tool called the ‘seizure meter’ to track the type, frequency, and severity of their seizures and code their symptom data in structured format. Such tools are developed and continuously updated via patient input enabling patients to closely monitor their own condition and even discover new conditions, the outcome of which is more self-awareness and disease literacy: I’ve gained a lot of insight from mood tracking tools, seeing patterns in my mood cycles and how that relates to factors like medication changes and weight gain. (Patient59, Mood). I’ve gained a lot of insight from mood tracking tools, seeing patterns in my mood cycles and how that relates to factors like medication changes and weight gain. (Patient59, Mood). Over the years I've had bouts of depression which I've added to my Mood Map…Then - whoa i l ki th h t I li d th t th bi i i lif h b Over the years I've had bouts of depression which I've added to my Mood Map…Then - whoa - in looking over the chart I realized that the bigger issue in my life has been compulsion…Having identified this I can now wrestle with how to live with it. (Pat62, Mood) Over the years I've had bouts of depression which I've added to my Mood Map…Then - whoa - in looking over the chart I realized that the bigger issue in my life has been compulsion…Having identified this I can now wrestle with how to live with it. (Pat62, Mood) In conventional surveillance, individuals lacked self-understanding, while information retention by the medical elite was somehow justified as ‘for their own good’ (Foucault, 1975). These self-surveillance practices are what PatientsLikeMe was primarily built for, and in seeking to increase awareness of their embodied self and disease literacy, they engage in self-datafication. This allows them to visibilize their disease and themselves: MS is not a dirty secret for me. It's a tiny part of who I am…I will not keep it a secret, and giving a face to MS makes it more personal and human to others (Patient66, MS). ---FIGURE 1 (see end of document) --- While data privacy was one of the key issues being discussed by PatientsLikeMe users and that initially drove our attention to study this platform, our findings yielded intriguing positions from the community members concerning how tracking tools were being used for surveillance purposes on various interrelated levels. Health data was being massively recorded and shared, arguments for such sharing outnumbered and outweighed privacy concerns, and a variety of stakeholders where ‘converging’ on the platform. This is when we realized that health-tracking tools were used for surveillance purposes (e.g. patients surveilled their health but also others’), and patients were not the sole targets of surveillance, which was also being directed to experts, and pharmaceutical companies (taking surveillance beyond the platform). This intriguing observation made us think of surveillance as a way to make sense of our data (with 5 second-order codes to categorize the surveillance practices across micro, meso and macro levels) and tempted us to engage in a critical reading. However, the more we immersed in it, and despite the iterative loops with critical literature on the risks of pervasive surveillance in the digital age, we realized it would be imposing a reading that didn’t quite fit. We agreed that the themes that emerged were telling a different story, pointing towards other outcomes than capitalistic exploitation. We identified 2 second-order codes to categorize such outcomes: patient empowerment and multi-stakeholder engagement in medical knowledge generation, indeed challenging traditional power hierarchies in healthcare. At that point, we moved to the axial/conceptual coding and returned abductively to literature calling for more attention being paid to other more ‘positive’ outcomes of surveillance and particularly Haggerty and Ericson’s (2000) notion of surveillant assemblage that could account for the converging flows of surveillance in and through PatientsLikeMe. We finally made relational connections within the data structure more salient (Gioia et al., 2013) with 2nd order constructs pertaining to two derived theoretical dimensions: 1) multilevel flows of surveillance and 2) desires that ‘hold together’ the surveillant assemblage, each spread out across micro, meso and macro levels (summarized in figure 2). 11 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 In our next section, we unpack our findings using selected quotes from the various data sources that triggered and illustrate each theme. Findings: PatientsLikeMe’s surveillant assemblage Through our study, we identified that each actor had particular expectations or desired outcomes, which motivated the kinds of surveillance practices they engaged in. Ultimately, these practices unravelled at micro, meso and macro inter-related levels in and through the platform, making PatientsLikeMe the locus where such multi-level flows of surveillance converged and ‘worked together’, thereby constituting a surveillant assemblage, summarized in the form of a funnel by figure 2, and unpacked next. Micro level The more I talk about living with HIV, the easier it becomes to actually live with it. It also gives me a chance to put a face to the disease, humanizing it and hopefully dispelling some ignorance and fear (Patient67, HIV). Data sharing increases both individual and public literacy about diseases, which humanizes and makes them more ‘livable’. Data sharing increases both individual and public literacy about diseases, which humanizes and makes them more ‘livable’. 12 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 Intra-surveillance. PatientsLikeMe patients also desire to find others suffering from the same condition in disease-specific communities as its name suggests, and constitute support groups to generate and share data, aided by visualization tools once more: We're just aware of each other’s moods more on here through mood charts and can empathize with each other more (Patient61, Mood). Our analysis of video data also reveals that search tools and disease symptom charts enable patient analysis of disease data via matching algorithms to compare to other patients’ personal profiles and personalize his/her treatments accordingly. (https://www.youtube.com/watch?v=wch3GdWJiLc). The platform also licenses patient reported outcome measures under creative commons open for all to share and suggest improvements for personalized treatment protocol, including drug and supplement use, lifestyle and dietary changes, and communicating disease specific data (https://www.youtube.com/watch?v=XQZ5M9oLkXw). However, gazing on others’ profiles not only enhances disease literacy and better knowledge of the self through exchanging with peers but also triggers forms of empathic oversight: We had a patient whose weight was dropping precipitously, which can accelerate a patient's deterioration in ALS. Since patients track and share their important outcome measures, another patient could remind him of the importance of keeping his weight up” (Founder1, Community blog). Everyone snarling back at her is NOT going to help. Anybody looked at her mood map? Depression almost off the chart, same with compulsion. She is having a BAD TIME, people… (…) Doesn't that suggest that we should be rallying around her to support her, instead of further tearing her down??? (Patient 63, Mood) Everyone snarling back at her is NOT going to help. Anybody looked at her mood map? Depression almost off the chart, same with compulsion. She is having a BAD TIME, people… (…) Doesn't that suggest that we should be rallying around her to support her, instead of further tearing her down??? Micro level The highlight of every third day is that I get to do my mood map, which stops me from putting myself in hospital” This is where I come instead of self-harming or attempting suicide. The highlight of every third day is that I get to do my mood map, which stops me from putting myself in hospital” (Patient94, Mood). Self and intra-surveillance on PatientsLikeMe overcome the prior disconnection between patients in the traditional health system and yield betterment of social relations, where patients aim for self-datafication and personalization while engaging in caring surveillance relationships. Micro level (Patient 63, Mood) This empathy is further sustained by self-modulation and responsibilization in order to enable a smooth flow of data-sharing. Dialogues between patients and staff reveal this responsibilization as patients watch over each other, and endows community members with a licence to surveil and police in order to protect: Our community members are a very switched-on group. If anybody posts something suspicious or overtly commercial we normally hear about it in a matter of minutes and can respond appropriately. (SupportStaff3, Mood). Our community members are a very switched-on group. If anybody posts something suspicious or overtly commercial we normally hear about it in a matter of minutes and can respond appropriately. (SupportStaff3, Mood). Our observations of forum discussions also suggest that light-touch moderation discourse facilitates surveillance by pushing patients to share and update as much data as possible on their profiles in an effort to increase the authenticity of their profiles and the data harmonization: Patients who (…) sort of have an aggressive voice on the forum, who don't fill out their profile tend to get pushed by others in the community to do that, because it becomes a validator of who they are, which is an incredibly powerful part of our model (Founder1, Mood). Patients who (…) sort of have an aggressive voice on the forum, who don't fill out their profile tend to get pushed by others in the community to do that, because it becomes a validator of who they are, which is an incredibly powerful part of our model (Founder1, Mood). Sometimes, such responsibilization backfires, for example, when dealing with trolls or those who create chaos in the community and obstruct data generation. In such cases, PatientsLikeMe staff interferes and controls the situation for a continuous and smooth data generation and creates 13 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 a forum code of conduct. Overall, this also helps those new to the platform to feel confident enough to interact, being welcomed by older community members. a forum code of conduct. Overall, this also helps those new to the platform to feel confident enough to interact, being welcomed by older community members. To sum up, by engaging in self and intra-surveillance, knowledge and caring function in an intricate way with potentially vital consequences: This is where I come instead of self-harming or attempting suicide. on PatientsLikeMe, which I use as a reference and share with treatment providers (Patient37, Mood). We believe that openness is much more powerful concept than privacy in medicine, and one that gives patients the power to take control of their health. (Founder1, Community blog). A modern focus on privacy as a goal has moved the line to the point that medicine is slowed, treatments are delayed, and patients die for failure to have what they need when they need it. We have substituted real harm for mostly theoretical harm. We believe that openness is much more powerful concept than privacy in medicine, and one that gives patients the power to take control of their health. (Founder1, Community blog). PatientsLikeMe founders challenge the dynamics of the current healthcare market that functions on privacy as an absolute goal and a fundamental human right, a right to ‘not share’. Openness and transparency promoted as core organizational values work through people’s desires to improve and manage their care and making data anonymous provides feelings of safety for normalizing surveillance at the meso-level. Meso level Inter-surveillance. Surveillance practices also extended beyond the platform through its network of stakeholders, thereby challenging existing meso-level interactions. PLM engages physicians through clinical research and using the platform as a way of tracking patients between visits. Patients also bring ‘doctor visit sheets’ designed for tracking their condition to their doctors’ appointments, which destabilize traditional relational power dynamics: Drugs I’m currently on were never offered to me during my six years with MS, due to my inability to adequately describe my symptoms, and my doctor’s inability to think outside the box from what he defaulted to using. Through doctor visit sheets, I learned to describe my symptoms better, and shared with my doctor other treatments. With a little time, he became more open, and I communicated better. (Patient23, MS). In the past, we physicians had information power and (since it dictated our livelihood) we guarded it jealously. Patients didn’t have the ability (or desire) to read through complex medical texts to understand their diagnoses. Now, they routinely come to me armed with a printout from WebMd or PatientsLikeMe and more often than not they are spot on. As a physician, I am no longer one who hoards information but a consultant who provides experience, context, meaning and perspective to what the patient is experiencing (Physician1, Community blog). Some physicians in PatientsLikeMe now praise self-tracking tools and the resulting patient- generated data, perceived as a sign of increased patient literacy, and an opportunity to collaborate with them in clinical settings. This enhances inter-surveillance dynamics by carrying the flows of surveillance beyond the patient community and the platform. This is motivated by the desire of more personalized medicine and reducing medical errors. Patients can compare their physician’s decisions against their medical history recorded via tracking tools on PatientsLikeMe, which enables a smoother patient data flow and improved connection among healthcare providers: Almost every time I’ve been hospitalized, I’ve been thrown in with an unfamiliar psychiatrist who gives me a new diagnosis and a completely different set of medications. …I now keep a centralized record of my mood and treatment history via mood charts and doctor visit sheets 14 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 on PatientsLikeMe, which I use as a reference and share with treatment providers (Patient37, Mood). While concerns regarding personalization through surveillance practices remain, se reflexive accounts signal both the awareness of these issues and the mitigating role of the platform While concerns regarding personalization through surveillance practices remain, self reflexive accounts signal both the awareness of these issues and the mitigating role of the platform I think some patients may be their own worst enemy. For instance, doctor visit sheet and mood charts. I try very hard to be completely honest as I answer the survey questions, but I can easily see how I can manipulate the results to convince my doctor to prescribe me certain meds or diagnose me with something I may feel more comfortable with. Sometimes knowing you are being scrutinized, and specifically what about you is being scrutinized can affect your behavior (Patient97, Mood). I think some patients may be their own worst enemy. For instance, doctor visit sheet and mood charts. I try very hard to be completely honest as I answer the survey questions, but I can easily see how I can manipulate the results to convince my doctor to prescribe me certain meds or diagnose me with something I may feel more comfortable with. Sometimes knowing you are being scrutinized, and specifically what about you is being scrutinized can affect your behavior (Patient97, Mood). In destabilizing meso-level interactions in healthcare, PatientsLikeMe challenges the current constitution of privacy by making patient engagement in surveillance and sharing of private health data acceptable through framing data-sharing as a ‘human right’ and normalizing discourses such as transparency and de-identification. Furthermore, in carrying the flows of surveillance to clinical settings PatientsLikeMe promotes a transformation from proprietorship to partnership in disclosure and distribution of medical data, which challenge the very definition of privacy, that is, control over externalization of one’s personal information, which belongs to the person (Goodwin, 1991). For example, PatientsLikeMe co-authored the Declaration of Health Data Rights and launching HealthDataRights.org in June 2009. PatientsLikeMe founders also testified before the National Committee for Health and Vital Statistics at the Gov 2.0 Summit to challenge macro institutions’ reliance on secrecy and desire to have control over private information: A modern focus on privacy as a goal has moved the line to the point that medicine is slowed, treatments are delayed, and patients die for failure to have what they need when they need it. We have substituted real harm for mostly theoretical harm. Macro level Corporate and institutional surveillance. Other healthcare stakeholders such as pharmaceutical companies and research institutions are also involved in surveillance relationships in PatientsLikeMe. Data collected using tracking technologies are shared with them in order to bridge the gap between anecdote and evidence-based medicine, to inform and guide future clinical practice. This translates into pharmaceutical companies tracking PatientsLikeMe patients for research (e.g., Genentech was granted access to PatientsLikeMe data to study drug effectiveness for cancer treatment), recruiting patients for randomized clinical trials (e.g., Novartis has recruited 15 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 PatientsLikeMe MS patients), and analyzing existing PatientsLikeMe patient-led clinical research to validate their own research, test/generate hypothesis and inform their clinical trial protocol design. This aims to provide alternative solutions to existing ailments and improve existing solutions in a timely manner, as reflected in our analysis of news items and published medical research using PatientsLikeMe data. For example, PatientsLikeMe research scientists won the 2009 Journal of Medical Internet Research Medicine 2.0 Award for research concerning secondary uses of drugs or drugs that are off-patent and unlikely to be studied systematically. This enables pharmaceutical companies to have easy and inexpensive access to patient-generated data, potentially lower research costs and faster clinical research processes that provide real-time feedback from patients (cf. Arnst, 2008 in Table 1). Despite some data scraping incidents on the platform (Angwin & Stecklow, 2010) (our video data also suggests that PatientsLikeMe creates a contractual relation with industry partners in order not to reidentify or scrape data, which will have legal consequences (https://www.youtube.com/watch?v=pTuemJ7ISaM)), patients actually endorse the use of PatientsLikeMe data for improved patient care: We give Pharma real life experiences with medications they provide for us, and make them better with less side effects (Patient103, Mood). We give Pharma real life experiences with medications they provide for us, and make them better with less side effects (Patient103, Mood). Data collection is what helps us get drug firms pay attention to what works or does not work for MS (Patient104, MS). Data collection is what helps us get drug firms pay attention to what works or does not work for MS (Patient104, MS). Including corporate and institutional research partners in the surveillant assemblage has several outcomes. Traditionally, clinical trials have been dominated by medical authorities and academics, which resulted in information sequestration, privileging the medical elite (Epstein, 2007). Macro level Although expert-collected clinical data and patient-generated data may sometimes contradict due to methods and sampling (Kitchin, 2015), such discrepancies provide useful information as well, and on the whole this collaboration is destabilizing the disciplining rules/criteria of top-down medicine. One example is PatientsLikeMe’s MS rating scale, designed to exhibit symptom data in the patient’s own language, and made in collaboration with pharmaceutical companies and specialized healthcare providers to test the validity of these tools (see Kallinikos & Tempini, 2014): If there are inaccuracies, we can modify the tool. We did this in ALS community where one member indicated that ALS rating scale didn't pick up her changes having progressed to a very advanced stage of disability. An extension was designed to pick up on changes (SupportStaff2, MS). This tracking tool developed with patient input was validated by comparing the results to findings generated with standard clinical assessment methods used in MS (Bove et al., 2013). PatientsLikeMe also launched the Open Research Exchange hub to establish a collaboration ecosystem of industry partners and patients, thereby changing the way medical knowledge is generated. Another important outcome is that access, an important challenge for big data, as it is privately produced, is overcome to a greater extent (Kitchin, 2015). The PatientsLikeMeListen 16 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 service enables industry partners to receive patient-reported outcome data via tracking tools on drug effectiveness and side-effect information. PatientsLikeMe also performs sentiment analyses on patient discussions about drug effectiveness in community forums, and enables pharmaceutical access to aggregated sentiment data. Along with scientific expertise, patient experiential expertise has the potential to become a productive force in medical research. service enables industry partners to receive patient-reported outcome data via tracking tools on drug effectiveness and side-effect information. PatientsLikeMe also performs sentiment analyses on patient discussions about drug effectiveness in community forums, and enables pharmaceutical access to aggregated sentiment data. Along with scientific expertise, patient experiential expertise has the potential to become a productive force in medical research. State surveillance. By including State agencies in its web of interrelated actors, PatientsLikeMe contributes to change our traditional understanding of State surveillance and regulation in two ways. First, by inverting the surveillant gaze. 17 5 MedWatch is FDA’s voluntary drug adverse event reporting system largely used by healthcare providers, and inaccessible to patients until the partnership. 4 In the USA, this is led by the FDA, usually performed via clinical trials with small numbers of patients in controlled circumstances. Macro level Instead of monitoring patients with the aim to control them as in a Foucaldian scenario, PatientsLikeMe provides a way for enhancing state oversight over pharmaceutical companies by taking post-market surveillance (aka pharmacovigilance) to monitor drug safety after-market release4, a step further. Since 2008, PatientsLikeMe developed a partnership with the Food and Drug Administration (FDA) and its MedWatch pharmacovigilance system5 to self-generate, record and track individual reports, and aggregate data from the patient community to share with the FDA: Patients desperately need a way to collect reports of adverse effects from medications, a responsibility shirked by the FDA and subverted by the drug companies. (Patient16, MS). We launched a pilot program in our MS community, which helps patients submit treatment related adverse events directly to FDA through PatientsLikeMe. Understanding when these events occur helps FDA better regulate pharmaceutical and medical industries to protect consumer safety and bring safer, more effective products (Founder2, MS). We launched a pilot program in our MS community, which helps patients submit treatment related adverse events directly to FDA through PatientsLikeMe. Understanding when these events occur helps FDA better regulate pharmaceutical and medical industries to protect consumer safety and bring safer, more effective products (Founder2, MS). State collaboration with social media platforms and the use of patient-generated data on these platforms for pharmacovigilance is a fairly recent phenomenon, as State focus on patient voice for complementing the existing pharmacovigilance has been very limited (https://www.centerwatch.com/articles/16413). By connecting the patient directly to FDA to report drug side effects, PatientsLikeMe increases the responsibility of the State to further regulate the pharmaceutical industry (e.g. issue product recalls, warnings, safety messages) and compare patient-reported adverse effects with those of clinicians in randomized clinical trials in order to assess the risks and benefits of these reports. Such macro level responsibilization reduces pharmaceutical industry intrusion and influences how people evaluate and communicate the risks of existing medications (Cox et al., 2010). Second, PatientsLikeMe provides information to patients regarding state health-related initiatives. For instance, PatientsLikeMe imports data from a federal site called clinicaltrials.gov (where all US clinical trials are required to register) to inform patients about the ongoing active randomized clinical trials, for which they may be eligible. 17 4 In the USA, this is led by the FDA, usually performed via clinical trials with small numbers of patients in controlled circumstances. 5 MedWatch is FDA’s voluntary drug adverse event reporting system largely used by healthcare providers, and inaccessible to patients until the partnership. Macro level Other collaborations, for example, between the US Department of Veterans Affairs’ Epilepsy Centers of Excellence, PatientsLikeMe, 4 In the USA, this is led by the FDA, usually performed via clinical trials with small numbers of patients in controlled circumstances. 17 5 MedWatch is FDA’s voluntary drug adverse event reporting system largely used by healthcare providers, and inaccessible to patients until the partnership. 17 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 and UCB Pharmaceuticals for research into the factors that influence health outcomes for veterans with epilepsy, bring State bodies into the surveillant assemblage. and UCB Pharmaceuticals for research into the factors that influence health outcomes for veterans with epilepsy, bring State bodies into the surveillant assemblage. Overall, multiple stakeholder engagement in organizing big data generation via surveillance practices in and through PatientsLikeMe indicates that surveillance in platforms is not a unilateral or top-down process, but a multi-level flow of interrelated agencies and practices that ‘work together’ and are ‘held together’ by the outcomes desired by each party. All the stakeholders involved become knowingly both the agents and the targets of surveillance converging in a co- constructed and surveilled digital environment and it is these practices that constitute PatientLikeMe as a multi-level surveillance assemblage. Platforms as loci where multi-level surveillance phenomena converge Platforms as loci where multi-level surveillance phenomena converge Firstly, we show how platforms serve as digitally-mediated spaces for surveillance, constituting a dynamic crossroads of micro, meso and macro surveillance phenomena both within their online communities and beyond. Unpacking the multi-level complexity of surveillance in and through platforms reveals how such levels, practices and objects are held together in a digital environment, converging as a surveillant assemblage (Haggerty and Ericson, 2000). Figure 2 provides a visual representation of this convergence in the form of a funnel, illustrating multiple objects in flow (information, bodies, desires, institutions and processes) making several systems, directions, outcomes and levels of surveillance converge into a functional entity that transforms the purposes and hierarchies of surveillance. Our case reveals platforms’ potential for fostering and shaping a complex web of such surveillance flows, how and why all these converge and ‘work’ together and constitute a surveillant assemblage. Discussion Our study shifted analytical attention to aspects of surveillance often left understudied: what kind of surveillance practices emerge in and through platforms, which stakeholders are involved, and what outcomes are produced. By exploring how “individuals actively participate (wittingly or not) in their own visibility, thereby creating new potentialities of surveillance by others” (Brivot & Gendron, 2011, p. 140), we raise a scantly studied question about what happens when those who were once under surveillance, and thereby disciplined and controlled, now enact surveillance themselves. Concretely, we were able to highlight how surveillance operates at multiple inter-related levels that are ‘knotted together’, i.e. converging on the platform. This provides two main contributions and several potential directions for future research. ---FIGURE 2 (see end of document) ---- This conceptualization can revitalize the study of surveillance in and through platforms by allowing a more fine-grained analysis of its complex, rhizomatic and multi-level dimensions. It could also complement sociomaterial accounts of platforms (Orlikowski & Scott, 2014) with 18 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 whom surveillance studies share the notion of ‘assemblage’. However, Deleuze and Guattari, whose work Haggerty and Ericson relied on to theorize surveillant assemblages, use the French word agencement, which translates into English as ‘assemblage.’ This nevertheless neglects a key dimension in Deleuze and Guattari’s thinking rooted in its Latin origin: agens, i.e. its agentic, dynamic and processual dimension, somewhat present in Haggerty and Ericson’s notion of ‘desires’ that make the systems converge, but not as salient as in Deleuze and Guattari. This is of prime importance in sociomaterial studies of information technology (Cecez-Kecmanovic et al., 2014) and in studies of the role of human and non-human actors and interactions (Taupin, 2019) in yielding social and institutional change and alternative forms of organizing (Ouahab & Maclouf, 2019). This essential point should make us further refine our understanding of the surveillant assemblage not only as an sociomaterial assemblage of surveillance flows, but as an agencement process that sets such flows in motion and makes them converge and produce or perform particular outcomes – a key question for future studies to explore. Potentialities of the platforms’ surveillant assemblage Second, our paper answers calls to go beyond the risks of platform-related surveillance (e.g. Lyon, 1994, 2007; Albrechtslund, 2008; Pridmore, 2013; Hafermalz, 2020). Within the surveillant assemblage of PatientsLikeMe, surveillance becomes more relational and entangled than ever, and seems to be empowering patients’ license to heal by shifting traditional power dynamics (Haggerty, 2006) in healthcare. Data sharing and aggregation enables new forms of patient empowerment and knowledge generation ultimately influencing patient well-being, transforming roles, hierarchies of visibility and power, and relations among stakeholders in and beyond the platform. Our netnographic design is part of the novelty of our study of surveillance in platforms inclusive of elements relevant to the experience of surveillance on the receiving end (Hafermalz, 2020). Our findings hint towards the potentialities of surveillance no longer being just an omniscient exploitative practice exerting control over populations (Zuboff, 2015, 2019). Such risks do not disappear, but are subsumed into a more complex process where it is not so easy to either fully accuse or praise platforms’ surveillance effects. This highlights not only constraining but also enabling power dynamics on healthcare platforms. The question of access to health, being mediated by technology, has been criticized for stressing inequalities (Fotaki, 2009; Visser et al., 2019). However, PatientsLikeMe, while being fundamentally a platform of patients, brings an understanding of individual patients not as consumers of healthcare (since it does not offer healthcare services) but as a networking platform of multiple stakeholders. Gaining literacy and managing one’s disease’ fits a prosumer narrative but is sustained by a different ideology: ‘sharing as a fundamental human right’. There is indeed no such thing as a ‘universal patient’ (Visser et al., 2019), and PatientsLikeMe precisely seeks to harbour a myriad of different patient experiences, whose multiplicity should be made visible by the platform. This brings attention to “why individuals take on the responsibility of ‘visibilizing’ themselves, (…) due to an existential need to be seen as a legitimate member (…) to remain ‘on the inside’” (Hafermalz, 2020, p. 7) and not be cast out of society due to their disease. 19 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 Through the surveillant assemblage, all involved actors knowingly become both the agents and the targets of surveillance which is not fixed or unilaterally given by the technological environment but is enacted in practice. Potentialities of the platforms’ surveillant assemblage Patients engage in self-datafication that enables increasing caring and awareness of the self and others. Decisions people make concerning their care become more reflexive and confident, which then allows them to make more direct and deliberate demands from other entities on and beyond the platform. We find that both state and non-state actors, private actors, and communities gain relative power to perform surveillance activities, and greater control over being surveilled voluntarily. For patients, such voluntary visibilization of oneself is often framed as a reflexive, relational and existential necessity (Hafermalz, 2020) in order to connect with others (Iedema & Rhodes, 2010). For instance, practices were shifting previous conceptualizations of privacy and surveillance, and challenging their oppositional constitution. As already foreseen by Haggerty and Ericson (2000, p. 616): “Privacy is now less a line in the sand beyond which transgression is not permitted, than a shifting space of negotiation where privacy is traded for products, better services or special deals”. Concerns about privacy were omnipresent, and understandably so, but discourses about privacy on PatientsLikeMe were contrary to what we expected: such practices and their outcomes seemed to challenge the ‘in-house assumptions’ (Alvesson & Sandberg, 2011) dominating the literature on surveillance in platforms, which did not easily recognize the potentials for new developments. In less than two decades, platforms have achieved the prediction that Haggerty and Ericson (2000) had concluded their paper with: the ‘disappearance of disappearance’ in surveillance capitalism’s total reach, “making the Panopticon seem prosaic” (Galič et al., 2017, p. 25). However, even though the platforms’ rhetoric may constitute an order through which patient engagement in surveillance practices might be tailored to corporations’ self-interests (Rose, 1999), patients did not unilaterally equate to naïve dopes subjected to the exploitative gaze of surveillance capitalism (Zuboff, 2019) and did not seem to engage with PatientsLikeMe in a consumerist fashion, contrary to what other recent works on healthcare technologies have denounced (see Visser et al., 2019). PatientsLikeMe patients, who perceive benefits to self and others, and who feel ‘in charge’ of their data, tend to choose data sharing over privacy, and opt for personalization via collaborative modes of surveillance facilitated by new technologies (Topol, 2014). Hence, a communitarian perception of privacy as a shared liability based on crafting a balance between secrecy of individual information and responsibility for aiding common good prevails (Etzioni, 1999). Potentialities of the platforms’ surveillant assemblage 20 Overall, the effects of and responses to surveillance invite us to think beyond binary and reactive responses to surveillance and answer calls for a more encompassing view of surveillance as it unfolds in platform organizations (Majchrzak et al., 2013). One notable effect in this view of surveillance is responsibilization. In order to generate medical knowledge and effectively contribute to levelling the power dynamics in healthcare, surveillance in and through platforms warrants a coordinated approach and responsibilization (Shamir, 2008) at all ends of the spectrum, not only the neoliberal responsibilization of patients. The macro-level for data security and management of surveillance on one hand, and at the micro-level of patients on the other, as the sustainability of the system relies on patient literacy and expertise on providing their data and 20 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 updating it regularly. Data (il)literacy remains an important struggle, as it may obstruct decisions concerning patient care and scientific knowledge production, as well as power dynamics since disaccord may occur between patients and physicians in terms of the standards used in surveillance tools to track patient health (Bove et al., 2013). Another effect is personalization, which poses ambiguities in terms of data manipulation and discrimination that potentially risk patient care and transparency in patient-physician collaboration. While personalized medicine via platform surveillance is in its early stages, for it to work efficiently, the vast amount of de-identified data will require the necessary infrastructure, expertise, and systematic validation from involved parties and tools (Topol, 2014) in order to analyze and make sense of it, and allow patient control in enabling access to their specific health data that could cause stigma and discrimination in managing patient care, but in “a double movement of autonomization and responsibilization” (Rose, 1999, p. 174; Shamir, 2008). Overall, our findings point to the empowerment of patients and the inclusion of multiple stakeholders in medical knowledge generation through convergent surveillance practices in and beyond platforms. This moves the traditional loci of patient and disease tracking (from hospitals, laboratories, doctors’ offices) towards a personal and collective setting, which challenge the existing scientific research infrastructure and epistemological expert-based tradition of medical research (Kallinikos & Tempini, 2014). Potentialities of the platforms’ surveillant assemblage Although we find that PatientsLikeMe offers a great potential for patients to become literate in understanding and analyzing their illnesses, whether and how much this potential will be realized and what conditions are required for this realization are significant future research questions in PatientsLikeMe and other platforms. For instance, designing platforms in ways that promote literacy (Firat & Vicdan, 2008) and increased inclusion of patients in the design of surveillance tools, the constitution of surveillance practices and oversight on the disclosure and sharing of the data will be essential. Although platforms harbor the potential for increasing the scope and directionality of surveillance, avoiding some of the discriminatory effects of surveillance requires constant vigilance by all stakeholders, including governments, advocacy groups, companies, and citizens, and granting control of information flow among data partners to data subjects (Lyon, 2002). Funding No specific funding to declare. Acknowledgements The authors would like to thank Editor Thomas Roulet and the anonymous reviewers for their support and help in revising this work, as well as the members of OCE Research Centre and the MOTI seminar at GEM for their precious feedback on earlier versions. Concluding remarks As we witness the global reach of Covid-19 pandemic, we realize the astounding contemporary relevance of how “surveillance is driven by the desire to bring systems together, to combine practices and technologies and integrate them into a larger whole (…) providing for exponential increases in the degree of surveillance capacity” (Haggerty & Ericson, 2000, p. 210). In early April 2020, a dedicated forum in PatientsLikeMe included over 70,000 members and a very active Twitter account, where patients and health providers interacted to clarify questions about this outbreak, filling and refining the Covid symptoms tracker, reporting and comparing treatments received. Such initiatives based on citizen-generated data (e.g. eu-citizen.science) are burgeoning worldwide as a possible response to fast-evolving global sanitary crises for early detection (cf. Joshi et al., 2020) and monitoring the virus’ spreading (Knight, 2020). This is 21 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 providing a contemporary macro justification of the openness philosophy of sharing of micro health data defended by PatientsLikeMe and others, and is something that each individual can contribute to while still complying with lockdown measures. But this only seems possible through particular surveillant assemblages like we have described in our analysis, where flows of information on a variety of surveillance systems converge, geared at producing and distributing knowledge, while still remaining alert to potential manipulations and requiring new responsibilizations, as future research might continue to explore. References Alaimo, C. & Kallinikos J. (2017). Computing the everyday: Social media as data platforms. 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The age of surveillance capitalism: The fight for a human future at the new frontier of power. NY: PublicAffairs. Zuboff, S. (2019). The age of surveillance capitalism: The fight for a human future at the new frontier of power. NY: PublicAffairs. 27 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 Table 1 Data sources Table 1 Data sources Table 1 Data sources 28 Table 1 Data sources 28 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 29 Forthcoming in M@n@gement; check for updates on online publication, DOI: 10.37725/mgmt.v25i1.4586 FIGURE 1 DATA STRUCTURE FIGURE 2 PLM AS A SURVEILLANT ASSEMBLAGE FIGURE 1 DATA STRUCTURE FIGURE 1 DATA STRUCTURE FIGURE 2 PLM AS A SURVEILLANT ASSEMBLAGE FIGURE 2 PLM AS A SURVEILLANT ASSEMBLAGE FIGURE 2 PLM AS A SURVEILLANT ASSEMBLAGE 30
https://openalex.org/W2089401299	https://icono14.net/ojs/index.php/icono14/article/download/256/133, https://dialnet.unirioja.es/descarga/articulo/3301761.pdf	es		Evolución social de las relaciones públicas en el perú. Contexto académico y profesional de la comunicación organizacional	La Revista Icono 14	2,012	cc-by	8,998	REVISTA ICONO 14, 2010, Año 8, Vol. 2, pp. 243-263. ISSN 1697-8293. Madrid (España) Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú Recibido: 05/02/2010 – Aceptado: 28/03/2010 A8/V2 – REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS – ISSN: 1697 - 8293 Palabras clave EVOLUCIÓN SOCIAL DE LAS RELACIONES PÚBLICAS EN EL PERÚ Comunicación organizacional, relaciones públicas, teorías de la comunicación, entorno empresarial peruano, investigación sobre el impacto de los medios, opinión pública, públicos internos, públicos externos Key Words Contexto académico y profesional de la Organizational communication, public relations, communication theories, Peruvian business context, university education in Peru Abstract Comunicación organizacional Amybel Nila Walther Sánchez Profesora Doctora Facultad de Ciencias de la Comunicación. Universidad San Martín de Porres. Avda. Tomás Manzano, 242. Surquillo. Lima (Perú) – Tlfn.: (511)5136300 - Email: amybelsanchez@epu.edu.pe Resumen Se considera a las Relaciones Públicas como un sistema multidisciplinar clave para integrar a la organización empresarial y a sus públicos, mediante la aplicación de estrategias de comunicación que fomenten el intercambio de información (feedback) y la bidireccionalidad. Para lograr una mejor comunicación con los públicos internos y externos, no basta con intentar la aplicación irreflexiva y apresurada de las estrategias de comunicación, sino en reexaminar las fuentes bibliográficas y estructurar una teoría más cercana con las exigencias de la “sociedad del conocimiento” y la realidad nacional. 8/2 Public Relations are considered a key multidisciplinary system to integrate the business organization with its publics. However, applying communication strategies is not enough: there is also a need to re-examine bibliographic sources and to give a structure to a theory that better addresses the requirements of the “knowledge society”, and the particular status of each national reality. In this sense, the present article deals with the evolution of Public Relations in the Peruvian context, so as to determine if university curricular programs meet the requirements and needs of the labor market. A radiography depicting the performance of communication units from the main corporations and businesses in the country has been prepared as well. Although the communication that the public relationist performs is born within a business context, a new methodology allowing for the confluence of different social agents (State, corporations, citizenship, NGOs) can be promoted in order to REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-243 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú Si bien la comunicación que realiza el relaciones públicas nace dentro de un entorno empresarial, se puede inculcar una nueva metodología que permita la confluencia de los actores participantes (Estado, corporaciones, sociedad) y facilitar el diálogo, la concertación y finalmente la integración. easy the dialogue, agree upon and, finally, obtain integration. To attain this objective, there is a need to restructure current study plans in Peru, opening the door to more elective and specialized courses, in accordance with the severity imposed by a more globalized world Introducción Las Relaciones Públicas, así como todo acto comunicativo, es una actividad que intenta articular una Comunicación armónica entre las organizaciones y sus públicos, a fin de convertirlas en lazos simbióticos y provechos para ambas partes. Esta alianza estaría reflejada no sólo en la mejora de la calidad de vida (impulsada por las inversiones de las organizaciones) sino también en los programas de Responsabilidad Social y de administración de la controversia. Estos últimos contribuirían a la formación de un entorno público favorable y positivo. La importancia de esta correspondencia hace indispensable no sólo el contacto diario con las audiencias o conocer sus inquietudes, sino también con el enriquecimiento y ampliación de las bases teóricas de las Relaciones Públicas. La optimización de sus fundamentos favorecería la labor hacia las comunidades, una comprensión más cercana a la realidad social y los procesos de Comunicación en general. El desarrollo de estas Relaciones Públicas estará reflejado en la actividad de profesionales con un consolidado bagaje conceptual, metodológico y ético. En ese sentido, no hay duda que el dominio eficiente del conocimiento sigue siendo la cualidad diferencial dentro del competitivo mercado laboral del siglo XXI. Objetivos La primera parte del artículo científico establecerá la relación existente entre las definiciones de “Comunicación” y “Relaciones Públicas” a fin de determinar convergencias y distinciones básicas. Al puntualizarse las divergencias, buscará estructurar un concepto de RR. PP., así como su 8/2 misión y objetivos. La segunda parte emprenderá una revisión histórica de las definiciones teóricas en torno a las Relaciones Públicas, tanto en el extranjero (con énfasis en el caso estadounidense) como en el Perú. REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-244 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú Luego nos detendremos en analizar las estrategias de comunicación, evaluando sus rasgos planificadores y persuasivos. Dentro de ese apartado también se detallará el perfil del profesional de las Relaciones públicas y su desenvolvimiento práctico en el mundo empresarial peruano. Antes de llegar a las conclusiones, se identificarán las nuevas tendencias en el campo de las Rela- ciones Públicas: la reconfiguración de muchas tareas de esta profesión ha planteado retos y exigencias a los comunicadores, al contenido de los mensajes y a los tipos de canales que se emplean (TIC). En ese sentido, el valioso aporte de Internet contribuiría a plasmar con claridad los fines tanto de la organización como de la opinión pública. Metodología La investigación que se desarrollará en esta monografía tendrá un carácter teóricoacumulativo, a fin de establecer una plataforma conceptual para reflexionar acerca de las Relaciones Públicas. Por lo tanto, este trabajo analizará y estudiará fuentes bibliográficas a fin de ofrecer un panorama más amplio y diverso de las principales teorías referidas a la naturaleza de las Relaciones Públicas, presentes en la bibliografía española, americana y peruana. 1. Naturaleza de las Relaciones Públicas 1.1. Relación con las Ciencias de la Comunicación Existe una similitud entre las definiciones de la Comunicación y de las Relaciones Públicas. Sin embargo, la primera es un campo más amplio, que suele contener otras especialidades, entre ellas la del profesional. Etimológicamente el término “Comunicación” proviene de dos palabras latinas: del sustantivo communicatio y del verbo communicare. Ambos vocablos derivan, a su vez, 8/2 de communis “común”, que también significaría “consultar” o “discutir”. El Diccionario de la Real Academia de España/ DRAE define la Comunicación en su primera acepción: “Acción y efecto de comunicar o comunicarse” y, a su vez, define el verbo comunicar de las siguientes maneras: a) Hacer a otro partícipe de lo que uno tiene; b) descubrir, manifestar o hacer saber a alguien algo; c) conversar, tratar con alguien de palabra o por escrito; d) transmitir señales mediante un código común al emisor y al receptor. REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-245 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú Este último significado nos parece muy interesante, ya que consideramos la comunicación como un proceso dinámico entre personas, cuyos principales elementos son el emisor, el receptor, el canal y el mensaje. Este proceso es posible cuando comprendemos los significados de los símbolos que compartimos y utilizamos. Francisco Gil Tovar (1984: 11) al referirse a la comunicación como proceso, nos manifiesta lo siguiente: La acción de comunicar es todo un proceso. Es decir, se desarrolla en el tiempo y en el espacio. No es un acto súbito y simple sino algo que está dándose y que en su transcurso se modifica continuamente. De ahí que sea cambiante y viva, como vivos y cambiantes son los mismos seres que se comunican, sus circunstancias y las sociedades en que desempeñan sus actividades. También exige la capacidad de crear dichos símbolos, y que quienes los utilicen conozcan y acepten sus significados. Esta es una facultad propiamente humana. Sobre esto Salvador Mercado (2002: 37) remarca lo siguiente: “Sólo si las palabras (u otra forma de comunicación) son comunes tanto al comunicador como al receptor, es decir, significan lo mismo para los dos, existe realmente comunicación”. La mayoría de las acepciones inciden en el carácter bipartito de la comunicación, donde emisor y receptor se valen de un código común para hacer partícipe a la otra parte del saber que uno ostenta. Sin embargo, este término puede ser abordado 8/2 desde otra perspectiva. Martín Martín (2004: 44) expone su propia definición: Acción o proceso de creación, coordinación, definición, transmisión y difusión de mensajes o información entre personas o de una empresa o institución que utilizando las distintas herramientas y canales, consiguen alcanzar los objetivos de percepción propuestos entre sus públicos o audiencias. La Comunicación humana posee intencionalidad y el deseo de influir o persuadir a su interlocutor. Así Rojas Guzmán (2000: 55) comenta lo siguiente: La problemática de la comunicación es inherente al ser humano, puesto que todo acto humano es un acto comunicativo. El hombre busca agruparse, forma colectivos que tienden a la satisfacción de necesidades básicas, secundarias y de convivencia, para alcanzar objetivos comunes que a su vez interpretan requerimientos individuales que llevan a la propia realización. Lo último nos sugiere que la comunicación es una herramienta “bidireccional” (en cuanto al sentido de los mensajes) que nos permite relacionarnos entre seres humanos. Este panorama se reflejaría también en la dinámica social de las organizaciones. En ese sentido, la Asociación de Empresas Consultoras en Relaciones Públicas y Comunicación (ADECEC, 2002: 23-45) definió su especialidad como un “conjunto de estrategias para generar confianza entre la empresa y sus públicos”. Tres pasos serían REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-246 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú necesarios para informar y predisponer positivamente: Claudia Canilli (1995: 16) profundiza más al respecto: - Conseguir influir de manera que se cumplan los objetivos del cliente en los distintos públicos objetivos haciendo que conozcan, aprecien, defiendan y si es posible transmitan las ideas que nuestra compañía les ha hecho llegar. Tanto la actividad como la inactividad, las palabras como el silencio, tienen valor de mensaje: influyen a los otros y sobre ellos, y a su vez, los demás no pueden dejar de responder a estas comunicaciones, de modo que también ellos comunican. - Gestión de lo que opinan los stakeholders de una empresa creando opinión y/o motivando una reacción en ellos. Esta gestión significa trasmitir información y mensajes a los distintos públicos y audiencias ya sea de forma voluntaria o involuntaria. Coincidimos entonces con la conclusión de Canilli: “La comunicación, además de inevitable, muchas veces es también inconsciente” (Ibídem: 17). Mercado (2002: 37) es contundente al añadir que cada vez que alguien recibe un mensaje tenemos comunicación. En ese sentido, Alejandro Formanchuk (2001: web) sostiene lo siguiente: - Asesoramiento e implementación de campañas que permitan a los clientes la consecución de objetivos comerciales, utilizando herramientas diversas de Comunicación. Dada la importancia de la Comunicación organizacional, no podemos dejar nada al azar: el relaciones públicas debe tener cuidado al interpretar ciertos eventos que puedan crear desazón o incertidumbre en los públicos. Daniel Scheinsohn (1996: 27) señala que “toda ausencia de comunicación no es „no comunicación‟ sino comunicación negativa”. Si asumimos que todo acto humano es comunicación y que toda organización tiene un comportamiento, concluiremos que será imposible no comunicar, siendo así necesario poseer el mayor control sobre la comunicación “inconsciente”, pero real. 8/2 Es necesario que las organizaciones perciban la complejidad que envuelve a la acción de "comunicar" para que por fin le asignen el valor que verdaderamente tiene y encomienden su gerenciamiento (en verdad, el intento de dirigir la lectura dentro de los límites proyectados orientando la decodificación hacia un significado preferente) a un profesional. Así, para evitar ocultaciones o malinterpretaciones en la información, la organización debe saber lo que desea comunicar de sí misma; luego, planificar adecuadamente ésta comunicación, por lo que deberá encargársela a quien esté en condiciones de realizar un trabajo efectivo, es decir, el profesional de las Relaciones públicas. REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-247 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú 1.2. Reexaminando las definiciones Existe una gran diversidad bibliográfica y opiniones solventes en torno a las Relaciones Públicas. Según Reilly (Arceo Vacas, 1988: 20) la mayoría de las definiciones concuerdan en erigir una función gerencial diseñada para analizar, aconsejar y ejecutar programas de comunicación. También aparecen otros conceptos que resaltan la creación del consensoi, la integración entre la organización y sus públicos, así como el asesoramiento a la gerencia y la creación de una filosofía organizacional. Aparte de la definición que propondremos al final de este capítulo, queremos rescatar las coincidencias y particularidades substanciales que hemos encontrado en los autores consultados. De esta manera, podremos establecer un marco conceptual amplio y con parámetros claros. Por un lado, la palabra “relación” proviene del latín (refero) y designa el trato, conexión o comunicación entre personas o cosas. “Relacionarse” es vincularse, constituir una mutua correspondencia, conocerse. Para Niceto Blázquez (1994: 66) este término hace referencia al encuentro entre seres humanos constituidos en grupos. Así la profesión de relaciones públicas habría surgido por la necesidad de resolver conflicto. Por otro lado, “público” atiende al conjunto de personas reunidas en determinado lugar o que participan de unas mismas aficiones (Gil Tovar, 1984: 86). 8/2 De acuerdo con Noguero (1990: 43-51), las Relaciones Públicas comprende una serie de actividades que persiguen maximizar el acuerdo entre las organizaciones y sus públicos, mediante el uso de estrategias y tácticas científicas de información. Estos mensajes “planificados” serían, a la vez, “persuasivos” y capaces de influir sobre un público considerable (Marston, 1993: 3). Desde la perspectiva de Scott Cutlip y Allen Center (2000: 34), esta profesión contendría los siguientes atributos: Relaciones Públicas son la función directiva que evalúa las actitudes públicas, identifica las estrategias y procedimientos de un individuo u organización frente al interés público y planifica y ejecuta un programa de acción con el fin de conseguir la comprensión y aceptación pública. Esta definición encuentra eco en Arceo Vacas (1988: 21), quien interpreta las Relaciones Públicas como una “filosofía gerencial” que se hace tangible a través de una serie de acciones de comunicación persuasiva (de tipo interpersonal, o colectivo masivo y no masivo), presentadas en forma preponderantemente informativa, que pretenden conquistar o modificar la aceptación de una persona natural o jurídica por sus públicos. William Nielander (1973: 9) también centra su atención en el aspecto de la función directiva: Constituye una función continuada de dirección gracias a la cual una organización, a través de la investigación, evalúa sus relaciones con su público, desarrolla unas direc- REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-248 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú trices y unas prácticas justas y también unas comunicaciones y actividades efectivas, buscando con ello obtener y mantener la comprensión y la buena voluntad. El Institute of Public Relations (Harrison, 2002: 2-3) amplía la definición de las RR. PP. al dar cabida a un elemento esencial: “dirección de reputación”. Encontramos autores como Solórzano y Pirotte (2006: 111), quienes rescatan la actitud y el esfuerzo profesional antes que a la función directiva: Las Relaciones Públicas se ocupan de la reputación: el resultado de lo que uno hace, lo que dice, y lo que los demás dicen sobre uno [...] con el fin de lograr la comprensión y el respaldo, y de influir sobre la opinión y el comportamiento. Se trata de un esfuerzo planificado y sostenido por establecer y mantener la buena voluntad y la comprensión mutua entre una organización y sus públicos. Actitud y esfuerzo deliberado, continuo y planificado, destinado a mantener relaciones convergentes y armoniosas entre una institución o empresa, pública o privada, y sus diferentes públicos. Fraser Seitel (2004: 3) explicita en su definición la dirección que ha de tener el flujo comunicativo, subrayándose la necesidad imperativa de la bidireccionalidad como cualidad: Las Relaciones Públicas son un proceso planeado para influenciar la opinión pública, a través de un carácter sonoro y performance propia, basado en una satisfactoria Comunicación mutua de doble vía. La Public Relations Society of America (Guth y Marsh, 2006: 6) toma en cuenta la referencia el contexto posmoderno: Las Relaciones Públicas ayudan a nuestra compleja sociedad pluralista a tomar decisiones y a funcionar más eficientemente, al contribuir al entendimiento mutuo entre grupos e instituciones. Sirven para proporcionar armonio al público y las instituciones públicas. 8/2 La Asamblea Mundial de las Relaciones Públicas de 1978 (Wilcox y otros, 2001: 12) definió esta disciplina como una “ciencia”: La práctica de las Relaciones Públicas consiste en el arte y la Ciencia social de analizar tendencias, predecir sus consecuencias, asesorar a los líderes de las organizaciones y aplicar programas planificados de acción que sirven tanto a la organización como al público. En un afán de síntesis, Rex Harlow (1976: 36) analizó 472 definiciones y compuso la siguiente, que reunía las virtudes de las anteriores: Las Relaciones Públicas son una función directiva característica que ayuda a establecer y mantener líneas de Comunicación mutuas, entendimiento, aceptación y cooperación entre una organización y sus públicos; implica la gestión de problemas o temas, ayuda a los REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-249 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú directivos a mantenerse informados y sensibles hacia la opinión pública; define y enfatiza en la responsabilidad de los directivos de servir al interés público; ayuda a los directivos a mantenerse al frente de los cambios y utilizarlos de forma efectiva contemplándolos como un sistema de aviso para anticiparse a las tendencias y utiliza la investigación, la preparación y la Comunicación ética como sus herramientas. Las Relaciones Públicas son connaturales al ser humano. Todos vivimos en sociedad y en sociedad nos comunicamos. A tal punto que la vida social no se puede entender sin los lazos que se tienden en los diálogos y contactos que establecemos en cada oportunidad en la que uno se encuentra con los otros mediante actos de buena fe. Nos realizamos como personas al ser provechosos para la comunidad y en virtud de ello, obtenemos el reconocimiento y respeto de los demás. Podemos concluir entonces en que las Relaciones Públicas componen una actividad profesional propia de las organizaciones definida como un sistema multidisciplinar a cargo de la alta dirección y que apoyada en el conocimiento científico, investiga a los públicos con los cuales se relaciona a fin de planificar, elaborar y ejecutar estrategias de comunicación para alcanzar la integración y el consenso. 8/2 1.3. Misión y objetivos de las Relaciones Públicas El fin de las Relaciones Públicas se manifiesta en el deseo por impedir y solucionar los conflictos que se presentan entre una organización y sus públicos, salvaguardando en todo momento la cohesión (Aguadero, 1993: 22). Podríamos agregar que “la legitimidad última de las relaciones públicas está en satisfacer el derecho a la información del público” (Soria, 1997: 270) pues sólo así podrá lograr la integración que pretende, fortaleciendo el “bien común” y la confianza de la opinión pública. No hay duda que la misión de las RR. PP. radica esencialmente en la formación del consenso en el corto o mediano plazo. Solano Fleta (1999: 41) asegura que la finalidad de esta disciplina se basa en dos puntos: La realización y mantenimiento de la cohesión social, mediante la actuación de aquellos procesos de comunicación intragrupales e intergrupales susceptibles de generar y mantener un alto grado de solidaridad entre los sujetos que los realizan. La satisfacción de los públicos demanda de las organizaciones no sólo el apoyo gerencial a la labor del profesional, sino también el fomento de un espíritu cooperativo y solidario. Este contexto favorable debe armonizarse con objetivos importantes, tales como el asesoramiento a la dirección, REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-250 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú la permanente investigación y análisis de ambientes en los que actúa la organización. Autores como Carlos Bonilla Gutiérrez (1988: 82-85), Román Pérez Senac y Emilio Solórzano Hernández (1999: 75), Francisco Aguadero Fernández (1993: 23) y Tony Greener (1995: 13-14) hacen hincapié en el rigor científico de los objetivos que condicionan el desarrollo de las RR. PP. Hemos reunido sus aportaciones en el siguiente cuadro: Cuadro nº 1. Objetivos de las Relaciones Públicas Carlos Bonilla Gutiérrez Román Pérez y Emilio Solórzano Francisco Aguadero Tony Greener - Investigación: Se busca conocer las opiniones, actitudes y respuestas de los públicos que se ven afectados directamente por las acciones y políticas de la organización. - Planeación y programación: Relacionar las opiniones, ideas, actitudes y reacciones de los públicos con las políticas y programas de la organización. - Comunicación: Aquí se razona y argumenta el itinerario elegido ante los públicos interesados y cuya cooperación es primordial. - Evaluación: En esta etapa se miden y examinan los resultados alcanzados con la ejecución del programa, demostrándose la efectividad de las técnicas empleadas. - Identificar y analizar las tendencias de las organizaciones y sus públicos. - Diagnosticar situaciones y predecir o pronosticar comportamientos. - Asesorar a los niveles directivos de las organizaciones en la administración de los procesos relacionales y comunicacionales, con la consiguiente participación en la formulación de políticas y estrategias adecuadas. - Evaluar el Balance social y responsabilidad pública de las mismas, para realimentar el proceso. - Controlar las actitudes y opiniones que los diferentes públicos tienen sobre la organización. - Asesorar a la más alta jerarquía en materia de comunicación, conducta y cultura organizacional. - Mantener informada a la dirección sobre las tendencias y los cambios en el endo y exoentorno socioeconómico y político. - Determinar, por medio de la investigación, las necesidades de comunicación en todas las áreas de la Organización y de esta con sus públicos. - Elaborar políticas de comunicación para todas las áreas. - La presentación positiva de una organización a todo su público para influir en lo que los demás piensan de ésta. - Edificar el prestigio de empresas y entidades e individuos, para incrementar su reconocimiento. - Reforzar la credibilidad del posicionamiento público de la empresa o su valía. - Ejecutar programas para alcanzar objetivos concretos. Fuente: Elaboración propia Al mantener un profundo contacto con la problemática sociocultural, las Relaciones Públicas no pueden descuidar los principios éticos y del bien común. Al emprender programas de cohesión e integración social, el comunicador-relaciones públicas tiene la enorme responsabilidad de orientar ade- 8/2 cuadamente la controversia pública. Al promover la participación interdisciplinaria, las Relaciones Públicas estudian, interpretan y aplican conocimientos, proponiendo opciones estratégicas y técnicas de convivencia, para ayudar así al desarrollo de las organizaciones y de sus actividades. REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-251 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú 2. Desarrollo de la teoría 2.1. En América El término “Relaciones Públicas” fue utilizado por primera vez en 1882 por Dorman Eaton durante una conferencia titulada The Public Relation and the duties of Legal Profession (Mercado, 2002: 44), aunque se pueden hallar algunos antecedentes en los llamados press-agents (agentes de prensa) liderados por Amos Kendall, miembro destacado del “Gabinete Kitchen, durante el gobierno de Andrew Jackson (18291937). A comienzos del siglo XX aparecería la figura de Ivy Ledbetter Lee, el “Padre de las Comunicación organizacional y de las Relaciones Públicas”. Periodista y acucioso reportero, Lee legaría su famosa “Declaración de Principios” (Solano, 1995: 156157) para esta profesión: Esto no es una oficina de prensa secreta. Nuestra labor se expone a la vista de todos. Nuestra finalidad es la de proporcionar noticias [...] La labor que desarrollamos en nombre de las empresas comerciales y de las instituciones públicas consiste en facilitar a la prensa y al público de los Estados Unidos materia informativa, rápida y precisa, sobre todo asunto, cuyo valor e interés le haga merecedor de ser conocido por ellos. Cabe destacar la contribución de Edgard Bernays. Sobrino de Sigmund Freud, asimiló muchas teorías provenientes de la 8/2 psicología social para comprender a los públicos. Fue uno de los primeros académicos en considerar a las Relaciones Públicas como una “ciencia social aplicada” que busca guiar y orientar el pensamiento y el comportamiento de las audiencias. Luego de la Segunda Guerra Mundial, las Relaciones Públicas se extienden por toda Europa y desde ese entonces no ha dejado de ganar espacios académicos en diferentes partes del mundo. Pese a su relativa juventud, esta disciplina cuenta con numerosas asociaciones, instituciones regulativas y foros oficiales. Gruning (2000: 7274) afirma que la evolución histórica permite identificar cuatro modelos de Relaciones Públicas: - Modelo ‘agente de prensa’ o Publicity: De carácter propagandístico, se caracteriza por el flujo unidireccional de la información a cargo de personal empírico. En la actualidad, está más ligado al mundo del espectáculo, promotores artísticos, representantes de teatro, entre otros. - Modelo de información pública: Aunque continúa con el sentido unidireccional, transmite mensajes objetivos. El relaciones públicas suele ser un periodista ligado a la institución. - Modelo asimétrico bidireccional: Emplea las teorías de las ciencias sociales a fin REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-252 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú de analizar las actitudes y las conductas de los públicos. Se rescata la utilidad del feedback o retroalimentación. el tema minero en los Andes. Si bien la temática es peruana, toda la bibliografía empleada fue estadounidense. - Modelo simétrico bidireccional: Los relaciones públicas son los mediadores entre la organización y sus públicos. La investigación social ya no responde sólo a la persuasión de los públicos, sino para lograr la comprensión y el entendimiento mutuo. Por su contenido, puede inscribirse dentro del modelo de Comunicación simétrico bidireccional. Esto se desprende del prólogo (Waples, 1959: 7) realizado por los editores de la Universidad de San Marcos: 2.2. En Latinoamérica (Perú) A inicios del siglo XX en nuestro país aún no se ha consolidado el vínculo entre la gran empresa privada y la universidad. Por ello, no sorprende los pocos títulos que se han escrito sobre las Relaciones Públicas o políticas de Responsabilidad Social. El trabajo más antiguo que registra la Biblioteca Nacional del Perú sobre Relaciones Públicas corresponde a un folleto editado en 1958 por Alberto Joffré Cousido (1958: 4). Un año después, la Universidad Nacional Mayor de San Marcos publicó el libro Los problemas de la Comunicación pública en el Perú, del sociólogo Douglas Waples. El libro aborda el tema de la Comunicación Pública en un primer capítulo. Luego trata el tema de la huelga bancaria acaecida en 1958 y de las estrategias comunicativas que se siguieron para aplacarla, acercando a las partes en conflicto. El tercer capítulo aborda el tema de la productividad. El cuarto capítulo menciona la visita de Nixon a Lima y el quinto y último capítulo enfoca 8/2 Su técnica no consiste en aplicar unas reglas y unos modelos a experiencias analizadas con fría circunspección, sino en ‘escuchar’ voces y almas, sentimientos, quejas y deseos, con la delicadeza de un espíritu libre para quien el cultivo de la Ciencia no tiene objeto si no es para servir una causa humana. Un año después se publica Relaciones Públicas, del estadounidense Howard Stephenson, profesor visitante de la Universidad Católica del Perú. Al laborar en el servicio diplomático de su país, Stephenson vislumbró el alcance de las políticas de Comunicación persuasiva, pese a no ser periodista. Su libro consta de cinco partes: 1) Relaciones Públicas: Poder y prestigio. 2) La opinión pública. 3) Los públicos. 4) Las comunicaciones. 5) La aplicación de Relaciones Públicas. Esta obra también presenta fuentes netamente norteamericanas y pertenece al modelo simétrico bidireccional. Podemos realizar esta afirmación sobre la base de la lectura de propio texto (1960: 4-5). En resumen podemos decir que nuestro enfoque es triangular. Un vértice es la Opinión Pública, otro es la Empresa, y, enlazando es- REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-253 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú tos dos vértices, tenemos las Comunicaciones en un doble sentido: de la Empresa hacia el público y del público hacia la Empresa. En 1962 se publica la primera Tesis de bachillerato sobre Relaciones Públicas, titulada: “Organización y funcionamiento del departamento de Relaciones Públicas en una empresa minera”. Su autor fue Ubaldo Velasco Alvarado (1962: 16), quien se licenció por la Universidad Nacional Mayor de San Marcos. Este autor, empero, no pertenecía a la carrera de Ciencias de la Comunicación, sino más bien era alumno de la Facultad de Ciencias Económicas y Comerciales. En 1965 aparece la primera publicación peruana: Fundamentos de las Relaciones Públicas, de Francisco Flores Bao. Además de ser pionero en la materia, es el autor que más libros sobres Relaciones Públicas ha publicado en nuestro país. Sin embargo, no poseía formación de comunicador social: cursó estudios de Derecho, Filosofía y Ciencias Sociales en la Universidad Nacional Mayor de San Marcos. Efectuó, asimismo, un postgrado en Ciencias Administrativas en la Universidad de Roma. En este primer período es clara la influencia norteamericana, así como la presencia de científicos sociales, administradores y gente dedicada a las Ciencias Económicas, antes que a las Ciencias de la Comunicación propiamente dicha. La escasa producción bibliográfica respondió a la “mentalidad rentista” del empresario peruano, poco preocupado por sus públicos. En la siguien8/2 te década, el mercado estaría sobreprotegido y los empresarios se esforzaron poco para posicionar sus productos, ya que no tenían competencia externa. De esta forma, resultó innecesaria una relación óptima con el cliente, pues a éste no le quedaba otra que consumir productos peruanos, creando así en las empresas una idea de falsa viabilidad. En 1971, se publica el opúsculo Las Relaciones Públicas en la Guardia Civil del Perú, de Pérez Astete. En el siguiente año se seguiría otro a cargo de un organismo público: Relaciones Públicas en la Empresa de Saneamiento de Lima. En San Marcos se defiende la tesis “Investigación de Actitudes en la Dirección de Correos y Telégrafos”, de Temístocles Armendáriz Cuba de Piérola. Francisco José del Solar Rojas emprendería su producción bibliográfica con las obras Nuevos conceptos sobre Relaciones Públicas (1972) e Importancia de las Relaciones Públicas en el proceso de cambio (1973). Entre 1977 y 1978 aparecieron los primeros manuales de consulta de Javier Vela, Hernández Urbina, Alfieri Olcese Salvatecci y Gildomero Arista. Cabe destacar el Vocabulario de Relaciones Públicas, términos usuales en esta moderna especialidad (1978), de Roberto Bustamante Flores. Durante la década de 1980 aparecen con frecuencia las publicaciones de Flores Bao, Del Solar Rojas y otros autores, como Enrique Estremadoyro Escobar (Relaciones Públicas y la empresa) y Justo Avellaneda (Relaciones Públicas). REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-254 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú Durante la siguiente década, la disciplina experimentaría un acercamiento con las editoras universitarias, dotándolas de un carácter más científico y de metodologías de investigación más exhaustivas. La Universidad Nacional San Antonio Abad del Cuzco publica Relaciones Públicas en el contexto de la Comunicación (1990), de Ortiz Castillo. La Universidad de San Martín de Porres impulsa la publicación de Relaciones Públicas de Latinoamérica y del Perú, rumbo al siglo XXI (1993), de Emilio Solórzano Hernández. La Universidad Inca Garcilaso de la Vega hace lo propio con Protocolo, Ceremonial del Estado y Relaciones Públicas (1994), de José Francisco del Solar Rojas. A estos libros le seguirían Relaciones Públicas, calidad total y factor humano (1996), de Fernando Calderón Quino; La Ciencia de las Relaciones Públicas, de Francisco del Solar Rojas (1996); y Relaciones Públicas Superiores (1999), de Román Pérez Senac y Emilio Solórzano; así como la importante publicación de las actas del Seminario internacional “Relaciones Públicas: Herramienta Básica para la Gestión Empresarial Moderna” (1995), del primer y segundo Foro Iberoamericano de Relaciones Públicas (1996 y 1998). 3. Aplicación y desempeño 3.1. Perfil del Comunicador Relaciones públicas públicas es vital para la formación de una “filosofía grupal”, orientada a establecer comunicaciones efectivas y productivas. Como mediador e intérprete del mensaje, este profesional se aboca al moldeamiento de la imagen e identidad de la organización (Rojas Guzmán, 2000: 59). Asimismo, participa en el desarrollo de los objetivos generales de la empresa, teniendo la capacidad de proponer programas a la dirección general, gestionando su presupuesto y ejecutándolo. El acercamiento a los públicos es fundamental. Según Salvador Mercado (2002: 51), el profesional busca influir en la opinión pública mediante una comunicación atractiva y persuasiva. Mientras mayor sea el grado de entendimiento, habrá menos cabida para la desinformación, el desconocimiento o la malinterpretación de las intenciones organizacionales. En ese sentido, Philip Boiry (2000: 7) sostiene que el área de comunicaciones no puede quedarse relegada dentro del organigrama, puesto que la labor del relaciones Gil Tovar (1984: 87-88) lo sitúa entre los intereses generales de la empresa y la de los públicos, mostrando siempre un profundo conocimiento de la psicología y la idiosin- 8/2 REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-255 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú crasia de la comunidad en la que trabaja. Para proyectar una imagen “auténtica”, este profesional debe velar que la organización actúe siempre de manera consecuente y coherente con sus propios objetivos y creencias. 3.2. Estrategias de Comunicación organizacional Dentro de las Relaciones Públicas, el flujo comunicativo entre la organización y sus públicos es de carácter formal (el emisor es la gerencia y utiliza los canales establecidos), instrumental; (su propósito es que los receptores actúan o respondan al mensaje); directo (“cara a cara”), indirecto (que requiere el empleo de un medio técnico) y bidireccional Debido a la complejidad de los públicos y a la configuración particular de las empresas, las estrategias deben ser resultado de una investigación y responder a escenarios determinados, que exigen la planificación ante cualquier crisis o inconveniente (“comunicación preventiva”) o la resolución ante un problema (“Comunicación correctiva”). Así, mediante la programación, dirección y realización sistemática de acciones las Relaciones Públicas adquieren su verdadera dimensión e importancia dentro de la organización. Con esa metodología, advierte Rojas Guzmán (2000: 58), esta comunicación permitirá que se logren objetivos comunes y mensajes más significativos. 8/2 Según Barquero Cabrero (2001: 146-147) toda estrategia comunicativa dependerá de la disposición y graduación adecuada de ocho puntos (las “8 C‟s”) que detallamos a continuación: - Credibilidad: Dependerá de la fuente informativa, cuyo propósito principal es el servir al receptor. - Confianza: Consecuencia de lo anterior. No hay comunicación posible si el receptor no confía en el emisor. - Contexto: Conocimiento de las circunstancias y adaptarse a las mismas. - Contenido: el mensaje debe ser de interés e importancia para el receptor, debe significar algo para éste. - Claridad: El mensaje debe ser claro y sencillo. - Continuidad: El mensaje debe ser constante y coherente en sus propuestas. Puede cambiar su forma, pero no su contenido. - Canal de comunicación: Deben utilizarse aquellos que permitirán que el mensaje llegue al público objetivo. - Capacidad del auditorio: mientras menor es el esfuerzo que realiza el receptor para entender el mensaje, mayor será su efectividad. Para Salvador Mercado (2002: 52), el éxito dependerá de la carga persuasiva que quiera plasmarse en los objetivos finales. En ese sentido, el cambio de actitud será posible si se cumplen los siguientes puntos: REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-256 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú - Si existe armonía entre la propuesta y las normas y lealtades de grupo. - Cuando la propuesta toma en consideración las necesidades e impulsos circunstanciales de la personalidad. - Cuando se considera que sus fuentes son fidedignas y facultativas. - La propuesta presentada a través de los medios masivos, aunque reforzada por el contacto directo. - Cuando la propuesta va acompañada por otros factores determinantes de las creencias y de la actitud. La metodología (Ibídem: 37-38) sería la siguiente: a) un análisis del material preliminar, que consiste en el sondeo de opiniones, actitudes y reacciones de las audiencias ante los actos o política de la empresa; b) la elaboración de objetivos de comunicación; c) síntesis y análisis de los mensajes, a fin de evaluar sus efectos; y d) relación mutua entre los elementos (feed- back), ya que el público reconoce en el mensaje sus sentimientos, impulsos, entre otros. Los frutos de esta planificación son incalculables. Ricardo Homs (1990: 67-69) sostiene que además de la integración de los públicos, también se lograría cultivar un sano “sentimiento de orgullo” de los empleados (al sentirse identificados con la identidad de la organización), la práctica de un “trato humano” y personalizado que instituya un clima de comprensión entre quienes integran la empresa; la mejora de canales de comunicación internos y la administración efectiva de conflictos. Las metas más ulteriores girarían en torno a la profundización del conocimiento de la empresa; la agilización de la producción gracias a un mejor clima organizacional; moldear canales “horizontales” de comunicación, que permitan a cualquier trabajador a expresarse ante la dirección general. 4. Nuevas tendencias 4.1. Retos profesionales El objetivo de las Relaciones públicas no es fabricar imágenes simpáticas de las empresas y los empresarios para venderlos a la opinión pública. Ante esa simplificación teórica, creemos esta práctica multidisciplinaria intenta resolver los conflictos emergentes entre las instituciones y los 8/2 públicos. La promoción de la integración, en todos los niveles de la sociedad, ayudaría a alcanzar la productividad y la excelencia (en el entorno de la empresa), alentando el progreso y el bienestar en la nación. Un buen profesional es consciente de los siguientes factores: - El conflicto es inminente: La falta de información o cualquier descuido en el REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-257 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú tratamiento de la controversia puede desencadenar en crisis. tos de aprobación en los públicos, borrándose así las influencias desorientadoras. - Investigación continua: Para conocer el contexto y a las comunidades a tratar. En ese sentido, la configuración de una comunicación de calidad sería fundamental. No hay duda que la excelencia es vital para diferenciarse de la competencia dentro de un mercado relativamente homogéneo. Esta metodología gerencial busca aumentar el valor de los productos/servicios, así como alcanzar o exceder las expectativas del cliente. Se tendrían que adoptar los siguientes pasos: - Empleo de medios: Que faciliten el flujo informativo y el posicionamiento de la organización. En la actualidad, las Relaciones Públicas exigen sus profesionales que no se queden en el discurso: reclaman idoneidad necesaria para asesorar en la definición de los valores, filosofía, política y estrategias de integración. También, sería necesario contar con una adecuada formación académica y práctica, además de ser carismático y ser dueño de un tacto especial en casos urgentes y delicados. 4.2. Comunicación de calidad Si el público no recibe una información veraz, oportuna y directa de la empresa, el resultado será la proliferación de rumores y malentendidos que terminarán por lesionar las buenas relaciones que se pudieran haber logrado. Para evitar esa situación negativa, la comunicación debe procurar generar mensajes más persuasivos. Como hemos mencionado anteriormente, las Relaciones Públicas no sólo pretenden transmitir ideas con un propósito meramente informativo, sino que aspira a persuadir a los destinatarios. Al persuadir de forma positiva, se logra formar sentimien- 8/2 - Invertir en la formación del empleado y en los procesos de producción. - Conocer y mantenerse cercano a las reacciones de los clientes. - Fuerte liderazgo del equipo gerencial hacia la calidad. 4.3. Empleo de las TIC Las nuevas tecnologías de la información y la comunicación (TIC) son herramientas indispensables en el radio de acción del relaciones públicas. En esta “sociedad red”, Internet se convierte en un canal importante, ya que concentra la palabra escrita, imágenes, sonidos y vídeos. Además, tiene la capacidad de transmitir directamente sus mensajes a un número ilimitado de receptores en cualquier momento o lugar. Gracias a la “red de redes”, el siglo XXI se caracteriza por la velocidad en las transacciones, en el acceso inmediato a la información y en la distribución efectiva de la REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-258 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú misma a todos los miembros de la organización que la necesiten. El funcionamiento de este “sistema nervioso digital” ayudará a la gestión y a la toma de decisiones, así como a un contacto más cercano y atento con los clientes. - Convertir los trámites sobre papel en procesos digitales para agilizar la acción empresarial. Entre otros efectos, podemos mencionar los siguientes: En esta coyuntura, los profesionales de Relaciones Públicas son los responsables de familiarizar a los clientes y/o colegas en la empresa en el uso de esta nueva y espectacular herramienta de gestión, pues permite la comunicación permanente e interactiva. En consecuencia, se estaría cristalizando el modelo bidireccional: gracias al feedback, el contacto es de doble vía y personalizado. - Canalización de la comunicación interna por correo electrónico. - Analizar online los datos comerciales para detectar faltas y compartir las revelaciones con prontitud. Así se interpretarán las tendencias generales. - Usar sistemas digitales para encaminar inmediatamente los reclamos del cliente y mejorar el servicio. Conclusiones Al establecerse la red digital, la empresa combinaría los beneficios de la tecnología y el humanismo. Exhibiría las siguientes características: En síntesis, las Relaciones públicas pueden interpretarse como: - Estaría fundada en la dignidad de la persona. - Un nuevo concepto filosófico de cómo conducir una empresa, basado en la función social que cumple en la comunidad. - Tomaría decisiones interfuncionales y compromisos compartidos. - La humanización de la función administrativa. - Prevalecería la cultura sobre los procedimientos. - La información que crea conocimiento y confianza, mediante técnicas adecuadas. - Tomaría más atención a la percepción de la Comunicación que a la emisión. - Reconocería que su principal recurso es el capital humano. 8/2 - El proceso de la conducta humana basado en la integración, comprensión y buena voluntad. REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-259 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú - La aplicación radical de las modernas técnicas publicitarias y de las ramas afi- nes para hacer conocer una empresa o los servicios que presta. Referencias ADECEC (2002) La Comunicación y Relaciones Públicas en España. Radiografía de un sector. Barcelona, Pirámide BUSTAMANTE FLORES, R. (1978) Vocabulario de Relaciones Públicas, términos usuales en esta moderna especialidad. Lima, Unidas. AGUADERO FERNÁNDEZ, F. (1993) Comunicación Social Integrada: Un reto para la organización. Barcelona, Consejo Superior de Comunicación y Relaciones Públicas de España. CALDERÓN QUINO, F. (ed.) (1995) Relaciones Públicas: Herramientas para la gestión empresarial moderna. Lima, Universidad de San Martín de Porres. 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Recuperado (Fecha de acceso), de http://www.icono14.net REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-262 Amybel Nila Walther Sánchez: Evolución social de las Relaciones Públicas en el Perú NOTAS i De acuerdo con Jürgen Habermas (1998: 367-369), “consenso” sería el producto del entendimiento que se da gracias al diálogo entre interlocutores dispuestos a entenderse mediante el uso natural del lenguaje. 8/2 REVISTA ICONO 14 – A8/V2 – pp. 243/263 \| 07/2010 \| REVISTA DE COMUNICACIÓN Y NUEVAS TECNOLOGÍAS \| ISSN: 1697–8293 C/ Salud, 15 5º dcha. 28013 – Madrid \| CIF: G - 84075977 \| www.icono14.net 1-263
https://openalex.org/W2953071745	https://hal.science/hal-01806906/file/msy015.pdf	English	null	Codon Usage Bias in Animals: Disentangling the Effects of Natural Selection, Effective Population Size, and GC-Biased Gene Conversion	Molecular biology and evolution	2,018	cc-by	12,515	Codon Usage Bias in Animals: Disentangling the Effects of Natural Selection, Effective Population Size, and GC-Biased Gene Conversion Ni l G lti C ill R M j l i R ll J th R i i Codon Usage Bias in Animals: Disentangling the Effects of Natural Selection, Effective Population Size, and GC-Biased Gene Conversion Nicolas Galtier, Camille Roux, Marjolaine Rousselle, Jonathan Romiguier, Emeric Figuet, Sylvain Glemin, Nicolas Bierne, L. Duret Galtier, Camille Roux, Marjolaine Rousselle, Jonathan Romiguier, Emeric Figuet, Sylvain Glemin, Nicolas Bierne, L. Duret To cite this version: Nicolas Galtier, Camille Roux, Marjolaine Rousselle, Jonathan Romiguier, Emeric Figuet, et al.. Codon Usage Bias in Animals: Disentangling the Effects of Natural Selection, Effective Population Size, and GC-Biased Gene Conversion. Molecular Biology and Evolution, 2018, 35 (5), pp.1092 - 1103. 10.1093/molbev/msy015. hal-01806906 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01806906 https://hal.science/hal-01806906v1 Submitted on 16 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 Abstract Selection on codon usage bias is well documented in a number of microorganisms. Whether codon usage is also generally shaped by natural selection in large organisms, despite their relatively small effective population size (Ne), is unclear. In animals, the population genetics of codon usage bias has only been studied in a handful of model organisms so far, and can be affected by confounding, nonadaptive processes such as GC-biased gene conversion and experimental artefacts. Using population transcriptomics data, we analyzed the relationship between codon usage, gene expression, allele frequency distribution, and recombination rate in 30 nonmodel species of animals, each from a different family, covering a wide range of effective population sizes. We disentangled the effects of translational selection and GC-biased gene conversion on codon usage by separately analyzing GC-conservative and GC-changing mutations. We report evidence for effective translational selection on codon usage in large-Ne species of animals, but not in small-Ne ones, in agreement with the nearly neutral theory of molecular evolution. C- and T-ending codons tend to be preferred over synonymous G- and A-ending ones, for reasons that remain to be determined. In contrast, we uncovered a conspicuous effect of GC-biased gene conversion, which is widespread in animals and the main force determining the fate of AT$GC mutations. Intriguingly, the strength of its effect was uncorrelated with Ne. Key words: synonymous codon usage, GC-content, recombination, evolution, gene expression, nonmodel organisms. Key words: synonymous codon usage, GC-content, recombination, evolution, gene express us codon usage, GC-content, recombination, evolution, gene expression, nonmodel organisms Article Codon Usage Bias in Animals: Disentangling the Effects of Natural Selection, Effective Population Size, and GC-Biased Gene Conversion Nicolas Galtier,,1 Camille Roux,1,2,3 Marjolaine Rousselle,1 Jonathan Romiguier,1,2 Emeric Figuet,1 Sylvain Glemin,1,4 Nicolas Bierne,1 and Laurent Duret5 1UMR5554, Institut des Sciences de l’Evolution, University Montpellier, CNRS, IRD, EPHE, Montpellier, France 2Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland 3UMR 8198 – Evo-Eco-Paleo, CNRS, Universite de Lille—Sciences et Technologies, Villeneuve d’Ascq, France 4Department of Ecology and Genetics, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden 5Laboratoire de Biometrie et Biologie Evolutive, UMR 5558, CNRS, Universite de Lyon, Universite Lyon 1, Villeurbanne, France Corresponding author: E-mail: nicolas.galtier@univ-montp2.fr. Associate editor: Nadia Singh Downloaded from https://academic.oup.com/mbe/article/35/5/1092/4829954 by Bibliothèque Universitaire de médecine - Nîmes user on 16 June 2021 The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com ol. Biol. Evol. 35(5):1092–1103 doi:10.1093/molbev/msy015 Advance Access publication January 30, 201 Introduction Please note, however, that the population genetic analyses of codon usage bias so far have only been conducted in a relatively small number of species of animals. on synonymous positions can be very difficult to disentangle from coding sequence analysis only (Jackson et al. 2017, but see Clement et al. 2017). gBGC, however, is expected to apply more strongly to highly recombining regions, and to affect non coding, flanking sequences as well as coding ones, unlike translational selection. p Subramanian (2008) analyzed codon usage bias intensity across 20 species of eukaryotes and reported a higher bias in short generation time, presumably large-Ne species than in long generation time, presumably small-Ne ones. This author questioned the Ne hypothesis and rather suggested that s might vary among species. According to his hypothesis, the selective pressure on translation efficiency would be stronger in fast growing species. Interestingly, growth rate seems to be the main determinant of among-species variation in codon usage bias intensity in bacteria (Rocha 2004; Sharp et al. 2005; Vieira-Silva and Rocha 2010). On the other hand, Machado et al. (2017) argued that s is not necessarily constant across synonymous codons and mutations. Comparison of poly- morphism and divergence patterns in Drosophila mela- nogaster indeed suggested that both strong (4Nes 1) and weak (4Nes1) selection applies on synonymous sites in this species (Lawrie et al. 2013; Machado et al. 2017). It is also well established that in many species selection for optimal trans- lation is stronger in highly expressed than in lowly expressed genes (Gouy and Gautier 1982; Duret and Mouchiroud 1999), and even in humans there is documented evidence for a phenotypic effect of specific synonymous mutations (Sauna and Kimchi-Sarfaty 2011). The question “does codon usage affect translational efficiency in species X” should therefore probably be rephrased as “what fraction of synonymous mutations in species X is effectively selected?,” and “how does Ne influence this fraction?” Preferred codons are usually defined as codons used more frequently in high-expressed than in low-expressed genes (Duret and Mouchiroud 1999). This could be problematic in practice because DNA or cDNA libraries generated for high throughput sequencing are known to be biased with respect to sequence base composition (Dohm et al. 2008; Aird et al. 2011). The GC-richest and GC-poorest fractions of target DNA are typically underrepresented, and sequences of medium GC-content overrepresented, in Illumina data (Choudhari and Grigoriev 2017). Introduction This experimental bias, if not properly taken into account, could corrupt the definition of preferred codons in generating false correlations between codon usage and sequencing coverage. The bias apparently varies between experiments and libraries, which makes it dif- ficult to model and correct for (Benjamini and Speed 2012). GC-content is therefore a potential confounder of analyses of selection on codon usage, both biologically and methodologically. Current knowledge on codon usage biases in animals is therefore limited in at least two respects. First, published analyses have so far focused on a relatively small number of species—mainly model organisms—and an even smaller number of taxa—mainly drosophilids and vertebrates. Secondly, the confounding effects of gBGC and experimental biases have not always been taken into account. We therefore lack a global picture of the relative impact of selection, drift, and gBGC on codon usage evolution in animals. Here, we analyzed a data set covering 30 nonmodel species of animals. In each species, the transcriptomes of five to eleven diploid individuals plus one outgroup have been previously se- quenced (Romiguier, Gayral, et al. 2014). In principle transcriptome-based population, genomic data are ideal for codon usage bias analysis in providing access to codon usage tables, gene expression level, allele frequencies at polymorphic positions, and flanking UTR sequences. We focused our anal- ysis of translational selection on GC-conservative pairs of syn- onymous codons—that is, codons differing by a G$C or an A$T substitution—and separately analyzed the effect of gBGC. We found that translational selection on codon usage is only detectable in short-lived, large-Ne species, whereas gBGC is widespread across animals and of strength apparently independent of Ne. Besides selection and drift, patterns of codon usage might be influenced by neutral, directional forces such as mutation biases and GC-biased gene conversion (gBGC), a recombination-associated segregation bias that favors G and C over A and T alleles in high recombining regions (Duret and Galtier 2009; Mugal et al. 2015). The existence of gBGC has been experimentally demonstrated in yeast (Mancera et al. 2008; Lesecque et al. 2013), humans (Williams et al. 2015; Halldorsson et al. 2016), flycatcher (Smeds et al. 2016), and Daphnia (Keith et al. 2016). gBGC has been identified as the main driver of GC-content evolu- tion in vertebrates (Duret and Galtier 2009; Figuet et al. 2014; Glemin et al. 2015; Bolıvar et al. 2016) and several other taxa (Pessia et al. 2012; Glemin et al. Introduction particular, it is expected that, in small populations, the ran- dom fluctuations of allele frequencies due to genetic drift should decrease the efficiency of natural selection and lessen the efficiency of selection on codon usage. The theory pre- dicts that if Ne, the effective population size, is sufficiently small such that the 4Ne s product is much <1, s being the selection coefficient in favor of optimal codons, then the ef- fect of selection should be negligible. One question of interest, therefore, is whether selection shapes codon usage in large organisms, such as animals, the same way as in microbes, despite their presumably smaller Ne. The reasons why synonymous codons do not occur at equal frequencies in protein coding sequences have been puzzling molecular evolutionary researchers for decades (Duret 2002; Hershberg and Petrov 2008). One fascinating aspect is the early discovery that, in various microbial genomes, codon usage responds to natural selection. In Escherichia coli and Saccharomyces cerevisiae, for instance, the codons most com- monly observed in highly expressed genes match the most abundant tRNAs in the cell (Ikemura 1985), strongly suggest- ing that codon usage and tRNA content have coevolved in a way that optimizes translation—hence the term “translational selection.” These observations promoted codon usage bias as a textbook example of a weak selective pressure operating at molecular level, detectable from patterns of cod- ing sequence variation, but difficult to apprehend experimentally. Evidence for selection on codon usage has been reported in fruit flies (Shields et al. 1988; Akashi 1994; Bierne and Eyre- Walker 2006), in the nematode Caenorhabditis elegans (Duret and Mouchiroud 1999), and in the branchiopod Daphnia pulex (Lynch et al. 2017). In contrast, codon usage in mam- mals is primarily governed by within-genome variation in GC- content, and only weakly, if at all, correlated to gene expres- sion and tRNA content (Semon et al. 2006; Rudolph et al. 2016; Pouyet et al. 2017, see also Doherty and McInerney Because selection on codon usage is presumably weak, other evolutionary forces might contribute to explain its var- iation across genes and genomes (Sharp and Li 1986). In 1092 MBE Codon Usage Bias in Animals . doi:10.1093/molbev/msy015 2013). The effectiveness of selection on codon usage in small- sized invertebrates but not in large-sized vertebrates is super- ficially in agreement with the hypothesis of a Ne effect. Introduction 2014; Wallberg et al. 2015). In many respects, the expected impact of gBGC on patterns of sequence variation is similar to that of directional selection. For instance, the expected fate and frequency distribution of an allele promoted by gBGC is identical to that of a favorable allele under codominant selection (Nagylaki 1983). Importantly, the so-called “preferred” synonymous codons—that is, codons more frequently used in high- expressed genes—often end with C or G, D. melanogaster being an extreme example in which all the preferred codons are C- or G-ending (Duret and Mouchiroud 1999). This implies that the effects of translational selection and gBGC Results GC-Changing versus GC-Conservative Mutations In each of the 30 focal species, we correlated GC12, GC3, and GC_UTR across genes. The three measures of GC-content were strongly correlated with each other in nearly all species (supplementary table S1, Supplementary Material online). Analyzing polymorphism data, we calculated the mean allele frequency of the G or C allele at synonymous, AT$GC SNPs. 1093 MBE Galtier et al. . doi:10.1093/molbev/msy015 ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● 0.3 0.4 0.5 0.6 0.7 0.3 0.4 0.5 0.6 0.7 a. AT/GC SNPs, mean GC allele frequency synonymous UTR r2=0.43, p<10−5 ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ● ●●● ● ●● ● ● ● ● ● ● ● 0.3 0.4 0.5 0.6 0.7 0.3 0.4 0.5 0.6 0.7 b. A/T or G/C SNPs, mean CT allele frequency synonymous UTR r2=0.004, NS FIG. 1. Mean allele frequency at GC-changing and GC-conservative SNPs. Each dot represents a species. X axis: synonymous SNPs; Y axis: ﬂanking noncoding SNPs. (a) Mean allele frequency of G or C alleles at AT versus GC SNPs. (b) Mean allele frequency of T or C alleles at A versus T and G versus C SNPs. , mean GC allele frequency mean CT allele frequency Downloaded from https://academic.oup.com/mbe/article/35/5/1092/4829954 by Bibliothèque Universitaire de médecine - Nîmes user on 16 June 2021 synonymous FIG. 1. Mean allele frequency at GC-changing and GC-conservative SNPs. Each dot represents a species. X axis: synonymous SNPs; Y axis: ﬂanking noncoding SNPs. (a) Mean allele frequency of G or C alleles at AT versus GC SNPs. (b) Mean allele frequency of T or C alleles at A versus T and G versus C SNPs. be associated with promoters of active genes (Baker et al. 2017). In any case, be it artefactual or real, the correlation between GC-content and expression can confound the iden- tification of preferred codons. The mean frequency of GC alleles was >0.5 in a majority of species, which is not expected from sequences at mutational equilibrium (Glemin et al. 2015). We applied the exact same analysis to UTR sequences and found that the mean allele frequency of GC alleles in UTRs was strongly correlated to the mean allele frequency of GC alleles at synonymous sites across species (fig. 1a). Results This shows that the evolutionary fate of AT!GC and GC!AT mutations at third codon positions is primarily governed by forces similarly impacting noncoding DNA, that is, independent of selection on codon usage. We reproduced the analysis using G$C and A$T polymor- phisms instead of AT$GC ones and obtained a very different picture (fig. 1b): the frequency of C and T alleles showed no significant correlation between UTRs and third codon posi- tions. These observations are consistent with the hypothesis that the GC-content of genes can be affected by gBGC, both in UTRs and at synonymous sites and hence that this process has to be taken into account to investigate signatures of selection on codon usage. Preferred Synonymous Codons We therefore decided to focus our analysis of codon usage on synonymous codon pairs of the form XYA/XYT or XYG/XYC. There are 17 such GC-conservative pairs of synonymous codons in the standard genetic code—two per 4- or 6-fold degenerate amino acid, and the isoleucine-coding ATA/ATT pair. For each pair of GC-conservative codon and each CDS, we calculated the relative frequency of the C- or T-ending codon as fXYC=ðfXYC þ fXYGÞ or fXYT=ðfXYT þ fXYAÞ; Furthermore, one key point for investigating translational selection is to correctly characterize preferred codons. The classical approach consists in identifying codons whose fre- quency increases with expression level. However, in a majority of species, we observed significant relationships, either posi- tive or negative, between expression level (inferred from se- quencing depth) and GC_UTR (supplementary table S1, Supplementary Material online). The hypothesis of selection on codon usage bias does not predict any relationship be- tween GC_UTR and expression. As mentioned in Introduction, these correlations might reflect artefacts, result- ing from the well documented GC-bias in Illumina libraries (Benjamini and Speed 2012). It is also possible that these correlations reflect covariations between expression level and recombination rate (and hence intensity of gBGC). For instance, in humans, intragenic recombination rates (and GC3) correlate negatively with expression level in meiotic cells (Pouyet et al. 2017), whereas in vertebrate species lacking PRDM9, on the contrary, recombination hotspots tend to where fXYZ is the frequency of occurrence of codon XYZ in the considered CDS (X, Y, and Z in {A; C; G; T}). We correlated these with gene expression level. The corresponding correla- tion coefficient, hereafter called rPYR (for pyrimidine), were taken as measures of preference for codons XYC or XYT, compared with XYG or XYA. This was done independently in 17 GC-conservative codon pairs and 30 species, that is, 510 correlation analyses. Supplementary figure S1, Supplementary Material online, shows a heatmap of the distribution of rPYR across codon pairs and species. Of the 510 estimated rPYR, 441 (86%) were positive, indi- cating a general preference for C over G and for T over A at synonymous positions in animals. We arbitrarily called “preferred” those codons for which rPYR was >0.05 (or below 0.05) and significantly different from zero with a P value <0.001. We identified 117 preferred codons out of 510 codon pairs, of which 64 were C-ending, 43 were T-ending, seven were A-ending and three were G-ending. Codon Usage Population Genetics For each pair of GC-conservative, synonymous codons, we extracted the corresponding biallelic SNPs and recorded allele frequencies, independently in the 30 species. We first focused on the 117 preferred codons as defined above, and found that their average allele frequency, 0.546, was significantly >0.5 (standard error of the mean estimate: 0.015). In contrast, the mean allele frequency of codons weakly correlated to gene expression (abs(rPYR)<0.05 or P> 0.001) was 0.508 and not significantly different from 0.5. FIG. 2. Number of preferred codons among 17 GC-conservative co- don pairs. Each dot is for a species. Codons are called preferred when their prevalence is correlated with gene expression with correlation coefﬁcient >0.05 and P value <0.001. Plain dots: gene expression ¼ sequencing depth of coverage. Open dots: gene expression ¼ residual of the regression of sequencing depth of coverage on GC12. In order to extract information from the entire data set, we recorded MAJ, the binary variable equal to one when the frequency of the C or T allele was >0.5 and zero when it was <0.5, SNPs at which allele frequency was exactly 0.5 being disregarded. This was done independently in the 30 species. Then we pooled the data across SNPs, codons, and species and performed a logistic regression of MAJ on the correlation between codon usage and expression, rPYR. In this analysis, the number of data points equalled the total (across species and GC-conservative codon pairs) number of SNPs at which allele frequency was different from 0.5. Detailed data are provided in supplementary table S3, Supplementary Material online. The logistic regression is appropriate here because it accounts for unequal SNP sample size among species and codon pairs. be preferred over purines as far as GC-conservative synony- mous codons are concerned in animals. The number of pre- ferred codons varied across species from zero (in nine different species) to 13 (in C. brenneri), and was negatively correlated to species propagule size (n¼ 30, r¼0.59, P¼ 0.00054; fig. 2), suggesting that translational selection on codon usage is more efficient in large Ne species. This correlation was robust to a control for phylogenetic indepen- dence (P¼ 0.006). We performed a similar analysis this time focusing on the 29 synonymous codon pairs of the form XYG/XYA or XYC/ XYT. Preferred Synonymous Codons Pyrimidines tend to 1094 MBE Codon Usage Bias in Animals . doi:10.1093/molbev/msy015 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −2 −1 0 1 2 0 5 10 15 log10(propagule size) nb preferred codons ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● FIG. 2. Number of preferred codons among 17 GC-conservative co- don pairs. Each dot is for a species. Codons are called preferred when their prevalence is correlated with gene expression with correlation coefﬁcient >0.05 and P value <0.001. Plain dots: gene expression ¼ sequencing depth of coverage. Open dots: gene expression ¼ residual of the regression of sequencing depth of coverage on GC12. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −2 −1 0 1 2 0 5 10 15 log10(propagule size) nb preferred codons ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● the uncorrected one. The corrected and uncorrected num- bers of preferred codons were almost perfectly correlated across species (n¼ 30; r¼ 0.97; P< 1015), and both were significantly correlated with species propagule size (fig. 2 and supplementary fig. S2, left, Supplementary Material online). XYG/XYA and XYC/XYT codon pairs, however, behaved quite differently. GC12-corrected and uncorrected numbers of preferred codons were less strongly correlated with each other (n¼ 30; r¼ 0.73; P< 105), and not significantly re- lated to propagule size (supplementary fig. S2, right, Supplementary Material online), confirming the confounding effect of GC-content. Given the sensitivity of XYG/XYA and XYC/XYT codon usage to GC-biases, we below restricted our analysis of codon preference on GC-conservative codon pairs. Codon Usage Population Genetics 36years) and aver pN/pS (0089 vs 016) ● ● ● ● ● ● ● ● ● ●●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● −0.2 −0.1 0.0 0.1 0.2 0.2 0.4 0.6 0.8 b. Small Ne species codon usage/expression correlation mean allele frequency r2=0.04, NS ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● −0.2 0.0 0.1 0.2 0.2 0.4 0.6 0.8 a. Large Ne species codon usage/expression correlation mean allele frequency r2=0.50, p<10−11 FIG. 3. Intensity of selection on codon usage bias. Each dot represe a particular pair of synonymous codon in a particular species. O GC-conservative pairs of synonymous codons for which a minim of 20 SNPS are available are considered. X axis: rPYR, the correlat coefﬁcient between C or T usage and gene expression. Y axis: m frequency of the C or T allele. (a) Species in which propagule siz <0.2 mm. (b) Species in which propagule size is >2 mm. Red/da dots correspond to the nematode Caenorhabditis brenneri. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● −0.2 0.0 0.1 0.2 0.2 0.4 0.6 0.8 a. Large Ne species codon usage/expression correlation mean allele frequency r2=0.50, p<10−11 a. Large Ne species 20 species in which RNA had been extracted from at least four distinct tissues or the whole animal body (supplemen- tary table S1, Supplementary Material online). We repro- duced the above analyses and obtained very similar results (supplementary fig. Codon Usage Population Genetics S3, Supplementary Material online), indi- cating that our report of a link between propagule size, codon usage, and gene expression is not affected by tissue choice. Insect Data Analysis X axis: rPYR, the correlation coefﬁcient between C or T usage and gene expression. Y axis: mean frequency of the C or T allele. (a) Species in which propagule size is <0.2 mm. (b) Species in which propagule size is >2 mm. Red/darker dots correspond to the nematode Caenorhabditis brenneri. FIG. 3. Intensity of selection on codon usage bias. Each dot represents a particular pair of synonymous codon in a particular species. Only GC-conservative pairs of synonymous codons for which a minimum of 20 SNPS are available are considered. X axis: rPYR, the correlation coefﬁcient between C or T usage and gene expression. Y axis: mean frequency of the C or T allele. (a) Species in which propagule size is <0.2 mm. (b) Species in which propagule size is >2 mm. Red/darker dots correspond to the nematode Caenorhabditis brenneri. 8.5mm. The two groups of species were also markedly differ- ent in terms of average longevity (7.0 vs. 36years) and average pN/pS (0.089 vs. 0.16). The Ne effect on codon usage bias is illustrated in figure 3a, in which we plotted the average allele frequency of XYC or XYT codons against rPYR, separately in low propagule size (top) and high propagule size (bottom) species. Codon pairs for which <20 SNPs were available in the considered species were here excluded. Figure 3a shows a strong, positive corre- lation between codon usage bias and allele frequencies in low propagule size, large-Ne species. Red dots in figure 3a corre- spond to the nematode C. brenneri, in which codon usage bias is particularly pronounced (see supplementary fig. S1, Supplementary Material online). In contrast, no such relation- ship was uncovered in high propagule size, small-Ne species (fig. 3b). Insect Data Analysis It has been suggested that growth rate, not Ne, could be the main driver of codon usage bias intensity across species (Subramanian 2008). The two effects are not easy to disen- tangle since Ne was found to be strongly correlated to life history traits related to growth rate, such as fecundity, lon- gevity, and propagule size, in animals (Romiguier, Gayral, et al. 2014). To address this problem, we focused on insects and compared eusocial with solitary species. Eusocial species are characterized by a dramatic reduction in Ne, compared with solitary insects (Romiguier, Lourenc¸o, et al. 2014). Eusocial and solitary insects, however, share similar cellular and devel- opmental processes, so that the selective pressure for efficient protein translation can be assumed to be similar in the two groups of species. codon usage/expression correlation ● ● ● ● ● ● ● ● ● ●●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● −0.2 −0.1 0.0 0.1 0.2 0.2 0.4 0.6 0.8 b. Small Ne species codon usage/expression correlation mean allele frequency r2=0.04, NS b. Small Ne species We downloaded transcriptome data from 20 eusocial and 30 solitary insects and calculated in each species the 17 rPYR, our measure of preference for codons XYC (respectively, XYT). About 691 (81%) of the 850 estimated correlation coefficients were positive, similarly to our main data set. We considered the 850 rPYR as independent data points and tested the effect of eusociality on the absolute value of this variable (fig. 4a). We found that translational selection on codon usage is significantly stronger in solitary than in euso- cial insects (t-test, P< 1015). Eusocial insects belong either to Hymenoptera (ants, eusocial bees, and eusocial wasps) or to Blattodea (termites). To control, for taxonomy, the data set was split in five categories: eusocial Hymenoptera, solitary Hymenoptera, eusocial Blattodea, solitary Blattodea, and other solitary insects (fig. 4b). The effect of eusociality on rPYR was significant both within Hymenoptera (t-test, P< 103) and within Blattodea (t-test, P< 103). codon usage/expression correlation FIG. 3. Intensity of selection on codon usage bias. Each dot represents a particular pair of synonymous codon in a particular species. Only GC-conservative pairs of synonymous codons for which a minimum of 20 SNPS are available are considered. Codon Usage Population Genetics For each species and codon pair, we calculated the correlation coefficient between the frequency of occurrence of the G- or C-ending codon and gene expression level, rGC, 80% of which were positive. The number of preferred codons varied between zero and 26 across species and was not cor- related with species propagule size (n¼ 30, r¼0.16, NS). Large numbers of preferred XYG/XYA or XYC/XYT codons were found in Sauropsids Chelonoidis nigra (giant Galapagos turtle), Aptenodytes patagonicus (penguin), and Cyanistes caeruleus (great tit), a surprising result at odds with current knowledge on coding sequence evolution in vertebrates (Rao et al. 2011; Figuet et al. 2014). We found a significant, positive effect of rPYR on MAJ (P< 1015), indicating that synonymous codons more com- monly used in high-expressed genes tend to segregate at high population frequency, consistent with the hypothesis of translational selection on codon usage. The data set was split in four bins of species defined on the basis of propagule size, a variable negatively correlated to Ne (Romiguier, Gayral, et al. 2014), and the logistic regression was separately applied to the four bins. The relationship was strongly significant as far as the small propagule size bin was concerned (P< 1015), less strongly so in the second bin (P¼ 6104), and not signifi- cant in the large propagule size bins. Translational selection on codon usage, therefore, is apparently affected by variations in Ne among species. When species were analyzed separately, a significant relationship between MAJ and rPYR was detected in twelve species (supplementary table S1, Supplementary Material online). The mean propagule size across these twelve species was 0.69mm, whereas the mean propagule size across the 18 species for which no significant effect was detected was In an attempt to explicitly account for the confounding effect of GC-content, we computed the residual of the regres- sion of sequencing read depth on gene GC-content at first and second codon positions (GC12), here taken as a measure of gene expression level independent of GC-bias. We then correlated codon frequency to this measure of gene expres- sion level to identify preferred codons, as described earlier. As far as GC-conservative codon pairs were concerned, the GC12-corrected measure of codon bias was very similar to 1095 MBE Galtier et al. . doi:10.1093/molbev/msy015 8.5mm. The two groups of species were also markedly dif ent in terms of average longevity (7.0 vs. GC-Biased Gene Conversion Analysis (a) Distribution of the absolute value of rPYR, the correlation coefﬁcient between C- or T-ending codon frequency and gene expression, in 20 species of eusocial insects versus 30 species of solitary insects. (b) Eusocial species are split in Hymenoptera versus Blattodea; solitary species are split in Hymenoptera versus Blattodea versus other. FIG. 5. Evidence for GC-biased gene conversion. Each dot represents a species. Top: SFS analysis; X axis: propagule size (log scale); Y axis: estimated scaled gBGC coefﬁcient B; red/darker dots: signiﬁcant, pos- itive B; other dots: B not signiﬁcantly different from zero. Error bars reﬂect 95% conﬁdence intervals obtained by bootstrapping SNPs; Bottom: GC/recombination relationship; X axis: propagule size (log scale); Y axis: correlation coefﬁcient between bins of Rfgr and bins of GC; red/darker dots: signiﬁcant, positive correlation coefﬁcient; other dots: correlation coefﬁcient not signiﬁcantly different from zero. species belong to six of the eight metazoan phyla that were sampled. The estimated scaled gBGC coefficient, B, was not significantly correlated with pN/pS, propagule size, or longev- ity across species. SNP sample size was limiting in some spe- cies, as illustrated by error bars around estimated B in figure 5a. Still, even when only species with a narrow confi- dence interval are considered, no relationship between prop- agule size, and B is detectable. mutations increased with contig GC-content, again in agree- ment with the gBGC hypothesis (supplementary fig. S4, Supplementary Material online). The DAF of GC!AT muta- tions showed the reverse pattern—although less markedly— and the DAF of GC-conservative mutations was unaffected by contig GC-content. We investigated whether the GC-bias likely reflects the action of gBGC by analyzing the impact of recombination rate. We binned contigs according to their GC-content, and measured the average recombination rate (Rfgr) within each bin. We then correlated average Rfgr to average GC-content across bins in 29 species for which at least 800 loci were eligible for recombinant haplotype detection. The correlation coefficient was positive in 28 species, >0.75 in 21 species and significantly positive (P< 0.05) in 25 species from eight dis- tinct phyla (supplementary table S1, Supplementary Material online and fig. 5b). In one species (harvester ant M. capitatus), the correlation coefficient was negative but not significantly different from zero. The correlation coefficient between Rfgr and GC-content was not significantly correlated with propa- gule size (fig. 5b), longevity or pN/pS across species. GC-Biased Gene Conversion Analysis SFS analysis estimated B log10(propagule size) ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● −2 −1 0 1 2 0.0 0.4 0.8 b. Rfgr analysis recombination/GC correlation log10(propagule size) ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● FIG. 5. Evidence for GC-biased gene conversion. Each dot represents a species. Top: SFS analysis; X axis: propagule size (log scale); Y axis: estimated scaled gBGC coefﬁcient B; red/darker dots: signiﬁcant, pos- itive B; other dots: B not signiﬁcantly different from zero. Error bars reﬂect 95% conﬁdence intervals obtained by bootstrapping SNPs; ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● −2 −1 0 1 2 −4 −2 0 2 4 a. SFS analysis estimated B log10(propagule size) ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● −2 −1 0 1 2 0.0 0.4 0.8 b. Rfgr analysis recombination/GC correlation log10(propagule size) ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● FIG. 5. Evidence for GC-biased gene conversion. Each dot represents a species. Top: SFS analysis; X axis: propagule size (log scale); Y axis: estimated scaled gBGC coefﬁcient B; red/darker dots: signiﬁcant, pos- itive B; other dots: B not signiﬁcantly different from zero. Error bars reﬂect 95% conﬁdence intervals obtained by bootstrapping SNPs; Bottom: GC/recombination relationship; X axis: propagule size (log scale); Y axis: correlation coefﬁcient between bins of Rfgr and bins of GC; red/darker dots: signiﬁcant, positive correlation coefﬁcient; other dots: correlation coefﬁcient not signiﬁcantly different from zero. ● ● ●●●●●●●●● ● ● ●●●●● ● ●● ● ● eusocial solitary 0.00 0.04 0.08 a. Pooled rpyr ●● ● ● ●● ● ● ●●●●● ● ●● ● 0.00 0.04 0.08 b. Detailed rpyr Blattodea Hymenoptera other FIG. 4. GC-Biased Gene Conversion Analysis Codon usage bias in eusocial versus solitary insects. (a) Distribution of the absolute value of rPYR, the correlation coefﬁcient between C- or T-ending codon frequency and gene expression, in 20 species of eusocial insects versus 30 species of solitary insects. (b) Eusocial species are split in Hymenoptera versus Blattodea; solitary species are split in Hymenoptera versus Blattodea versus other. ● ● ●●●●●●●●● ● ● ●●●●● ● ●● ● ● eusocial solitary 0.00 0.04 0.08 a. Pooled rpyr ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● −2 −1 0 1 2 −4 −2 0 2 4 a. SFS analysis estimated B log10(propagule size) ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● −2 −1 0 1 2 0.0 0.4 0.8 b. Rfgr analysis recombination/GC correlation ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● −2 −1 0 1 2 −4 −2 0 2 4 a. SFS analysis estimated B log10(propagule size) ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● a. SFS analysis a. Pooled estimated B Downloaded from https://academic.oup.com/mbe/article/35/5/1092/4829954 by Bibliothèque Universitaire de médecine - Nîmes user on 16 Downloaded from https://academic.oup.com/mbe/article/35/5/1092/4829954 by Bibliothèque Universitaire de médecine - Nîmes user on 16 June 2021 log10(propagule size) ●● ● ● ●● ● ● ●●●●● ● ●● ● 0.00 0.04 0.08 b. Detailed rpyr Blattodea Hymenoptera other b. Detailed ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● −2 −1 0 1 2 0.0 0.4 0.8 b. Rfgr analysis recombination/GC correlation log10(propagule size) ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● recombination/GC correlation FIG. 4. Codon usage bias in eusocial versus solitary insects. GC-Biased Gene Conversion Analysis Finally, we calculated the average derived allele frequency (DAF) of AT!GC, GC!AT, and GC-conservative mutations in dis- tinct bins of contigs. We found that the DAF of AT!GC GC-Biased Gene Conversion Analysis In each species, synonymous SNPs were oriented by assuming that the outgroup species carries the ancestral allele. We fo- cused on the 28 species for which at least 500 oriented syn- onymous SNPs were available. We calculated the frequency of the derived alleles and found that in 27 species out of 28, the average frequency of alleles resulting from an AT!GC mu- tation was above the average frequency of alleles resulting from a GC!AT mutation (supplementary table S1, Supplementary Material online), in agreement with the gBGC hypothesis. The unfolded SFS for AT!GC SNPs, GC!AT SNPs, and GC-conservative SNPs, respectively, were built in each species using all contigs. A mutation/ drift/gBGC model was fitted to the three SFS (Glemin et al. 2015). A significantly positive segregation bias in favor of GC alleles was detected in 19 species out of 28 (supplementary table S1, Supplementary Material online and fig. 5a). These Different tissues have been used for RNA extraction in distinct species of our sample, and this might affect our results. In particular, our estimate of gene expression level could be less relevant in species where RNA has been extracted out of a single tissue, compared with multiple tis- sues. To control for this problem, we focused on the subset of 1096 MBE Codon Usage Bias in Animals . doi:10.1093/molbev/msy015 ● ● ●●●●●●●●● ● ● ●●●●● ● ●● ● ● eusocial solitary 0.00 0.04 0.08 a. Pooled rpyr ●● ● ● ●● ● ● ●●●●● ● ●● ● 0.00 0.04 0.08 b. Detailed rpyr Blattodea Hymenoptera other FIG. 4. Codon usage bias in eusocial versus solitary insects. (a) Distribution of the absolute value of rPYR, the correlation coefﬁcient between C- or T-ending codon frequency and gene expression, in 20 species of eusocial insects versus 30 species of solitary insects. (b) Eusocial species are split in Hymenoptera versus Blattodea; solitary species are split in Hymenoptera versus Blattodea versus other. ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● −2 −1 0 1 2 −4 −2 0 2 4 a. Discussion Selection versus gBGC: Methodological Aspects Disentangling the effect of natural selection from that of neu- tral forces such as gBGC is a difficult task (Ratnakumar et al. 2010). Clement et al. (2017) introduced a model of codon usage accounting both for gBGC, which is assumed to affect AT$GC mutations, and selection, which is assumed to affect mutations between preferred and non preferred codons. Their approach was successful in discriminating between the two evolutionary forces in eleven species of plants (Clement et al. 2017). Here, we faced the additional problem that, in a substantial number of species, gene GC content was correlated to gene expression irrespective of codon usage, so that even defining preferred codons was challenging. We 1097 MBE Galtier et al. . doi:10.1093/molbev/msy015 therefore addressed the problem by restricting our analysis of selection on codon usage to GC-conservative pairs of synon- ymous codons, which are supposedly unaffected by gBGC. One consequence is that we did not analyze 2-fold degener- ate codons, and among 4- and 6-fold did not compare all synonymous codons with each other, thus potentially missing a part of the signal. Of note, the classical approaches, which focus on the comparison between preferred and nonpre- ferred codons, also have their limitations in that, as far as 4- and 6-fold codons are concerned, the nonpreferred category is a mixture of several codons between which no distinction is made. For instance, published analyses of codon usage bias in Drosophila have hardly considered the preferences between T-ending over A-ending codons at 4-fold sites, since in this group all such codons fall in the nonpreferred category (but see Zeng 2010). synonymous mutations. In C. brenneri, for instance, no biased usage or skewed allele frequency distribution was detected for the proline-coding CCC versus CCG pair, whereas strong effects were detected for, for example, GCT versus GCA (Ala) and CGT versus CGA (Arg). Besides such differences between synonymous codon pairs, the effect of a particular type of synonymous mutation should also vary depending on which gene and which position is affected (Akashi 1994; Zhou et al. 2009; Machado et al. 2017), whereas we are here mea- suring the average strength of selection across sites, for any given pair of synonymous codons. Our analysis specifically targets selective effects on codon usage that are related to gene expression and the efficacy of translation—either fidelity or speed. Selection on Codon Usage in Animals: A Global Picture We report a significant effect of translational selection on codon usage in animals. Codons showing a higher prevalence in highly expressed genes tend to segregate at higher popu- lation frequency. We found that C-ending codons tend to be preferred over G-ending codons, and T-ending codons over A-ending ones. This is, to our knowledge, the first report of a general preference for pyrimidines over purines at third co- don positions in animals. We checked from previously pub- lished data (Duret and Mouchiroud 1999; Lynch et al. 2017) that the trend is also found in C. elegans (with the same exceptions as in C. brenneri), D. melanogaster, and D. pulex. We would expect codon preference to be quite stable over evolutionary times since switching to a new preferred codon should impose a high genetic load by simultaneously modi- fying the selection coefficient at many synonymous positions. This does not explain why pyrimidines would be preferred over purines, though. In C. elegans and perhaps more gener- ally, C-ending and T-ending synonymous codons are trans- lated by the same tRNA—the so-called wobble effect— whereas each A-ending and G-ending codon has its specific tRNA (Duret 2000; Percudani 2001). For this reason, one should probably expect correlated preferences for C and T at third codon positions—that is, a frequent usage of both C- and T-ending codons when their shared tRNA is abundant, infrequent usage otherwise. Again, we see no obvious reason why the existence of a shared tRNA for C- and T-ending codons would explain that these are generally favored over A- and G-ending codons. gBGC Is Widespread across the Metazoan Phylogeny We detected a significant effect of gBGC in a majority of species of the data set. gBGC manifested itself via a higher average allele frequency of GC over AT alleles both at synon- ymous and flanking regions, a significant difference between AT!GC and GC!AT oriented SFSs, and a correlation be- tween the long-term recombination rate and GC-content. Here, scaled recombination rate was approached at contig level using an approximate method derived from the four- gamete rule. The approach is suboptimal in several respects. First, we analyze spliced sequences and have no information on intron length, so that normalization by contig length is inexact. Secondly, we analyzed unphased data, thus loosing power compared with data sets consisting in experimentally phased haplotypes. Discussion It is important to keep in mind that third codon positions can be affected by translation-independent selective pressures, for example, ow- ing to mRNA splicing (Wu and Hurst 2015). These could in principle be distinguished by separately analyzing codons near versus far away from intron/exon boundaries. However, information on the position of introns is lacking in most of the species we have analyzed. Selection on Codon Usage in Animals: A Global Picture Why a Ne Effect on Codon Usage Bias but Not on gBGC? Why a Ne Effect on Codon Usage Bias but Not on gBGC? The average per base recombination rate is known to vary among metazoans, from 0.1 cM/Mb to>15 cM/Mb (Wilfert et al. 2007). There is also evidence that the length of gene conversion tracts (l) varies across species. For instance, in mammals, l is of the order of 400bp for crossovers and 50bp for noncrossover recombination events (Cole et al. 2014), whereas l is 2,000bp for both type of events in bud- ding yeast (Mancera et al. 2008). In drosophila, noncrossover gene conversion tracts are on an average 440bp long (Miller et al. 2016), that is, 8 times longer than in mammals. More data are crucially needed to characterize more thoroughly the variation in l among animals. Repair bias b0, finally, was esti- mated to be 0.014 in yeast (Mancera et al. 2008), 0.12 in Daphnia (Keith et al. 2016), 0.18 in flycatcher (Smeds et al. 2016), and up to 0.36 in humans (Halldorsson et al. 2016). This so far limited sample suggests that b0 varies substantially among species and could be inversely correlated with Ne, perhaps explaining the absence of a Ne effect on gBGC inten- sity in our analysis. We detected evidence for translational selection on codon usage only in the small propagule size, large-Ne fraction of the species we sampled. The estimated efficiency of selection was strongest in the nematode C. brenneri, whereas no evidence for translational selection on codon usage was found in large mammals, birds, reptiles. This is in agreement with the nearly neutral theory of molecular evolution (Ohta and Gillespie 1996). Of note, the distinction between small-Ne and large- Ne species does not perfectly fit the vertebrates/invertebrates contrast: we detected significant evidence for translational selection on codon usage in common vole Microtus arvalis, but not in cuttlefish Sepia officinalis, for instance. It has been suggested that among taxa variation in codon usage bias intensity is determined by variation in selective pressure, not Ne, with short generation time, high growth rate species having stronger requirement for efficient protein synthesis (Subramanian 2008). To distinguish between the two hypothesis, we compared codon usage bias in eusocial versus solitary insects, which differ in terms of Ne but share similar developmental processes. We found a strong effect of eusociality on codon usage bias, strongly suggesting that Ne, not variable selective pressure, explains the among-taxa var- iation we detect. Selection on Codon Usage in Animals: A Global Picture Thirdly, the calculation only partially accounts for allele frequencies and the probability of detect- ing recombinant haplotypes when they exist—for example, Rfgr can only increase as sample size increases. Despite these many approximations, a strong and significant correlation between Rfgr and GC-content was identified in>80% of the species we sampled, which is indicative of a prominent and widespread effect of gBGC in animals. SFS analysis corrobo- rated this finding in uncovering a significant segregation bias in favor of G and C alleles in a majority of species. Among the 29 species for which sufficient polymorphism data was avail- able, 28 yielded evidence for gBGC in either the SFS or the recombination rate analysis—only in the oyster Ostrea edulis did both approaches fail to identify a significant signal. Of note, our SFS analysis captures the effect of both gBGC and, potentially, selection on GC-ending versus AT-ending codon usage. UTR sequence analysis demonstrates the impact of gBGC (fig. 1), but selection on GC-changing synonymous mutations might also be at work in some or many of the analyzed species. The effect of translational selection on GC-conservative codons is significant but weak, and only detectable in a subset of species and codon pairs. This is perhaps surprising knowing that strong phenotypic effects of codon usage on expression levels of single genes have been experimentally reported in various systems, including fruit flies (Carlini et al. 2001; Carlini and Stephan 2003). Our approach, however, relies on poly- morphic sites and can only detect relatively weak effects— sufficiently weak such that deleterious alleles are segregating in natural populations. Our results are indeed consistent with the existence of a broad distribution of fitness effect of In animals, gBGC had so far been identified in vertebrates (Figuet et al. 2014), bees, and ants (Kent et al. 2012; 1098 MBE Codon Usage Bias in Animals . doi:10.1093/molbev/msy015 Wallberg et al. 2015), and Daphnia (Keith et al. 2016), but not in D. melanogaster (Robinson et al. 2014), albeit on the X chromosome (Galtier et al. 2006; Haddrill and Charlesworth 2008). We here considerably expand the range of species and taxa in which gBGC is documented, adding annelids, echino- derms, tunicates, nemertians, cnidarians, lepidopterans, gas- tropod and bivalve molluscs, decapod and isopod crustaceans. gBGC is obviously widespread among animals. Selection on Codon Usage in Animals: A Global Picture It significantly impacts the population frequency and fixation probability of AT$GC mutations in a majority of species and should be considered as a potential confounder of molecular evolutionary studies, particularly studies of molecular adap- tation, not only in mammals and vertebrates (Ratnakumar et al. 2010; Corcoran et al. 2017) but more generally in Metazoa. This study adds to the growing evidence that gBGC is a nearly universal process affecting a wide range of organisms (Pessia et al. 2012; Long et al. 2018). and Abatus cordatus (brooding sea urchin), for instance, whereas the detected effect was weaker in the presumably large-Ne M. galloprovincialis (mussel) and Ciona intestinalis (tunicate) despite large numbers of SNPs available in the lat- ter two species (supplementary table S1, Supplementary Material online). A similar pattern was recently reported in plants, based on a data set of eleven species (Clement et al. 2017). This result is somewhat surprising in that, just like selection, gBGC should only be effective if of magnitude well above that of drift. The intensity of the signal for gBGC is expected to be determined by the product of four param- eters, namely Ne, the effective population size, r, the per base recombination rate, l, the length of gene conversion tracts, and b0, the repair bias in favor of GC. The rlb0 product is often denoted as b (Glemin et al. 2015). Our results rule out the hypothesis that b is constant—or a Ne effect should be detected. r, l and/or b0 must therefore vary substantially across species, and/or be inversely related to Ne. Why a Ne Effect on Codon Usage Bias but Not on gBGC? Subramanian (2008) noted that under the Ne hypothesis one would expect a step-like relationship be- tween codon usage intensity and Ne, since the theory predicts no biased usage for every Ne well below 1/4s and almost perfect codon usage (i.e., 100% of preferred codons) for every Ne well above 1/4s, s being the selection coefficient in favor of preferred codons. This rationale, however, implicitly assumes that a constant selection coefficient applies to every synonymous mutation, which is unlikely to be true. If one rather assumes a distribution of s across synonymous muta- tions then a gradual effect of Ne on the intensity of codon usage bias is expected, consistent with our and Subramanian’s (2008) results. We can think of two possible reasons why b0 would scale inversely with Ne. First, gBGC is generally speaking a deleteri- ous process in that it promotes G and C alleles irrespective of their effect on fitness (Galtier et al. 2009; Glemin 2010; Necs¸ulea et al. 2011; Lachance and Tishkoff 2014). It might be that the molecular machinery involved in recombination is more efficiently selected to minimize b in large Ne species. A formal model would be required to validate this verbal hy- pothesis, though. Secondly, Lesecque et al. (2013) demon- strated that in yeast, when several SNPs are part of the same conversion tract, these are most often converted in the same direction—same donor and same recipient chromosomes—the direction only being influenced by SNPs located at the extremities of tracts. This implies a me- chanical decay of the average GC bias as the number of SNPs per tract increases, since AT versus GC SNPs located in the middle of a conversion tract are converted in either direction with probability 0.5. This mechanism, if effective in animals too, might contribute to explaining the lack of a Ne effect on gBGC intensity, SNP density being positively correlated with Ne. Of note, the evolution of genomic GC-content has been In contrast, no relationship was detected between the in- tensity of gBGC and Ne in our analysis. Significant gBGC was detected in the presumably small-Ne Lepus granatensis (hare) 1099 MBE Galtier et al. . doi:10.1093/molbev/msy015 associated with traits related to Ne in mammals and birds (Romiguier et al. 2010; Weber et al. 2014). Transcriptome Assembly and Annotation p y Transcriptome assembly, open reading frame (ORF) predic- tion, orthology prediction, and alignment between focal and outgroup coding sequences were achieved using the Abyss v1.3.4, Cap3 v10/15/07, Trinity_ORF, BLAST, and MACSe v1.02 programs, as previously described (Gayral et al. 2013; Romiguier, Gayral, et al. 2014, http://http://kimura.univ- montp2.fr/PopPhyl). We only retained contigs containing a predicted coding sequences (CDS) longer than 200bp. The median number of contigs per species was 3,480 (supplemen- tary table S1, Supplementary Material online). Contig expres- sion level was measured as the per base pair read depth, averaged across individuals. For each contig of each species, we calculated GC-content at first and second codon positions (GC12), third codon positions (GC3) and UTR (GC_UTR), and the frequency of the 61 sense codons. Conclusions Translational selection is a significant determinant of codon usage patterns in large-Ne species of animals, but is weak or absent in small-Ne ones, such as large vertebrates and social insects. In contrast, gBGC is widespread across animals and of strength independent of Ne. gBGC is therefore a major con- founder that must be seriously taken into account in any analysis of codon usage bias. This study uncovered two unexpected results that remain to be elucidated, that is, a general preference for C- and T-ending codons over G- and A-ending ones, respectively, and an inverse relationship be- tween the recombination-associated GC repair bias and Ne. Why a Ne Effect on Codon Usage Bias but Not on gBGC? This might be explained by b being fairly homogeneous within groups, but much more variable across distantly related taxa, so that B would only respond to Ne at a relatively small time scale. It might also be the case that the relationship between GC-content dynamics and life-history traits in mammals and birds is not (entirely) mediated by Ne—but rather by, for example, the mutation rate in a nonequilibrium situation (Romiguier et al. 2010; Bolıvar et al. 2016). the analyzed species. pN/pS is expected to be negatively cor- related with Ne due to the decreased efficiency of purifying selection against slightly deleterious nonsynonymous alleles in small populations (Lanfear et al. 2014). SNP and Genotype Calling Genotypes and single nucleotide polymorphisms (SNPs) were called using the reads2snp v1.0 program, which was designed for genotyping based on RNAseq data (Tsagkogeorga et al. 2012; Gayral et al. 2013; Ballenghien et al. 2017). This method models read counts at each position as a multinomial distri- bution determined by allele frequencies, genotype frequen- cies, sequencing error rate, and cross-contamination rate. Allele frequencies are estimated a priori from read counts across all individuals. Genotype frequencies are assumed to follow the Hardy–Weinberg prior. The method first estimates the error rate by maximum likelihood, then the posterior distribution of genotypes in the empirical Bayesian frame- work (Tsagkogeorga et al. 2012). Contamination rate (Flickinger et al. 2015; Ballenghien et al. 2017) was here set to 0.2. This parameter likely captures a combination of effects leading to overdispersion of read counts and spurious calls of heterozygote genotypes (Ballenghien et al. 2017). A filter for false SNPs due to hidden paralogy was applied posterior to genotyping. Species Sampling We used recently published Illumina transcriptome data from population samples of non model animals (Romiguier, Gayral, et al. 2014; Rousselle et al. 2016; Ballenghien et al. 2017; Romiguier et al. 2017), which covered 32 distinct families of Metazoa. In each family, we selected the species with the largest number of individuals, provided this number was five or more. Mosquito Culex pipiens (Culicidae) and trumpet worm Pectinaria koreni (Pectinariidae) were excluded because transcriptome assembly in these species yielded a small num- ber of very short contigs (Romiguier, Gayral, et al. 2014). Harvester ant Messor barbarus (Formicidae) was excluded because of its peculiar mating system, which dramatically departs the Hardy–Weinberg assumption (Romiguier et al. 2017). Its sister species Messor capitatus was rather included, despite a lower number of sampled individuals. The final data set included 30 species, of which seven vertebrates, six insects, five molluscs, three crustaceans, three echinoderms, two tuni- cates, one annelid, one nematode, one nemertian, and one cnidarian (supplementary table S1, Supplementary Material online). Five to eleven individuals per species were analyzed. For each of these focal species, one outgroup from the same family was selected. The tissues from which RNA has been extracted, which differ across species, are provided in supple- mentary table S1, Supplementary Material online. Correlation Analyses Pearson’s correlation coefficient and associated P values were calculated in R. Phylogenetic control analyses were performed using the independent contrasts method as implemented in the CAPER package. Effect of Recombination Rate We approximated the population-scaled recombination rate of each locus via a calculation based on the four-gamete rule (Hudson and Kaplan 1985). For every pair of SNPs in a locus, haplotypes were identified from individuals homozygous at both SNPs, and from individuals heterozygous at one SNP and homozygous at the other SNP. In these two situations, linkage relationships between alleles can be determined with certainty even from unphased data. Individuals carrying a heterozygous genotype at both SNPs were disregarded here. When the four possible haplotypes were found to be segre- gating in the sample, a recombination event was inferred. The total number of recombination events per contig was recorded by summing across pairs of SNPs, taking care of only counting once events supported by non independent pairs of SNPs. We defined Rfgr (for “four-gamete rule”) as the ratio of total number of inferred recombination events by contig length. This was calculated, in each species, for each contig carrying at least one pair of SNPs, excluding singletons, such that four haplotypes or more could be inferred. Contigs departing these conditions were not considered eligible for recombination analysis, and missing data was recorded. Species in which <500 eligible contigs were available were not considered. We performed simulations to assess whether Rfgr could be used as a proxy for Rho. These simulations showed that Rfgr is affected by SNP density. However, for a given level of polymorphism and for the range of rho ob- served in animals, Rfgr strongly covaries with Rho (see supple- mentary fig. S5, Supplementary Material online). Thus, given the relatively limited variation in SNP density withing genomes, Rfgr appears to be a good indicator for intragenomic variation in recombination rate. Supplementary data are available at Molecular Biology and Evolution online. Acknowledgments We thank Thomas Bataillon for helpful suggestions regarding statistics and the Montpellier Bioinformatics and Biodiversity platform for computational resources. This work was sup- ported by European Research Council grant 232971, Swiss National Foundation grant CRSII3_160723, and Agence Nationale de la Recherche grant DaSiRe ANR-15-CE12-0010. References Aird D, Ross MG, Chen W-S, Danielsson M, Fennell T, Russ C, Jaffe DB, Nusbaum C, Gnirke A. 2011. 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(2015), which accounts for the effect of muta- tion bias, gBGC, and drift and assumes constant gBGC among sites. Two versions of the model were considered, accounting or not for SNP orientation error. We estimated B¼ 4 Ne b, the scaled gBGC coefficient, under both models and used as our point estimate a weighted average between estimates from the two models, weights being derived from Akaike’s Romiguier, Gayral, et al. (2014) reported significant corre- lations between life history traits, such as species longevity, fecundity, and propagule size, and population genomic vari- ables theoretically related to Ne, such as the synonymous diversity, pS, and the ratio of nonsynonymous over synony- mous heterozygosity, pN/pS. In this study, we used longevity, propagule size, and pN/pS as markers of the long-term Ne of 1100 MBE Codon Usage Bias in Animals . doi:10.1093/molbev/msy015 Information Criterion as suggested by Posada and Buckley (2004). 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https://openalex.org/W2895844540	https://discovery.ucl.ac.uk/id/eprint/10064211/1/Variation%20between%20European%20eel%20Anguilla%20anguilla.pdf	English	null	Variation between European eel Anguilla anguilla (L.) stocks in five marshes of the Thames Estuary (United Kingdom)	Wetlands ecology and management	2,018	cc-by	5,877	Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) Kristen Steele . Sally Chadwick . Alison Debney . Matthew Gollock Received: 10 December 2017 / Accepted: 21 September 2018 / Published online: 8 October 2018 The Author(s) 2018 Abstract The European eel (Anguilla anguilla, L.) was historically widely distributed throughout the United Kingdom, in coastal waters, lakes, rivers and wetlands. Recruitment has declined in recent decades and the species is now listed as ‘Critically Endan- gered’ on the International Union for Conservation of Nature and Natural Resources (IUCN) Red List. Management of suitable wetland habitats may con- tribute to species recovery; however, little is known about the stocks in these areas. In this study, yellow (adult stage [ 300 mm) eels were sampled in ditches in ﬁve marshes bordering the Thames Estuary in England, UK. Ecological variables, including ditch characteristics, invertebrate abundance and water quality parameters were measured. Habitat features were also observed and recorded, including access, land use and water management regimes. Eels were found in all marshes, but at varying catch-per-unit- effort (CPUE). There were no signiﬁcant correlations between CPUE and the ecological variables, except ditch width. However, a signiﬁcant difference in CPUE was found between two of the marshes, which may be explained by variations in local habitat management. Mean lengths showed a high proportion of females and mean body condition of four of the marshes was also found to be greater than in three rivers in the same region. These ﬁndings suggest that the marshes are potentially favourable eel habitats and that factors inﬂuencing habitat quality, such as land use and water management, may affect eel abundance, production of females and body condition. Effective management of such wetlands may therefore con- tribute to the conservation of European eel. Keywords North Kent marshes Thames RBD CPUE Yellow eel Ecological variables K. Steele (&) Royal Veterinary College, 4 Royal College Street, London NW1 0TU, UK e-mail: kristen.steele.15@ucl.ac.uk K. Steele (&) Royal Veterinary College, 4 Royal College Street, London NW1 0TU, UK e-mail: kristen.steele.15@ucl.ac.uk K. Steele UCL Anthropology, 14 Taviton Street, London WC1H 0BW, UK While the continental life stage of European eel (Anguilla anguilla, L.) has attracted substantial research interest over the last century, the majority of published studies have focused on eels in freshwater ecosystems, such as rivers and lakes (Aprahamian and Walker 2009; ICES 2009; Jacoby et al. 2015). Wetlands Ecol Manage (2018) 26:1181–1188 https://doi.org/10.1007/s11273-018-9628-5 (0123456789().,-volV)(0123456789().,-volV) SHORT COMMUNICATION Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) (2000) conducted a similar review of studies on ﬁsh assemblages in six tidal marshes in Europe, which included two sites in the UK. The only area where no eels were recorded was the Welwick marsh of the Humber estuary in Northeast England, despite eels having previously been captured in the adjacent tidal river. While there had been anecdotal reports of eels in the marshes around the Thames, no formal studies had previously been carried out. It was unknown whether eels remained in these marshes and, if so, where and in what densities. In 2010, the Environment Agency set three double-ended fyke nets (designed to catch eels [ 300 mm) at three sites in one North Kent marsh in the Thames RBD and caught 41 yellow eels (Chadwick 2010). It was noted that most of these eels appeared to be in particularly good body condition, all over 550 mm in length and some approaching a metre, which suggested a high proportion of females given that males tend to mature at shorter lengths (\ 45 cm) (Dekker et al. 1998; Tesch 2003). The study reported here revisited this location in 2011 and extended the research to four previously un-sampled marshes. The aims were to establish the presence or absence of eels, assess their relative body condition, and compare variations in relative abundance, as catch-per-unit- effort (CPUE), between the ditch systems of each marsh to local ecological variables. Due in part to dramatic declines in recruitment recorded across Europe, the European eel is now listed as ‘Critically Endangered’ on the IUCN Red List (Jacoby and Gollock 2014). Habitat loss and degra- dation are among the numerous threats facing eels during their lifecycle (Jacoby et al. 2015; Miller et al. 2016) and protecting, restoring and ensuring access to quality habitats is seen as an important conservation and management measure (Feunteun 2002). The European Eel Regulation (EC) No 1100/2007, adopted by the European Commission in 2007 mandates the creation of Eel Management Plans (EMPs) for each member country with eel habitats within their national borders (ICES 2013). Despite the potential for brackish and freshwater marshes to be productive habitats for the European eel, only a small number of studies have examined adult stocks in these areas, which Tomlinson et al. (2010) emphasise as a conspicuous research gap given the current conservation status of the European eel and its prey value to other species. Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) However, it is thought that coastal wetlands, including estuaries, lagoons and marshes may provide S. Chadwick Environment Agency, Kings Meadow House, Kings Meadow Road, Reading RG1 8DQ, UK A. Debney M. Gollock Conservation Programmes, Zoological Society of London, Outer Circle, Regent’s Park, London NW1 4RY, UK 12 3 Wetlands Ecol Manage (2018) 26:1181–1188 1182 productive habitat for eels for several reasons, includ- ing proximity to the sea (Laffaille et al. 2004), diverse and abundant food sources (ICES 2009; Van Lief- feringe et al. 2012) and preferred habitat features, such as soft, muddy substrate and densely vegetated margins (Knights 2003). Eels in saline environments also tend to have faster growth rates and reduced loads of the swimbladder parasite Anguillicoloides crassus than those in freshwater (Jakob et al. 2009). depth and density of aquatic vegetation. In the Lippenbroek, a tidal marsh in Belgium, Van Lief- feringe et al. (2012) investigated the foraging behaviour and body condition of yellow eels. They found that eels in the marsh had a more mixed diet than those sampled in the nearby River Schelde, with prey diversity being about 12 times higher. Although not statistically signiﬁcant, the marsh eels were on average heavier than the river eels, suggesting a higher fat content, which is essential for successfully completing the spawning migration. The European eel is a semelparous and catadro- mous species, which hatches in the Sargasso Sea and reaches European shores after a lengthy migration, metamorphosing from leptocephelus to glass eel in the process. During the subsequent pigmented elver stage they take up residence in coastal, estuarine and river habitats, adopting sedentary or semi-migratory habits (Edeline et al. 2005). Their diet shifts from plankton to macro-invertebrates, eventually incorporating ﬁsh and larger prey, including other elvers, as they mature into yellow eels (Tesch 2003). The yellow eels will spend anywhere from three to more than 20 years in continental waters until metamorphosing into silver eels and returning to the Sargasso Sea for spawning (Naismith and Knights 1993). Published studies on eels in marshes in the United Kingdom are also limited. A 2010 compilation of ﬁsh assemblage sampling over more than 20 years in the Norfolk Broads found eels in certain fens and marshes, notably in drainage dykes and reed bed habitats, but in declining numbers (Tomlinson et al. 2010). Mathieson et al. 123 Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) The marshes are intended to be maintained as freshwater systems and the tidal ﬂaps designed to operate in response to water pressure: opening to let freshwater ﬂow out of the marshes at low tide and closing as the tide rises. Water ﬂow within the marshes is further voluntarily controlled by land owners and managers with pumps, sluice gates and/or board structures located throughout the ditch system. Impairment of tidal ﬂap function, due to siltation, rust and/or vandalism, is not uncommon in the study marshes and they may remain fully open or closed regardless of tide. Connectivity of the ditches is further compro- mised by culverts and inﬁll for vehicle and livestock passage. Despite their designation as freshwater marshes, the majority of sampled sites were brackish (Table 1) due to tidal ﬂap malfunction and hydrolog- ical changes, both natural and managed. Land use and protection status varied substantially between the marshes, ranging from intensive grazing and unmon- itored public access (Marsh A) to the management of the area as a private shooting reserve (Marsh E). (To minimise the risk of eel poaching, individual marshes are not identiﬁed by name, but are designated alpha- betically from west to east.) Ditch width and depth were measured upon setting the nets and data for the following ecological variables were gathered over a 10 day period in mid-July. Benthic macro-invertebrates were collected using a 3 min pond net sampling method (Environment Agency 2009) near the bank within the 100 m fyke- netting area. Samples were immediately transferred into a white tray, where log abundance of combined invertebrates was estimated. Any sites with distinctly dominant taxa were noted. Dissolved oxygen (%), temperature and salinity (ppt) were measured with a YSI ‘Professional Plus’ water quality probe. Liquid assay kits were used to test for levels of Ammonium, Nitrite and Nitrate (JBL GmbH & Co, Germany) and pH (sera GmbH, Germany). Potential obstacles to eel passage were counted through a combination of walking along the ditches and examining satellite maps where ground access was not possible. Obstacles noted included tidal ﬂaps, board structures to control water ﬂow, inﬁll for vehicle passage and damaged culverts. Distance from the seaward side of the outfall to the nearest fyke net at each site was measured on satellite maps. Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) Laffaille et al. (2004) sampled eels of multiple size classes in a reclaimed marsh on the Atlantic coast of France and found that distribution and habitat use depended on a combina- tion of three environmental factors: ditch width, silt The ﬁve study marshes are located in North Kent, Southeast England along the Southern banks of the Thames Estuary. Reclamation of these marshlands began in medieval times with the original network of 123 Wetlands Ecol Manage (2018) 26:1181–1188 1183 ditches, protected by a sea wall, constructed in the 12th and 13th centuries to drain the land for agricultural use. Although an estimated 65% of grazing marsh has been lost in this area in the last hundred years (Hollis 1998), many of the remaining ditches have been maintained and the ﬁve study marshes are still used, at least partially, for arable crops and livestock grazing. Recognised by the government as an ‘Environmen- tally Sensitive Area’, these marshes are also important as habitats for other wildlife species, including the protected European water vole and marsh harrier. where ditches were wider than the width of the entire net, the far cod end was staked into the substrate. Nets were left in place overnight and retrieved the follow- ing day. Captured eels were transferred into buckets, then individually measured and weighed (to the nearest 5 mm and 50 g, respectively) and returned to the water immediately. Density of ditches to land area was observed to be similar across the marshes. As the land area containing the sampling sites in Marshes A and B each is approximately 200 hectares and Marshes C, D and E are roughly twice as large, nets were set in three sites each in Marshes A and B and six sites each in Marshes C, D and E to gain an equivalent sampling intensity between marshes. Sites were chosen using a numbered grid and random number generator. The character of the ditches was highly variable with some regularly dredged, straight, open, and the edges mowed, while others were unmaintained, meandering, heavily silted, and shaded by dense vegetation. p p Each ditch system is connected to the river by one or more ‘outfalls’, which consist of a tidal ﬂap and a penstock mechanism (usually a sluice gate). Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) Sampling took place over 6 weeks in June and July 2011, using double-ended fyke nets with an opening diameter of 52 cm with 6 m long leader, ﬁtted with an otter guard and a mesh size of 10 mm (15 mm stretched). This mesh size is expected to retain only eels over 30 cm (Bark et al. 2007). Three nets (a total of six cod ends) were set 50 m apart from each other, stretched diagonally across the width of the ditch, with one cod end staked into the near bank and the other staked as far away as the ditch width would allow, usually to the far bank. However, in several sites, Eel catch data at each site were divided by number of net ends to derive catch-per-unit-effort (CPUE) in number of eels caught per night (Naismith and Knights 1993). As histogram inspections showed non-normal distributions and unequal variances of CPUE, these data were log-transformed. Statistical signiﬁcance was 12 3 3 1184 Wetlands Ecol Manage (2018) 26:1181–1188 Table 1 Ecological variable measurements at each site and marsh-speciﬁc features Marsh Site Dissolved oxygen (%) Distance from outfall (metres) Invertebrates (log abundance) Dominant taxa Potential obstacles (number of) Salinity (ppt) Ditch depth (to substrate surface, in cm) Ditch width (metres) Outfall type Land use and marsh-speciﬁc features A 1 61 115 1–10 Gammarus 1 8.5 50 4 Steel tidal ﬂap Horse grazing. Publicly accessible, apparent vandalism, waste dumping in ditches. A 2 43 573 10–100 Dytiscus 3 0.35 100 5 A 3 15 951 100–1000 5 0.47 100 3 B 1 45 663 10–100 1 0.52 100 4.5 2000 mm square steel tidal ﬂap Arable crops, motocross track, shooting range. Limited access via road, gate closed and locked at night. B 2 68 1628 10–100 3 0.46 70 4.5 B 3 54 2510 100–1000 7 0.46 100 5.5 C 1 89 58 10–100 1 6.52 125 5 500 mm circular steel tidal ﬂap Sheep grazing, hunting reserve. Inundation sluice is thought to be permanently non-functional due to siltation. Limited access via road, multiple gates. Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) An analysis of variance (ANOVA) with a post hoc Tukey’s test was performed for comparison between the marshes, according to Belpaire et al. (2009). No further analysis of variance was conducted at the site level due to the high variability and small numbers of eels at most sites. After examination of the initial multivariate results, a separate regression analysis was used to detect correlations between invertebrate abundance and the other independent variables. set at P \ 0.05 and all analyses were conducted with R 2.13.1. Spearman rank correlations were performed on each ecological variable separately and conﬁrmed by univariate linear regression to identify effect size (R2) of both signiﬁcant and apparently non-signiﬁcant variables. Signiﬁcant ecological variables, as well as those that were slightly above the signiﬁcance thresh- old (P [ 0.05, but \ 0.1), but with an effect size of R2 [ 0.07 were included in a ﬁrst multivariate linear regression model. Variables were eliminated individ- ually until an optimal model was obtained, which was conﬁrmed by the R stepAIC (Akaike information criterion) procedure (Acou et al. 2010). In all multi- variate analyses, ‘marsh’ was included as an explana- tory variable, thereby minimising the potential for the ‘marsh effect’ (sites within one marsh having greater commonality with each other) to be a confounder. An analysis of variance (ANOVA) with a post hoc Tukey’s test was performed for comparison between the marshes, according to Belpaire et al. (2009). No further analysis of variance was conducted at the site level due to the high variability and small numbers of eels at most sites. After examination of the initial multivariate results, a separate regression analysis was used to detect correlations between invertebrate abundance and the other independent variables. Fig. 1 CPUE (eels net end-1 night-1) by marsh. Numbers of eels caught are in brackets. Error bars represent standard error and asterisks indicate signiﬁcant difference therefore excluded from the analysis. Other variables are presented in Table 1. Univariate linear regression showed all other independent variables to be not signiﬁcant in predicting eel CPUE, with the exception of ditch width (P = 0.000617, adjusted R2 = 0.3936). Dissolved oxygen was also close to the signiﬁcance threshold (P = 0.0732, adjusted R2 = 0.09949) and was included in the ﬁrst multivariate model. The best model (P = 0.001934, adjusted R2 = 0.5214) included only marsh and ditch width. Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) C 2 147 1000 10–100 Ephemeroptera 1 4.6 70 5.5 C 3 127 1430 10–100 5 2.58 120 4 C 4 96 330 100–1000 2 2.99 200 6.5 C 5 93 3925 10–100 5 2.38 170 5 C 6 85 967 100–1000 4 3.49 90 3 D 1 64 58 1000–10,000 1 2.44 130 4 Inundation sluice: two 2000 mm circular ﬂaps, one 1000 mm square ﬂap Cattle grazing, hunting reserve. Same access as March C. D 2 53 326 100–1000 3 1.65 55 2.5 D 3 49 4119 100–1000 2 1.29 95 4 D 4 40 4040 100–1000 4 1.31 140 10 D 5 98 1085 100–1000 2 2.15 165 12 D 6 116 3573 100–1000 2 2.12 120 9 E 1 122 1504 100–1000 3 5.78 120 6.5 HDPE plastic 600 mm tidal ﬂap, with smaller ﬂoated ‘eel ﬂap’ Hunting reserve, low density cattle grazing. Gated and manned access with regular surveillance. Tidal ﬂap newly installed (less than 3 months before study). Abundant wildlife. E 2 178 602 100–1000 2 6.42 160 39 E 3 157 125 10,000–100,000 1 8.04 135 41 E 4 167 2743 1000–10,000 Daphnia 3 4.71 90 21 E 5 168 3040 1000–10,000 4 4.38 90 23 E 6 148 4268 1000–10,000 4 5 110 20 12 Wetlands Ecol Manage (2018) 26:1181–1188 1185 Fig. 1 CPUE (eels net end-1 night-1) by marsh. Numbers of eels caught are in brackets. Error bars represent standard error and asterisks indicate signiﬁcant difference set at P \ 0.05 and all analyses were conducted with R 2.13.1. Spearman rank correlations were performed on each ecological variable separately and conﬁrmed by univariate linear regression to identify effect size (R2) of both signiﬁcant and apparently non-signiﬁcant variables. Signiﬁcant ecological variables, as well as those that were slightly above the signiﬁcance thresh- old (P [ 0.05, but \ 0.1), but with an effect size of R2 [ 0.07 were included in a ﬁrst multivariate linear regression model. Variables were eliminated individ- ually until an optimal model was obtained, which was conﬁrmed by the R stepAIC (Akaike information criterion) procedure (Acou et al. 2010). In all multi- variate analyses, ‘marsh’ was included as an explana- tory variable, thereby minimising the potential for the ‘marsh effect’ (sites within one marsh having greater commonality with each other) to be a confounder. Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) With the exception of Marsh A, mean body condition in the marshes was consistently higher than in the three rivers (Fig. 2). ANOVA, using log- transformed K values, showed a signiﬁcance of P = 0.01441. Post-hoc Tukey analysis showed signif- icant differences between Marsh E and the River Darent (P B 0.0001) and Marsh E and the River Thames (P = 0.00342). mesh. With the exception of Marsh A, mean body condition in the marshes was consistently higher than in the three rivers (Fig. 2). ANOVA, using log- transformed K values, showed a signiﬁcance of P = 0.01441. Post-hoc Tukey analysis showed signif- icant differences between Marsh E and the River Darent (P B 0.0001) and Marsh E and the River Thames (P = 0.00342). 2010). The size selectivity of the fyke net method means any juvenile eels in narrower ditches would have been missed, potentially skewing CPUE towards the wider ditches. As eel density has been found to decrease and size increase with distance from the tidal limit in rivers (Naismith and Knights 1993; Aprahamian and Walker 2009), distance from the outfall was expected to inﬂuence eel CPUE in the marshes. The lack of correlation between CPUE and distance in this study may have been due to the relatively small scale of the marshes; even the farthest site is less than 5 km from the outfall and all outfalls are within the Thames tidal zone. As the ﬁrst formal investigation into yellow eel stocks in the North Kent marshes, this study yielded several important ﬁndings. Most importantly, it determined that adult eels are still present in these areas, suggesting that, despite variable access through the outfalls, the marshes continue to provide favour- able habitat for the species. The study has also highlighted some of the differences between marshes in the numbers of eels as measured by CPUE. These differences were not entirely explained by the eco- logical variables measured, although, as a single variable, ditch width was signiﬁcantly correlated with CPUE. In a study by Laffaille et al. (2004), ditch width was also found to be signiﬁcant in relation to eel density. However, in the marshes, wider ditches may also mean less vegetative cover, greater abundance of invertebrates and a larger volume of water sampled over the 100 linear metres, all of which may act as confounding factors. Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) Furthermore, smaller eels have been found to prefer less open and more densely vegetated areas (Knights 2003; Tomlinson et al. Surprisingly, CPUE was unaffected by the number of obstacles between the main Thames and the sampled sites. Since most sites were brackish it seems the tidal ﬂaps do not close fully at high tide and therefore may not be an obstacle to the recruitment of smaller eels. It may be that obstacles present more of a barrier to escapement, larger eels being less able to pass through small cracks and climb vertical surfaces (White and Knights 1997). Even further inland, seemingly impassable barriers do not always stand in the way of recruitment. Site B3 appears to be entirely cut off from the outfall by a well-vegetated inﬁll, yet three individuals (53–76 cm) were found there. Although the timing of the inﬁll was unknown, this obstacle looked to be long-established. While it was possible that these eels entered the site before access was impeded, they may have also found their way there as smaller eels migrating partially overland through the shelter of the vegetation. Fig. 2 Mean Fulton’s body condition scores (K) for each marsh in comparison with three rivers in the Thames RBD. Actual K ﬁgures are displayed on second line. Error bars represent standard error and asterisks indicate signiﬁcant differences The wide variation in CPUE between the marshes may be explained by the differences in land use in each marsh. The lowest CPUE was found in Marsh A despite high salinity levels, which suggests good access through the tidal ﬂap. However, Marsh A is a high trafﬁc area, accessible to the public via the Thames Path. At the time of sampling, it was being used for horse grazing and was notorious for vandal- ism, as well as illegal dumping of waste. Many of the most accessible ditches were choked with household and industrial debris. It was the only marsh where there were no signs of water voles, but large numbers of invasive Chinese mitten crabs were caught in the fyke nets. Nevertheless, invertebrate abundance and diversity were comparable to the other marshes. In contrast, Marsh E, which had the highest CPUE, is Fig. 2 Mean Fulton’s body condition scores (K) for each marsh in comparison with three rivers in the Thames RBD. Actual K ﬁgures are displayed on second line. Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) Simple linear regression showed invertebrate abundance to be signiﬁcantly correlated with dissolved oxygen (P = 0.0418, adjusted R2 = 0.13) and ditch width (P = 0.00123, adjusted R2 = 0.3565). Invertebrate sampling at most sites found a heterogenous combination of anticipated invertebrate taxa, including Gammarus, Daphnia, Hydropsychidae, Ephemeroptera, Anisoptera, Cran- gon, Dytiscus and Helobdella with samples from only four sites showing dominance of one taxa ([ 25% of sample) (Table 1). Fulton’s body condition factors [K = (100,000 9 W)/L3 where W is weight in g and L is length in mm] were calculated for each eel caught, as well as mean K for each marsh (Nash et al. 2006). These were compared against mean K for eels caught during Environment Agency surveys on the main tidal Thames (Lundberg 2009 Unpublished Master’s thesis) and two freshwater rivers in the Thames RBD, the Darent (in 2006) and the Wandle (in 2009). Mean scores were plotted together for visual comparison and one-way ANOVA with Tukey’s post hoc analysis conducted. Lengths of eels suggested a predominance of females among the eels captured. Aggregated samples from Marshes A and B showed overall shorter yellow eels (n = 2, mean = 46 cm, s = 5 cm and n = 35, mean = 50.35 cm, s = 8.89 cm, respectively) than Marsh C (n = 21, mean = 80.9 cm, s = 13.09 cm), Marsh D (n = 22, mean = 81.45 cm, s = 9.3 cm) and Marsh E (n = 86, mean = 76.45 cm, s = 8.66 cm). However, given the mesh size was designed to catch only eels longer than 30 cm, it is possible that more males were initially captured, but escaped through the A total of 166 eel were caught over the ﬁve marshes. Marked variation was observed in the number of eels caught at each site and between marshes. In the univariate regression model at the site level, the ‘marsh effect’ explained 14.5% of the variation in CPUE. A signiﬁcant difference was also found between CPUE in Marshes A and E (ANOVA, P = 0.0439) (Fig. 1). Ammonia, nitrite and nitrate levels were at low levels at all sites (1 mg/L or lower). There was also little variation in pH (7–8 units). These variables were 12 3 3 Wetlands Ecol Manage (2018) 26:1181–1188 1186 mesh. Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) Error bars represent standard error and asterisks indicate signiﬁcant differences 12 3 Wetlands Ecol Manage (2018) 26:1181–1188 1187 privately owned and managed as a nature reserve for shooting, with only a small amount of cattle grazing. The land manager regularly monitored and manipu- lated the water ﬂow throughout the marsh to maintain habitat quality for a variety of wild species. Until 2010, the tidal ﬂap had been largely non-functional, impairing both recruitment and escapement of eels. This could have led to eels becoming trapped and contributed to the large size of the eels found there and the lack of smaller eels. However, the outfall had been functional for more than a year when this study took place. would be relatively simple measures that could increase both recruitment and escapement. Modifying land use practices in Marshes A, B, C and D may also improve these otherwise favourable habitats. Acknowledgements This study was funded by the Environment Agency, the Fishmongers Company and the Royal Veterinary College, and was carried out in cooperation with the Zoological Society of London (ZSL) and the Environment Agency. We would like to acknowledge the guidance and input of T. Sainsbury, M. Waters, R. Chang, D. Clifton-Dey, T. Cousins, J. Durkota, P. Howe, D. Kew, M. Rowcliffe and K. Tung. Special thanks are also due to those who assisted with the fyke-netting and ﬁeld surveys: E. Long, A. Thompson, S. Smith, G. Williams, B. Lord, M. Rogers, R. Lucas, L. Parker, D. Williams, A. Andrews, F. Clare, E. Hazard, J. Roche, C. Just, J. Dean, R. Pyper and G. Titheridge. Thanks also to A. Cliffe, D. Curnick, D. Hull, R. Jones, M. Stephens, C. Vance and everyone at the Northﬂeet depot for assistance with equipment, storage space and logistics. Instruction, information and support were provided by D. Clifton-Dey, T. Cousins, J. Durkota, P. Howe, D. Kew, M. Rowcliffe and K. Tung. Finally, we would like to thank the estate manager at Marsh E and the farmers and landowners at Marshes B, C and D for providing a hospitable habitat for eels and researchers alike. The skew towards larger, female eels is noteworthy given that preferential production of females is considered a priority in ensuring the survival of the species (Hildge 2006). Variation between European eel Anguilla anguilla (L.) stocks in ﬁve marshes of the Thames Estuary (United Kingdom) The body condition of the eels found in the marshes relative to those in the river surveys also suggests the marshes are favourable habitat, especially the signiﬁcant difference in body condition between marsh E and the two rivers. However, there is uncertainty regarding the quality of the unpublished data from the river surveys. Recording errors for several eels were noted in the Thames dataset and the Wandle dataset used weights derived from the National Fisheries Laboratory stan- dard length–weight measurement (Britton and Shep- herd 2005) rather than actual observed weights. These variations and discrepancies underline problems in data quality in eel monitoring practice. Nevertheless, the consistency of higher body condition across marshes B through E relative to the rivers may be biologically signiﬁcant. Belpaire et al. (2009) suggest a minimum of 20% body fat is required to complete the oceanic spawning migration with further stores required for gonad maturation and egg production. Distances from continental habitats vary by up to 4000 km so body condition may be even more important in Northern latitudes in relation to con- tributing viable spawners to the overall European stock (Clevestam et al. 2011). 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 unre- stricted 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 Com- mons license, and indicate if changes were made. Funding Royal Veterinary College, MSc student grant. Fishmongers Company, small grant for equipment. Environment Agency, in-kind funding for supplies and staff time. Funding Royal Veterinary College, MSc student grant. Fishmongers Company, small grant for equipment. Environment Agency, in-kind funding for supplies and staff time. References Acou A, Rivot E, Van Gils JA et al (2010) Habitat carrying capacity is reached for the European eel in a small coastal catchment: evidence and implications for managing eel stocks. Freshw Biol 56:952–968. https://doi.org/10.1111/j. 1365-2427.2010.02540.x This study provides evidence of continued use of these marshes by European eels and suggests they are quality growth habitats. It also reveals substantial variability in abundance of larger yellow eels between the marsh ditch systems, which may be explained by land and water management practices. 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https://openalex.org/W4256498768	http://ojs.unemi.edu.ec/index.php/cienciaunemi/article/download/174/177	Spanish; Castilian	null	Editorial	Ciencia UNEMI/Ciencia UNEMI (En línea)	2,015	cc-by	453	La producción científica y la acreditación de carreras de Producción regional se considerarán los artículos publicados o aceptados para publicación en Emerald, Pro-Quest, EBSCO, JSTOR, SCIELO, REDALYC, LI- LACS, OAJI, DOAJ. P P or lo dispuesto en la Ley Orgánica de Educa- ción Superior (LOES) vigente en el Ecuador, se cumplió con la acreditación de las Univer- sidades, proceso que culminó en noviembre de 2013 y en el cual se categorizó a las Universidades de “A” a “D”; en la actualidad es necesario que las carreras sean evaluadas para su posterior acreditación. Para la acreditación de carreras el Consejo de Evalua- ción, Acreditación y Aseguramiento de la Calidad de la Educación Superior (CEAACES) ha emitido, en marzo del 2015, el modelo genérico de evaluación del entorno de aprendizaje de carreras presenciales y semipresenciales de las universidades y escuelas politécnicas del Ecuador. Ante las exigencias de publicación, que deberán cumplir los profesores del sistema de educación su- perior del país, nos encontramos que en el Ecuador son escasas las revistas que se ubican dentro de las bases de datos exigidas por el CEAACES. Por ejem- plo: el ente acreditador para revistas del catálogo SCIELO, en el portal SCImago Journal & Coun- try Rank (Scopus: http://www.scimagojr.com), evidencia que al año 2014, de Ecuador solo están indexadas 2 revistas, activa solo 1 de ellas, por lo tanto como país a esa fecha no teníamos revistas en esa base de datos. En el modelo genérico para la evaluación de las ca- rreras se considera a la Investigación en el criterio deno- minado Producción Académica, el cual engloba los indi- cadores: producción académico – científica, producción regional, libros o capítulos de libros y ponencias. Por lo expuesto, en nuestra institución con el apoyo de las principales autoridades, se trabaja en la creación de revistas especializadas, donde los pro- fesores podrán dar a conocer sus investigaciones, el proceso para llegar a los catálogos exigidos nos to- mará varios años, por el momento nuestra revista Ciencia UNEMI ya se encuentra en formato digital (www.unemi.edu.ec/ojs), a mediano plazo estaremos solicitando el ingreso y pasaremos a formar parte de las bases de datos exigidas por CEAACES, para publi- caciones regionales. De los indicadores establecidos es importante resaltar los estándares que se utilizarán en la eva- luación de la producción académico – científica y producción regional. Editorial Editorial Editorial La producción científica y la acreditación de carreras Para la Producción académico – científica, solo se considerarán trabajos publica- dos o aceptados para su publicación en revistas que figuran en las bases de datos SCIMAGO (Scopus), o en las bases del ISI Web of Knowledge. En el caso Director Departamento de Investigación, Desarrollo Tecnológico e Innovación Ing. Richard Ramírez Anormaliza, PhD (c) │ 7
https://openalex.org/W2998072429	https://hess.copernicus.org/articles/24/1691/2020/hess-24-1691-2020.pdf	English	null	Turbulence in the stratified boundary layer under ice: observations from Lake Baikal and a new similarity model	Hydrology and earth system sciences	2,020	cc-by	14,826	Correspondence: Georgiy Kirillin (kirillin@igb-berlin.de) Correspondence: Georgiy Kirillin (kirillin@igb-berlin.de) Received: 12 November 2019 – Discussion started: 9 December 2019 Revised: 2 March 2020 – Accepted: 5 March 2020 – Published: 8 April 2020 Received: 12 November 2019 – Discussion started: 9 December 2019 Revised: 2 March 2020 – Accepted: 5 March 2020 – Published: 8 April 2020 Abstract. Seasonal ice cover on lakes and polar seas creates seasonally developing boundary layer at the ice base with speciﬁc features: ﬁxed temperature at the solid boundary and stable density stratiﬁcation beneath. Turbulent transport in the boundary layer determines the ice growth and melting conditions at the ice–water interface, especially in large lakes and marginal seas, where large-scale water circulation can produce highly variable mixing conditions. Since the bound- ary mixing under ice is difﬁcult to measure, existing models of ice cover dynamics usually neglect or parameterize it in a very simplistic form. We present the ﬁrst detailed observa- tions on mixing under ice of Lake Baikal, obtained with the help of advanced acoustic methods. The dissipation rate of the turbulent kinetic energy (TKE) was derived from corre- lations (structure functions) of current velocities within the boundary layer. The range of the dissipation rate variability covered 2 orders of magnitude, demonstrating strongly tur- bulent conditions. Intensity of mixing was closely connected to the mean speeds of the large-scale under-ice currents. Mix- ing developed on the background of stable density (temper- ature) stratiﬁcation, which affected the vertical structure of the boundary layer. To account for stratiﬁcation effects, we propose a model of the turbulent energy budget based on the length scale incorporating the dissipation rate and the buoy- ancy frequency (Dougherty–Ozmidov scaling). The model agrees well with the observations and yields a scaling rela- tionship for the ice–water heat ﬂux as a function of the shear velocity squared. The ice–water heat ﬂuxes in the ﬁeld were the largest among all reported in lakes (up to 40 Wm−2) and scaled well against the proposed relationship. The ultimate ﬁnding is that of a strong dependence of the water–ice heat ﬂux on the shear velocity under ice. The result suggests large errors in the heat ﬂux estimations when the traditional “bulk” approach is applied to stratiﬁed boundary layers. It also im- plies that under-ice currents may have much stronger effect on the ice melt than estimated by traditional models. 1 Introduction The demand for a better quantitative description of the for- mation, evolution, and decay of seasonal ice has grown re- cently because of large-scale trends toward a shortening ice season in the Northern Hemisphere and the drastic decrease of the Arctic sea ice extent. Closure of the global mass bud- get of the Arctic seasonal ice is a complex problem, related, apart from the atmospheric and terrestrial heat sources, to the upward transport of heat stored in the under-ice water body. An important role in the heat budget of seasonal ice is played by the storage of the solar radiation in the under- ice water, which is subsequently transported to the ice base by the under-ice currents. The effect of currents on ice melt is particularly apparent in the Arctic ocean, where the loss of ice mass in spring and summer occurs mainly from the ice bottom (McPhee, 1992; Perovich et al., 2011; Carmack et al., 2015; Peterson et al., 2017). Apart from the polar oceans and seas, seasonal formation of ice cover is an essential feature of high-latitude freshwater lakes. Physics of seasonal ice cover Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 https://doi.org/10.5194/hess-24-1691-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 https://doi.org/10.5194/hess-24-1691-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. giy Kirillin1, Ilya Aslamov2, Vladimir Kozlov3, Roman Zdorovennov4, and Nikolai Granin2 Georgiy Kirillin1, Ilya Aslamov2, Vladimir Kozlov3, Roman Zdorovennov4, and Nikolai Granin2 1Department of Ecohydrology, Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany 2Department of Hydrology and Hydrophysics, Limnological Institute, Siberian Branch of Russian Academy of Sciences, Irkutsk, Russia 3Institute for System Dynamics and Control Theory, Siberian Branch of Russian Academy of Science, Irkutsk, Russia 4Northern Water Problems Institute (NWPI), Karelian Research Center, Russian Academy of Sciences, Petrozavodsk, Russia Siberian Branch of Russian Academy of Sciences, Irkutsk, Russia 3Institute for System Dynamics and Control Theory, Siberian Branch of Russian Academy of Science, Irkutsk, Russia 4Northern Water Problems Institute (NWPI), Karelian Research Center, Russian Academy of Sciences, Petrozavodsk, Russia G. Kirillin et al.: Turbulence under ice In addition to the storage of the heat from the short- wave radiation penetrating the ice, convective mixing in the SML entrains the warmer water from the deep layers (Kir- illin et al., 2012). The convective SML is separated from the ice base by a stably stratiﬁed interfacial layer (IL) with an upward temperature drop down to the freezing point of freshwater. At low salinities, water temperatures remain al- ways higher than that of ice. Hence the IL with a down- ward temperature increase always exists under the ice base; its thickness depends on the strength of radiative heating and the temperature of the SML underneath (Barnes and Hobbie, 1960). The strong stratiﬁcation in the IL prevents convective mixing despite the negative buoyancy production by the de- crease of the solar radiation with depth and reduces convec- tive transport of heat to the ice–water interface. As a result, only a small amount of the heat is available for ice melt, de- spite strong convection in the SML (Kirillin et al., 2018). The situation is akin to formation of a stably stratiﬁed layer (SL) beneath the ice base and the near-surface temperature maxi- mum in marginal polar seas, driven by freshening of the sur- face waters due to runoff or accelerated sea ice melt (Jackson et al., 2010, 2012). p y The intensity of turbulence produced by velocity shear in the boundary layer and the resulting heat transport from wa- ter to the ice base may vary depending on the current velocity, ice structure, and density stratiﬁcation under ice. In order to estimate the effect of under-ice circulation on the ice–water heat ﬂux in lakes, we performed a ﬁeld experiment combin- ing temperature measurements with high temporal and ver- tical resolution within the ice cover and ﬁne-scale registra- tion of current velocities under the ice base. The temperature observations were subsequently used for estimation of the heat budget at the ice–water interface and derivation of the ice–water heat ﬂuxes. The data on ﬁne-scale velocity ﬂuctu- ations provided information on variability of mean currents under ice as well as on the characteristics of turbulent mixing in the ice–water boundary layer in the form of the dissipation rate of the turbulent kinetic energy (TKE). G. Kirillin et al.: Turbulence under ice on lakes has gained particular attention, as an essential part of climate change research (Magnuson et al., 2000; Kirillin et al., 2012). A shorter seasonal ice cover as a result of global warming may produce a positive feedback due to increase of greenhouse gas emission and changing the global carbon budget (Tranvik et al., 2009). Hence, estimation of the con- sequences of phenological changes on inland waters requires quantiﬁcation of the physical mechanisms that control the formation and melting of ice. The heat and mass transfer at the ice–water interface (IWI) is the least studied among these mechanisms (Kirillin et al., 2012; Aslamov et al., 2014a). inﬂuence of the Siberian atmospheric pressure maximum, Lake Baikal has steady ice cover over the entire lake for 3– 5 months of the year. Consequently, the seasonal ice regime plays a crucial role in hydrodynamics and ecosystem func- tioning of the lake. Aslamov et al. (2014a, b) reported high water-to-ice heat ﬂuxes in Lake Baikal during the period of ice growth. The ﬂuxes were apparently related to the sur- face water circulation pattern beneath the ice cover (Zhdanov et al., 2017). The observed ice-to-water heat ﬂuxes exceeded ﬂuxes measured in small lakes (Kirillin et al., 2018) by up to an order of magnitude. Free convection due to penetrat- ing solar radiation was not strong enough to produce upward heat release at these rates. Hence, the high turbulence level was tentatively attributed by the authors to the shear mixing produced by the water circulation under the ice surface. The seasonal ice cover on lakes, especially on large ones, shares many basic features with the seasonal sea ice. Storage of the heat from solar radiation in the surface mixed layer (SML) and its subsequent release to the ice base is the ma- jor mechanism of the ice cover melt in lakes (Kirillin et al., 2012) as well as in the ocean (Perovich and Richter-Menge, 2009). However, in contrast to seawater, lakes possess some speciﬁc physical features affecting formation and melting of ice. Water temperatures in ice-covered freshwater lakes are below their value of maximum density. Therefore, solar heating of upper layers produces free convection, which is the major mechanism of the SML formation (Mironov et al., 2002). G. Kirillin et al.: Turbulence under ice Below, both out- comes of the ﬁeld experiment are combined to analyze the characteristics of the turbulent boundary layer and to ana- lyze the effect produced by turbulent mixing on the ice cover thickness. The overarching goal of the presented study is to establish the scaling relationships linking the under-ice circu- lation with the seasonal ice cover dynamics and suitable for parameterization of the ice–water heat exchange in regional and global models of seasonal ice. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 1692 G. Kirillin et al.: Turbulence under ice does not typically exceed several millimeters. The temper- ature gradient dT dz−1 at z = 0 is barely detectable by the traditional observation methods and varies continuously de- pending on the mixing and temperature conditions in the un- derlying water column. ice–water interface in “rotten” freshwater ice subject to inter- nal melting, especially in the presence of impurities (Bluteau et al., 2017). In the majority of freshwater lakes, the afore- mentioned effects are negligible during most of the ice sea- son. In particular, in Lake Baikal, due to cold winters and low snow precipitation, practically 100 % of the ice cover consists of clear congelation (“black”) ice, which grows at the ice–water interface and has homogeneous crystal struc- ture and a much smaller amount of impurities than sea ice or river ice (Kirillin et al., 2012). Hence, the heat balance at the IWI can be expressed as the sum of conductive (molecular) boundary ﬂuxes and the heat release/consumption due to the phase change (freezing or melting) (Aslamov et al., 2014a): y g The TKE under ice is supplied by the free convection in the underlying convectively mixed layer (Mironov et al., 2002) and/or by the mean horizontal current (McPhee, 1992; Aslamov et al., 2014a). In the latter case, the vertical turbu- lent transport of momentum τ =< u′w′ >= u2 ∗is created by the current velocity shear S = ∂Umean∂z−1 at the ice base. Hence, close to the IWI, the distance from the ice base z is a major parameter, determining the turbulent mixing char- acteristics. 2 Heat budget of seasonal ice cover and scaling of the under-ice boundary layer To a good approximation, the base of the lake ice can be rep- resented as a rigid boundary on top of a ﬂuid; i.e., the verti- cal heat transport at the ice–water interface is close to being purely conductive on both ice and water sides, governed by molecular forces within the ice cover and within a thin “con- duction” layer of water. It should be noted that the assump- tion generally holds true for solid freshwater ice with a small amount of impurities: Saltwater ice undergoes brine extrac- tion, which can induce convection by mass ﬂux at the bound- ary and markedly increase the heat transport. Similarly, the increase of water ﬂow can destroy the conduction layer at the The ice–water interaction becomes more complex when a freshwater lake becomes essentially large compared to the Rossby radius of deformation (Gill, 1982). The condition suggests long-lasting water circulation under ice, which, sim- ilarly to the ocean circulation, is able to produce signiﬁ- cant velocity shear at the ice base and thus accelerate the upward heat transport (McPhee, 1992). Among such lakes, Lake Baikal – the largest lake by volume on earth – most closely resembles the Arctic Ocean with regard to seasonal ice dynamics. Thanks to the strong winter cooling under the www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 1693 G. Kirillin et al.: Turbulence under ice Assumption of the proportionality between the mixing length scale and the distance from the solid boundary leads to the relationships for the neutral (logarithmic) bound- ary layer, Qiw = Qci −ρiLf dhi dt , (1) (1) where the vertical coordinate is directed downwards with the origin zi at the IWI; dhidt−1 is the rate of basal ice melting (growth); ρiLf is the product of the ice density and latent heat of fusion; Qiw is the conductive heat ﬂux from/to the water, S = ∂Umean ∂z = u∗ κz, (4) (4) Qiw = Cpwρwκw dT dz zi+0 ; (2) Umean(z) = u∗ κ ln z z0 , (5) (2) (5) and Qci is the conductive heat ﬂux from/to the ice, where z is the turbulent pulsation length scale equal to the distance from the lower boundary of the ice, z0 is the rough- ness length at the ice bottom, κ ≈0.4 is the empirically de- termined von Kármán constant, and u2 ∗is the turbulent stress (shear velocity squared) produced by the vertical shear of the mean velocity S. Qci = Cpiρiκi dT dz zi−0 . (3) (3) Temperature at the IWI is ﬁxed at the melting point of 0 ◦C, corresponding to the following thermodynamic characteris- tics: the molecular heat diffusion coefﬁcient for freshwater κw ≈1.4 × 10−7 m2 s−1, the molecular heat diffusion coefﬁ- cient for ice κi ≈1.1×10−6 m2 s−1, the product of the water heat capacity and density is Cpwρw ≈4.18 × 106 JK−1 m−3, and the same product for ice is Cpiρi ≈1.96×106 JK−1 m−3 (see, e.g., Leppäranta, 1983). The logarithmic velocity distribution in the ice cover vicin- ity makes possible estimation of the momentum ﬂux based on mean velocity values only using a direct relationship de- rived from Eq. (5) (“bulk” formula), u2 ∗= CZU2 z , (6) u2 ∗= CZU2 z , (6) Equation (1) can be applied for reliable estimation of the ice–water heat ﬂux Qwi if the temperature proﬁle within the ice cover and the time variations of the ice thickness dhi dt−1 are known. This approach was used for estimation of the heat ﬂuxes in Lake Baikal by Aslamov et al. (2014a), who recorded the temperature proﬁle within the ice cover and the variations of the ice thickness dhidt−1 with high temporal resolution. www.hydrol-earth-syst-sci.net/24/1691/2020/ G. Kirillin et al.: Turbulence under ice water column of thickness hS. The buoyancy ﬂux is derived from the heat transport equation with the radiation term S = u∗ κz 1 + CN zN3/2 ε1/2 , (11) (11) BR = β  I(0) + I(hS) −2h−1 S hS Z 0 I(z)dz  . (8) (8) with the corresponding expression for the TKE production rate P being (cf. Eq. 7) with the corresponding expression for the TKE production rate P being (cf. Eq. 7) (8) P = u2 ∗S = u3 ∗ κ 1 z + CN LN . (12) Here, the assumption of a height-constant warming rate within the convective layer was used (Mironov et al., 2002). Here, the assumption of a height-constant warming rate within the convective layer was used (Mironov et al., 2002). β = gαT (T ) is the buoyancy parameter; αT is the ther- mal expansion coefﬁcient. The latter is generally not con- stant in freshwater due to non-linearity of the equation of state at temperatures close to the maximum density value Tmd ≈3.98 ◦C (αT (T ) ≈0.825 × 10−5(T −Tmd) K−1; see, e.g., Farmer and Carmack, 1981). (12) β = gαT (T ) is the buoyancy parameter; αT is the ther- mal expansion coefﬁcient. The latter is generally not con- stant in freshwater due to non-linearity of the equation of state at temperatures close to the maximum density value Tmd ≈3.98 ◦C (αT (T ) ≈0.825 × 10−5(T −Tmd) K−1; see, e.g., Farmer and Carmack, 1981). Close to the boundary, z ≪LN, Eq. (11) approaches the neu- tral scaling relationship 4. At large distances from the bound- ary, z ≫LN, Eq. (11) turns to a “z-less” scaling S = CN u∗ κLN , (13) S = CN u∗ κLN , (13) The ice–water boundary layer in freshwater lakes is rarely neutrally stratiﬁed: a distinctive feature of the layer is the ﬁxed temperature at the IWI. As a result, the water adjacent to the IWI in fresh or brackish environments is always subject to stable stratiﬁcation, with deeper waters being warmer and thereby heavier. Stratiﬁcation may alter the turbulent length scale, affecting Eqs. (4)–(7). Stratiﬁcation counteracts the shear production of turbulence and in the asymptotic case of a strongly stratiﬁed layer is the sole mechanism of turbu- lence damping. G. Kirillin et al.: Turbulence under ice This effect can be accounted for by introduc- ing an additional length scale apart from the distance to the boundary z, as expressed by a simple formula following from dimensional analysis, which in turn yields the N scaling for P which in turn yields the N scaling for P P ∝u3 ∗L−1 N . P ∝u3 ∗L−1 N . In stably stratiﬁed conditions, production of TKE is counter- acted by two major loss processes, dissipation ε and work against the stability forces BSt. The latter can be expressed in the form BSt = KρN2, where Kρ is the diapycnal diffu- sivity. From analysis of dimensions, the turbulent diffusivity can be scaled as Kρ ∝u2 ∗N−1 (see, e.g., Monin and Ozmi- dov, 1981). Then, the TKE budget can be approximated as u3 ∗ κ 1 z + CN LN = CBu2 ∗N + ε, (14) (14) S = u∗ κz 1 + Cx z Lx , (9) (9) with coefﬁcients CN and CB subject to estimation from em- pirical data, or where Lx is the stratiﬁcation length scale and Cx is an em- pirical coefﬁcient. Equation (9) replaces Eq. (4) in strat- iﬁed conditions with corresponding changes in Eqs. (5)– (7). For conditions dominated by the stabilizing buoyancy ﬂux at the boundary Bs = βQiw(cpwρw)−1, the stratiﬁcation length scale turns to the well-known Monin–Obukhov length scale LMO = u3 ∗B−1 s , with the empirically determined coefﬁ- cient Cx ≈5 (Stull, 2012), building the basis for the Monin– Obukhov similarity theory (MOST). u2 ∗N Ri−1/2 −CB = ε, (15) (15) where Ri is the gradient Richardson number, Ri = N2 S2 , (16) (16) expressing the relative importance of stratiﬁcation and veloc- ity shear for the vertical transport. Its critical value, Ricr ≈ 1/4 (Turner, 1979), marks the boundary between turbulent conditions, at which the shear can destroy the stratiﬁca- tion, and quiet conditions, at which strong N ultimately sup- presses any turbulent motions. Hence, for turbulence to ex- ist at weakly supercritical Ri, 0 < CB < 2 is required in Eq. (15). In the following we tentatively assume CB ≈1. Another scaling relationship relevant to the turbulent mix- ing on the background of stable stratiﬁcation is the buoyancy Reynolds number, If stratiﬁcation is created outside the boundary layer, its effect on boundary mixing is independent on the surface buoyancy ﬂux. G. Kirillin et al.: Turbulence under ice where the bulk transfer or drag coefﬁcient CZ corresponds to the depth Z of the current speed measurements and is deﬁned as where the bulk transfer or drag coefﬁcient CZ corresponds to the depth Z of the current speed measurements and is deﬁned as as as CZ = κ lnz −lnz0 2 . For a non-stratiﬁed steady-state turbulent boundary layer, the TKE budget tends to be the local balance of the largest terms in the TKE transport equation, production and dissipa- tion: However, direct estimation of Qiw in the absence of de- tailed data on the ice cover dynamics and temperature is less straightforward. The bulk of the water column under the ice is turbulent: while ice-covered waters are isolated from the direct inﬂuence of wind, vertical heat transport remains higher than the purely molecular one, intensiﬁed by convec- tive mixing due to solar radiation penetrating the ice cover and due to shear turbulence produced by under-ice currents. As a result, the thickness of the “diffusive” layer in the im- mediate vicinity of the ice base, where Eq. (2) holds true, ε = u2 ∗S ∝u3 ∗ z , (7) (7) (7) where ε is the TKE dissipation rate. where ε is the TKE dissipation rate. The second factor inﬂuencing the buoyancy ﬂux at the IWI is the destabilizing buoyancy ﬂux BR due to volumetric ab- sorption of solar radiation I(z) within the convectively mixed www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 1694 G. Kirillin et al.: Turbulence under ice G. Kirillin et al.: Turbulence under ice Two-dimensional electromagnetic current meters, “INFINITY-EM” (JFE Ad- vantech Co., Ltd.), were used to measure the current veloc- ities (velocity range: ±5ms−1; resolution: 0.02 cms−1; ac- curacy: ±1cms−1). The current meters were positioned at a distance of 1 m from the surface of the ice cover. Three additional current meters were deployed at station S1 at dis- tances of 0.6, 0.8, and 1.4 m from the ice surface. where 1T is the temperature difference across the layer hT beneath the ice base, often assumed in models as water tem- perature at the vertical grid point closest to the ice. The ex- pression is sometimes used in the form of the bulk formula- tion, assuming direct relationship between the friction veloc- ity and the main current speed u∗∝Umean (cf. Eq. 6): (19) Qiw = CQUmean1T, (19) where 1T is the temperature difference across hT , and CQ is an empirical bulk heat transfer coefﬁcient. The values of CQ were reported in the range [0.8 ± 0.3] × 10−3 (Hamblin and Carmack, 1990; Nan et al., 2016); stratiﬁcation effects on CQ were not investigated. When the same scaling considerations are adopted as in Eqs. (14)–(15), the heat ﬂux at the IWI Qiw in strongly strat- iﬁed conditions may be assumed to depend on the work of turbulence against the stability, Qiw = Kρ ∂T ∂z ∝u2 ∗N−11T h−1 T , or, in terms of buoyancy ﬂux Biw, or, in terms of buoyancy ﬂux Biw, Biw = gαT Qiw ∝u2 ∗N. (20) (20) Herewith, a strongly stratiﬁed case is characterized by the ﬂux dependence on the shear velocity squared and a weaker dependence on the stratiﬁcation, expressed by the exponent 1/2 at the vertical density gradient (as revealed by the direct proportionality to the buoyancy frequency N). Characteristics of turbulent mixing in the under-ice bound- ary layer were obtained with the help of the high-resolution Doppler current velocity proﬁler HR-Aquadopp (Nortek AS, Norway). The proﬁler was deployed for 48 h successively at each of the two stations, on 5–7 March 2017 at station S1 and on 8–10 March at station S2. The proﬁler was frozen into the ice facing downward, with the acoustic head 2 cm be- neath the ice base (veriﬁed with a remotely operated vehicle (ROV) camera). G. Kirillin et al.: Turbulence under ice which becomes important at distances from the solid bound- ary shorter than characteristic length scales of turbulence; its critical value is reported to be O(101) (Gargett et al., 1984). tonomous stations were installed in the vicinity of a quasi- stationary alongshore current (Fig. 1). Under-ice currents with characteristic velocity and spatial scales of 10−2– 10−1 ms−1 and 104–105 m, respectively, have been regularly observed in this region during the ice cover period (Aslamov et al., 2014a, 2017). The scales of the ﬂow suggest that away from boundaries it is balanced mainly by the Coriolis force, while the forcing may be attributed to density gradients cre- ated by in- and outﬂows, and topographic effects. In neutral conditions, the coefﬁcient of turbulent heat transfer KZ ∝u∗z (assuming the turbulent Prandtl number is approximately 1), and the corresponding relationship for the ice–water heat ﬂux Qiw (Eq. 2) can be written as Qiw ∝u∗1T ∝u∗hT gαT N2, (18) (18) Station S1 was installed 4.5 km away from the lake shore (51◦51.923′ N, 105◦4.779′ E) in the area of quasi-stationary jet-like alongshore current. Station S2 was located 3.5 km to the south of station S1. The total water depth in the vicin- ity of both stations amounted to ≈1400m. Each station reg- istered temperature at 30 vertical levels distributed within the ice cover, the water boundary layer, and the air above the ice. The distance between temperature sensors was 5 cm within the ice and in the under-ice boundary layer, increas- ing up to 10–50 cm at larger distances from the ice bound- ary in the water and in the air. Three short-wave solar ra- diation sensors were deployed vertically to measure the de- cay of solar radiative ﬂuxes across the air–ice–water system. Ice thickness was measured by a 330 kHz echo sounder, de- ployed upward-looking at a ﬁxed distance from the ice sur- face. The resolution of the system was 0.002 ◦C for tempera- ture, 0.1 Wm−2 for solar radiation, and 0.1 mm for ice thick- ness (operational range of 0.2–2.8 m). The system collected data for a period of 2 min, logged them internally, and sent data several times a day via a cellular network to a remote Internet server (see Aslamov et al., 2017, for a detailed de- scription of the ice station conﬁguration). G. Kirillin et al.: Turbulence under ice Three components of current velocity were registered with a time interval of 2 s and a spatial resolution of 15 mm in the pulse-to-pulse coherence (high-resolution) mode. Summarizing the considerations above, validation of the Dougherty–Ozmidov (D–O) scaling (Eqs. 10–14) for the ice boundary layer and the ice–water ﬂux parameterization (Eqs. 18–20) is possible when ﬁeld data are available on both the TKE dissipation rates and the mean ﬁelds of governing forces (production of convective instability by radiative heat- ing and/or mean horizontal ﬂow due to under-ice currents). G. Kirillin et al.: Turbulence under ice A characteristic length scale for turbulence in stratiﬁed media was independently proposed by Dougherty (1961) and Ozmidov (1965) as LN = ε1/2N−3/2, (10) LN = ε1/2N−3/2, (10) where where N = g ρ ∂ρ ∂z 1/2 ≈ β ∂T ∂z 1/2 N = g ρ ∂ρ ∂z 1/2 ≈ β ∂T ∂z 1/2 Reb = ε νN2 , (17) (17) is the buoyancy frequency and ρ is water density under the assumption of negligible salinity effects. Replacement of Lx by LN in Eq. (9) yields in this case where ν = O(10−6) m2 s−1 is the water viscosity. Reb refers to the work of turbulence against stratiﬁcation and viscosity, www.hydrol-earth-syst-sci.net/24/1691/2020/ www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 1695 3 Study site and ﬁeld methods The values of short-period ﬂuctuations of the ﬂow veloc- ity were used to calculate dissipation rates of TKE based on Kolmogorov’s 1941 hypothesis on the self-similarity of the velocity structure functions using the method described by Wiles et al. (2006). ε was derived as a coefﬁcient in the semi- The ﬁeld study was performed in February–March 2017 in the southern part of Lake Baikal. Two custom-made au- www.hydrol-earth-syst-sci.net/24/1691/2020/ www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 1696 G. Kirillin et al.: Turbulence under ice Figure 1. (a) Geographical location of the study site. Ice conditions in the southern part of Lake Baikal on (b) 9 February and (c) 12 April 2017 and locations of the autonomous measurement stations. The satellite images are from the Irkutsk Center of Remote Sensing (sput- nik.irk.ru, 2019). Note the stronger ice melt in the area of the jet current around station S1, visible as a dark area in (c). Figure 1. (a) Geographical location of the study site. Ice conditions in the southern part of Lake Baikal on (b) 9 February and (c) 12 April 2017 and locations of the autonomous measurement stations. The satellite images are from the Irkutsk Center of Remote Sensing (sput- nik.irk.ru, 2019). Note the stronger ice melt in the area of the jet current around station S1, visible as a dark area in (c). empirical equation for the velocity structure function Di(r) along the ith acoustic beam, 4.1 Atmospheric conditions and ice cover thickness Di(r) = Noise + Cvε2/3r2/3, (21) (21) The ice cover formed on Lake Baikal during the second half of January 2017, with several periods of ice break-up and re- freeze. The autonomous stations were installed on 1 Febru- ary 2017 and provided background information on the major forces driving the ice cover development. The temperatures of the ice surface remained below the freezing point of water during the entire observation period, varying between −14 and −2 ◦C with a slight warming trend (Fig. 2a). The initial ice thicknesses were nearly the same at both stations: 23 cm at station S1 and 24 cm (a day later) at station S2. During the ﬁrst 2 weeks of February the ice grew at a nearly constant rate of 1.2–1.3 cmd−1 (Fig. 2b). During this period, the ice surface temperatures at both stations were nearly equal and followed closely the air temperatures at a height of 1.5 m. This quasi-neutral stratiﬁcation in the air–ice boundary layer lasted until the end of February, caused apparently by con- vective heat ﬂux from the ice surface due to release of the latent heat of ice formation. Later, the ice thickness at sta- tion S1 (the one with strong under-ice currents) remained which includes noise estimation (Noise) due to instrumen- tal noise and non-turbulent velocity ﬂuctuations. Here, the constant Cv = 31/3 (see, e.g., Lien and D’Asaro, 2002). The velocity structure function was calculated from the measured along-beam velocities vi(z) at the distance z from the instru- ment’s head as which includes noise estimation (Noise) due to instrumen- tal noise and non-turbulent velocity ﬂuctuations. Here, the constant Cv = 31/3 (see, e.g., Lien and D’Asaro, 2002). The velocity structure function was calculated from the measured along-beam velocities vi(z) at the distance z from the instru- ment’s head as Di(r) = D (vi(z) −vi(z + r))2E . (22) (22) A quality check was performed based on values of Noise in Eq. (21); the ε values from three beams were compared for similarity and averaged. The detailed procedure of data post- processing and the quality check is described by Kirillin et al. (2018) and Volkov et al. (2019). Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 www.hydrol-earth-syst-sci.net/24/1691/2020/ G. Kirillin et al.: Turbulence under ice 1697 Figure 2. 4.1 Atmospheric conditions and ice cover thickness Background data on the Lake Baikal ice regime during the observations: (a) daily averaged temperatures of ice surface (tice) and air temperatures at 1.5 m above the ice (tair), (b) ice thickness, (c) incoming solar radiation, and (d) penetrated solar radiation. In (a) and (b) solid lines correspond to station S1 and dotted lines are for station S2. 1697 G. Kirillin et al.: Turbulence under ice G. Kirillin et al.: Turbulence under ice Figure 2. Background data on the Lake Baikal ice regime during the observations: (a) daily averaged temperatures of ice surface (tice) and air temperatures at 1.5 m above the ice (tair), (b) ice thickness, (c) incoming solar radiation, and (d) penetrated solar radiation. In (a) and (b) solid lines correspond to station S1 and dotted lines are for station S2. Figure 3. Temperature and current velocity vector proﬁles mea- sured from 14:00 to 16:00 LT (local time) on 6 March 2017 at (a) station S1 and (b) station S2. nearly constant, while basal ice at station S2 continued to grow at a slow rate of ≈0.3 cmd−1 (Fig. 2b). In mid-March, a stable stratiﬁcation developed in the air above the ice, with air temperatures dropping down to −16 ◦C. Whereas the temperature at the ice surface of station S2 also decreased following the air temperature trend, the ice surface at sta- tion S1 remained relatively warm, suggesting, together with the nearly constant ice thickness, a balance between the heat release to the atmosphere and the heat supply from the water column. 4.2 Mean currents, temperatures, and stratiﬁcation Figure 3. Temperature and current velocity vector proﬁles mea- sured from 14:00 to 16:00 LT (local time) on 6 March 2017 at (a) station S1 and (b) station S2. The thermal structure was similar at both stations S1 and S2 (Fig. 3): under ice, the water temperatures slightly in- creased with depth. The mean vertical gradient of 0.6 ◦C over the upper 10 m of the water column was about an order of magnitude weaker than those typically observed in shallow ice-covered lakes (Kirillin et al., 2018). Below 10 m depth the water column was well mixed vertically down to 30 m. Closer to the ice base, two horizontal layers could be distin- guished: a < 0.5m thin layer adjacent to the ice with a tem- perature difference of ≈0.3 ◦C across it. Underneath, a layer with nearly linear temperature increase of ≈0.03 ◦Cm−1 spread down to 10 m depth. In terms of stability, the two-layered thermal structure can be described by two nearly constant buoyancy frequen- cies: Nδ ≈2 × 10−2 s−1 in the layer 0 ≤z < δ and NS ≈ www.hydrol-earth-syst-sci.net/24/1691/2020/ 4.3 Solar radiation The solar radiation ﬂux at the ice surface doubled within the 2 months of observations (Fig. 2c), contributing to the deceleration of the ice growth. The light conditions under ice were estimated from continuous measurements of photo- synthetically active radiation (PAR) I(z) at the single depth z = 1.5m under the ice surface assuming one-band exponen- tial decay of radiation ﬂux (Beer’s law) I(z) = I0 exp(−γ z). We estimated the decay rate of radiation within the wa- ter column (light attenuation coefﬁcient) γ using PAR pro- ﬁles collected in previous studies in 2011, using the evi- dence that year-to-year variations of water transparency of Lake Baikal are small (Hampton et al., 2008). The light attenuation coefﬁcient was estimated from nine PAR pro- ﬁles as γ ≈0.17 ± 0.01m−1. The radiation ﬂux at the ice bottom amounted to ≈1–18 % of the surface radiation and varied depending on the snow conditions at the ice surface (Fig. 2d). The mean daily under-ice short-wave radiation was I0 = 9.7 Wm−2 with maximum values of up to 23.5 Wm−2. The drop of the under-ice solar radiation after 23 February (Fig. 2d) was caused by a (relatively light, < 0.5cm) snow- fall, which prevented light penetration through the otherwise transparent congelation ice. Variations in the under-ice radi- ation could have affected the temperature distribution under ice by slowing down or even canceling the warming in the convectively mixed layer at depths below 10 m. y y The diurnal and synoptic variations of the ice and water temperatures were similar to those observed in the previ- ous years (Aslamov et al., 2014a, 2017). The diurnal tem- perature oscillations driven by the solar radiation cycle were apparent in both water column and ice cover, with ampli- tudes decaying towards the ice–water interface. The begin- ning of the melt phase after 26 March 2017 was indicated by homogenization of the ice cover temperature at the melting point of 0 ◦C. Earlier, occasional increases of the air temper- ature, for instance on 25 February, provoked deceleration of the ice growth or short-term melting periods at both stations (Fig. 4). Relevant to the matter of the present study, a re- markable increase of the ice temperatures was observed at both stations during the period of turbulence measurements on 6–12 March. The warming was not correlated with the air temperature: the latter dropped signiﬁcantly during the same time (Fig. 2). G. Kirillin et al.: Turbulence under ice 4 × 10−3 s−1 in the layer δ ≤z < hS, where the thickness of the sub-ice layer δ ≈0.4m and the lower boundary of the stratiﬁed IL hS ≈10m. shore, at station S2, the currents, as measured during the next 2 d, revealed lower variability with time and had lower mag- nitudes of 1 to 4 × 10−2 ms−1. shore, at station S2, the currents, as measured during the next 2 d, revealed lower variability with time and had lower mag- nitudes of 1 to 4 × 10−2 ms−1. The two-layer structure was less distinct and the mixed- layer temperature was slightly higher at S2 (Fig. 3b); S1 had in turn a stronger vertical gradient close to the ice base. These temperature differences between the two stations suggested a stronger upward heat transport at S1 due to stronger verti- cal mixing caused by water ﬂow. Current speeds were indeed almost twice as high at S1 than at S2 (Figs. 3 and 5). The currents in the upper 20 m of the water column had a uni- form WSW direction aligned with the shoreline (see velocity vectors in Fig. 3). A weak, 10–15◦anticlockwise rotation of the current vector was detectable within the 1–2 m thin layer adjacent to the ice base, suggesting some effect of the Corio- lis force on the boundary layer currents. www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 1698 4.3 Solar radiation At S1, the warming was strong enough to pro- duce a decrease of the ice thickness (Fig. 4a), while the effect at S2 was too weak to cause any ice melt (Fig. 4b). With γ and I0 known, we estimated the theoretical thick- ness of the stratiﬁed interfacial layer δR. For a single-band exponential decay of the short-wave solar radiation within the water column I(z) = I(0)exp(−γ z), the steady-state so- lution of the radiation–conduction balance in a layer of thick- ness δR can be written as (Barnes and Hobbie, 1960) The mean currents obtained with the acoustic Doppler pro- ﬁler at a time interval of 2 s and a spatial resolution of 15 mm (Fig. 5a and b) agreed remarkably well with the records from the ﬁve electromagnetic velocity loggers at coarser tempo- ral and spatial resolution (Fig. 5a and b). The result allowed later extension of the boundary layer turbulence analysis to the whole period of electromagnetic velocity measurements, after a relationship between the mean ﬂow characteristics and the turbulent energy production was established from the short-term acoustic proﬁling. γ κTm + I0 1 + γ δR e−γ δR −1 = 0, (23) (23) which represents a transcendental equation with respect to δR. When substituted in Eq. (23), the values of I0 and γ yield δR ≈0.2–0.4m, adopting the temperature of the well-mixed layer of 0.6 ◦C for Tm. The estimate coin- cides well with the observed thickness of the ice-adjacent gradient layer δ (Fig. 3). The non-zero vertical tempera- ture gradient beneath this layer is in contrast to the typ- ical picture of convection in ice-covered lakes and sug- gests that the part of the convectively mixed layer δ < z < hS is altered by the turbulent shear due to under-ice cur- rents. Based on this suggestion, the under-ice radiation val- ues were used to estimate the destabilizing buoyancy ﬂux from Eq. (8) across the linearly stratiﬁed layer δ < z < hS as BR = gαIR = gα I(δ) + I(hS) −2h−1 S R hS δ I(z)dz . The resulting estimations are IR ≈2 Wm−2 and BR ≈2.5× which represents a transcendental equation with respect to δR. When substituted in Eq. (23), the values of I0 and γ yield δR ≈0.2–0.4m, adopting the temperature of the well-mixed layer of 0.6 ◦C for Tm. 4.3 Solar radiation The estimate coin- cides well with the observed thickness of the ice-adjacent gradient layer δ (Fig. 3). The non-zero vertical tempera- ture gradient beneath this layer is in contrast to the typ- ical picture of convection in ice-covered lakes and sug- gests that the part of the convectively mixed layer δ < z < hS is altered by the turbulent shear due to under-ice cur- rents. Based on this suggestion, the under-ice radiation val- ues were used to estimate the destabilizing buoyancy ﬂux from Eq. (8) across the linearly stratiﬁed layer δ < z < hS as BR = gαIR = gα I(δ) + I(hS) −2h−1 S R hS δ I(z)dz . The resulting estimations are IR ≈2 Wm−2 and BR ≈2.5× The mean current velocities from the two neighboring stations demonstrated different water ﬂow patterns. At sta- tion S1, current velocities in the upper 1 m of the water col- umn had mean values ≥5×10−2 ms−1. The magnitudes un- derwent variations on synoptic timescales, changing at 1 m under the ice from ≈10 × 10−2 to ≈3 × 10−2 ms−1 within 48 h (Fig. 5a). The event coincided with melting of the ice cover (Fig. 4a), suggesting that the upward heat transport by the currents is the mechanism of ice heating in this case de- spite the low air temperatures (Fig. 2a). Farther from the lake www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 G. Kirillin et al.: Turbulence under ice 1699 Figure 4. Temperature maps in the ice boundary layer and within the ice cover at station S1 (a) and station S2 (b). Note the different color scales for ice and water. G. Kirillin et al.: Turbulence under ice 1699 Figure 4. Temperature maps in the ice boundary layer and within the ice cover at station S1 (a) and station S2 (b). Note the different color scales for ice and water Figure 4. Temperature maps in the ice boundary layer and within the ice cover at station S1 (a) and station S2 (b). Note the different color scales for ice and water. Figure 5. Horizontal current speeds at station S1 (a, c) and station S2 (b, d) measured by the acoustic Doppler proﬁler Aquadopp (a, b) and the electromagnetic loggers INFINITY (c). 4.3 Solar radiation (a)–(c) are the time–depth maps; (d) shows the horizontal ﬂow speed measured by a single INFINITY logger at 1 m under ice (thin red line) and the Aquadopp velocity record from the same depth (thick blue line). Figure 5. Horizontal current speeds at station S1 (a, c) and station S2 (b, d) measured by the acoustic Doppler proﬁler Aquadopp (a, b) and the electromagnetic loggers INFINITY (c). (a)–(c) are the time–depth maps; (d) shows the horizontal ﬂow speed measured by a single INFINITY logger at 1 m under ice (thin red line) and the Aquadopp velocity record from the same depth (thick blue line). 4.4 Turbulence intensities in the ice–water boundary layer 10−10 m2 s−3. The characteristic scale of convective veloci- ties (Deardorff, 1970) w∗= (BRhS)1/3 ≈1.3mms−1, which agrees well with previous reports on radiative convection under lake ice (Mironov et al., 2002; Kirillin et al., 2018; Volkov et al., 2019). These estimates of the convective ve- locities suggest that radiation was of minor importance for the mixing conditions in the boundary layer compared with the mean ﬂow (cf. Fig. 5), especially after 23 February, when the subsurface radiation level dropped signiﬁcantly. There- fore, shear and stratiﬁcation in the boundary layer appeared to be the major factors determining water–ice heat transport. Fluctuations of current velocities around their means were characteristic of developed turbulence: the structure func- tions (Eq. 22) scaled well as the distance to the power of 2/3 clearly demonstrating the existence of the inertial inter- val in the wavenumber domain (Fig. 6a). According to the 2/3 power scaling, the upper boundary of the inertial inter- val reached 0.1–0.3 m, which can be treated as a character- istic size of turbulent eddies. In low-turbulence conditions www.hydrol-earth-syst-sci.net/24/1691/2020/ www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 1700 G. Kirillin et al.: Turbulence under ice Figure 6. Turbulence-related characteristics of the boundary layer: (a) velocity structure functions for high (circles) and low (asterisks) levels of TKE dissipation rates. Solid lines are the approximations by Eq. (22). (b) Vertical proﬁles of the reciprocal TKE dissipation rates κε−1 for conditions of high (circles) and low (asterisks) turbulence. Solid lines are the data approximations by Eq. (7). Horizontal dashed line in (b) marks the depth equal to the mean Ozmidov length LN ≈0.8m 1700 G. Kirillin et al.: Turbulence under ice Figure 6. Turbulence-related characteristics of the boundary layer: (a) velocity structure functions for high (circles) and low (asterisks) levels of TKE dissipation rates. Solid lines are the approximations by Eq. (22). (b) Vertical proﬁles of the reciprocal TKE dissipation rates κε−1 for conditions of high (circles) and low (asterisks) turbulence. Solid lines are the data approximations by Eq. (7). Horizontal dashed line in (b) marks the depth equal to the mean Ozmidov length LN ≈0.8m Figure 7. The TKE dissipation rates in the area of the jet stream (station S1, a) and in the region of weak currents (station S2, b). Figure 7. The TKE dissipation rates in the area of the jet stream (station S1, a) and in the region of weak currents (station S2, b). ε < 10−9 m2 s−3, the TKE dissipation rates were at their min- imum at the depth of ≈0.8m and increased towards the ice base (asterisks in Fig. 6b), supporting the scaling ε ∝z−1 (Eq. 7). The scatter of ε around the straight line ε−1 ∝z in- creased with the distance from the ice z, starting from z ≈Le. During periods of high turbulence (ε > 10−8 m2 s−3), the re- ciprocal of the TKE dissipation rate ε−1 increased with depth more homogeneously. Nevertheless, a small local extreme in the line ε−1(z) and a slight change of the slope were recog- nizable at the same critical distance z ≈Le ≈0.8m from the ice (circles in Fig. 6b). stead, an apparent correlation existed between the turbu- lence intensity ε and the temporal variations of the mean ﬂow velocities (Fig. 7): the highest TKE dissipation rates of O(10−7)m2 s−3 were observed during current intensiﬁcation up to O(10−1) ms−1 at S1. p The maximum values of the D–O length scale (Eq. www.hydrol-earth-syst-sci.net/24/1691/2020/ G. Kirillin et al.: Turbulence under ice Kirillin et al.: Turbulence under ice Figure 8. Ice boundary layer structure: (a) friction velocity u∗at station S1 as determined from the quasi-neutral production–dissipation balance (Eq. 7, thick solid line with symbols) and from the Dougherty–Ozmidov length scale (Eq. 13, thin lines) at a distance from the ice base nearly equal to the mean Ozmidov length LN ≈0.8m, (b) mean vertical proﬁles of u∗, and (c) vertical velocity proﬁles for strong (circles) and weak (asterisks) currents. The two proﬁles in (c) correspond to those in Fig. 6. Horizontal dashed line in (b, c) marks the depth equal to the mean Ozmidov length LN ≈0.8m Figure 8. Ice boundary layer structure: (a) friction velocity u∗at station S1 as determined from the quasi-neutral production–dissipation balance (Eq. 7, thick solid line with symbols) and from the Dougherty–Ozmidov length scale (Eq. 13, thin lines) at a distance from the ice base nearly equal to the mean Ozmidov length LN ≈0.8m, (b) mean vertical proﬁles of u∗, and (c) vertical velocity proﬁles for strong (circles) and weak (asterisks) currents. The two proﬁles in (c) correspond to those in Fig. 6. Horizontal dashed line in (b, c) marks the depth equal to the mean Ozmidov length LN ≈0.8m of the stratiﬁcation-based turbulence scaling (Eqs. 10–13) against the quasi-neutral law-of-the-wall (LOW) relationship (Eqs. 5–7). At z < zcrit, both “neutral” relationships (Eqs. 4 and 7) produced similar estimations of the friction veloci- ties u∗with 20–30 % higher values produced by u∗estima- tions from ε (Eq. 7). While the value of the von Kármán con- stant in the neutral LOW scaling κ ≈0.4 is relatively well known from tunnel experiments and numerical simulations, and it is supported by ﬁeld data, the proportionality constant in Eq. (13) is not well established. Therefore, for z-less D– O scaling (Eq. 13), the values of u∗were calculated from Eq. (13) assuming a unity coefﬁcient of proportionality CN. On average, the “stratiﬁed” scaling produced generally lower values of u∗at farther distances from the ice bottom and vice versa. The D–O scaling with CN = 1 and LOW demonstrated nearly perfect agreement at z = zcrit = LN (Fig. 8a). This fact justiﬁed the balance between the shear production at the boundary and the stratiﬁed production of turbulence at this distance from the wall, as well as supporting the choice of the unity constant in the D–O scaling. www.hydrol-earth-syst-sci.net/24/1691/2020/ 10), averaged over the period of observations, decreased with the distance from the boundary from LN ≈1.5m at z ≈0.2m to LN ≈0.9m at z ≈0.9m. The decrease in LN followed the decrease of ε. Here, the mean NS in the layer with quasi- linear stratiﬁcation of 0.5–10 m beneath the ice base was used as a characteristic value of the buoyancy frequency in the D– O scaling. At larger distances from the ice base, LN remained nearly constant, varying between 0.8 and 0.9 m. Hence, the value zcrit = LN = 0.85 can be treated as a boundary be- tween the “quasi-neutral” and strongly stratiﬁed z-less lay- ers, with the turbulent length scale deﬁned by the distance z closer to the ice base, and by LN at farther distances. In the area with weak under-ice currents at S2, the TKE dissipation rates ε varied around a value of 10−9 m2 s−3, close to the threshold between turbulent and laminar con- ditions. In turn, the average ε in the vicinity of the jet-like under-ice current at S1 was 2 orders of magnitude higher (Fig. 7). In contrast to the under-ice water temperatures, neither TKE dissipation rates nor friction velocities demon- strated any diurnal variations, suggesting a minor effect of the radiation-driven convection on turbulence generation. In- An important insight into the mechanisms of turbu- lence generation under ice is provided by comparison www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 1701 G. Kirillin et al.: Turbulence under ice G. Kirillin et al.: Turbulence under ice G. Kirillin et al.: Turbulence under ice Accordingly, CN = 1 was adopted for later application of the combined log-linear scaling (Eqs. 12 and 14). The combined log-linear scaling (Eq. 12) with CN = 1 produced u∗close to the neutral value in the vicinity of zcrit and decreased towards both IWI and the open water column (Fig. 8b). Like the TKE dissipation rates, the mean current speeds demonstrated behavior characteristic of the stationary boundary layer, i.e., ﬁtted well to logarithmic proﬁles at dis- tances from the IWI less than zcrit (Fig. 8c). Farther from the ice base the mean velocity proﬁles were nearly linear, with the slope close to zL−1 N . 2 The integral balance between the TKE loss terms u2 ∗N +ε and the turbulent energy production u2 ∗S (Eq. 7) held true within the 1.5 m thick layer covered by measurements of ε: the mean difference between the two terms integrated over the entire layer did not exceed 0.2 %; temporal variations of www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 1702 G. Kirillin et al.: Turbulence under ice Figure 9. TKE production–dissipation balance in the ice boundary layer. (a) TKE production and dissipation rates at the depth of 0.85 m beneath the ice base. Bold lines are the values ﬁltered by a moving average with a 6 h window. (b) Vertical proﬁles of the TKE budget components in Eq. (14) averaged over the 2 d period of measurements. Data are presented for station S1 only; the S2 results are qualitatively the same. G. Kirillin et al.: Turbulence u 1702 G. Kirillin et al.: Turbulence under ice G. Kirillin et al.: Turbulence under ice G. Kirillin et al.: Turbulence under ice Figure 9. TKE production–dissipation balance in the ice boundary layer. (a) TKE production and dissipation rates at the depth of 0.85 m beneath the ice base. Bold lines are the values ﬁltered by a moving average with a 6 h window. (b) Vertical proﬁles of the TKE budget components in Eq. (14) averaged over the 2 d period of measurements. Data are presented for station S1 only; the S2 results are qualitatively the same. cation – the effect described above and discussed in more details below. the dissipation rate followed closely those of the shear ve- locity (Fig. 9a). G. Kirillin et al.: Turbulence under ice The balance was disturbed only at current speeds < 0.02 ms−1, with a corresponding drop of ε down to < 10−9 m2 s−3 at station S2 on 9 March 2017 (not shown). During this same period, the vertical ﬂow proﬁles showed a signiﬁcant deviation from the logarithmic form, indicating laminarization of the boundary layer under these conditions. The boundary value of the friction velocity for the transi- tion to a turbulent regime was u∗≈1.0mm s−1. The mean balance between the production loss terms in Eq. (14) var- ied, however, with the distance from the ice base (Fig. 9b): close to the ice–water interface the production signiﬁcantly exceeded dissipation, while below the depth of ≈0.8m the dissipation prevailed above the production. Remarkably, this transition depth agreed well with the thickness of the layer where ε ∝z−1. 4.5 Heat budget at the ice base and relation of ice–water heat ﬂux to under-ice mixing The heat balance at the IWI was calculated by Eqs. (1) and (3) using data on temperature within the ice cover and ice thickness variability measured by the echo sounder. The ice– water heat ﬂux was generally stronger at station S1, corre- lated with stronger currents and mixing intensities. Already at the beginning of the observation period in February, the upward conductive heat loss Qci of up to 80 Wm−2 was com- pensated to 30–50 % by the heat supply from the water col- umn Qiw. The latter signiﬁcantly reduced the ice growth rate and latent heat release (cf. red and blue areas in Fig. 11). During 24 February–7 March the snow cover reduced the heat release to the atmosphere, which also lowered the con- ductive ﬂux at the ice base Qci (Fig. 11). As a result, the heat ﬂux from water to the ice at S1 (Fig. 11a) exceeded that from the ice to the atmosphere, producing melting at the ice base (negative QL) despite continuing surface cooling (Qci remained positive with values of up to 40 Wm−2). After 25 March, ice cover started to melt at both stations, coincid- ing with an increase of air temperatures above the freezing point (Fig. 2). Quantitatively, the ice–water heat ﬂuxes at S2 were in the range of 5–10 Wm−2, which agrees with esti- mations from earlier lake studies. However, Qiw at S1 had appreciably higher values, reaching up to 40 Wm−2 at their peaks. The good agreement of the measured velocity proﬁles with the logarithmic approximation at z < zcrit allowed estimation of the roughness of the ice bottom surface z0 from Eq. (5). The mean z0 amounted to 1.00 mm with a maximum of 3.5 mm and minimum of 0.2 mm. The roughness had a sig- niﬁcant (Pearson’s coefﬁcient of −0.52, p ≪0.01) negative correlation with the mean velocity as z0 ≈1.2 × 10−4U−1 mean. Our estimations of z0 and u∗yielded the following pa- rameters for the bulk formula Eq. (6): C1 m ≈3.4 × 10−3 and CZ = C1 mZ−1, where C1 m is the bulk transfer coef- ﬁcient for the momentum ﬂux at 1 m depth. The indepen- dent measurements of current velocities at four depths made by the single-point 2-D horizontal current loggers INFIN- ITY demonstrated good agreement with Eq. (6) when scaled against u∗from the HR-Aquadopp measurements (Fig. 10). G. Kirillin et al.: Turbulence under ice G. Kirillin et al.: Turbulence under ice 1703 G. Kirillin et al.: Turbulence under ice 1703 Figure 10. Bulk characteristics of the ice boundary layer: (a) bulk transfer coefﬁcient for momentum CZ as a function of distance from the IWI, where dots are values calculated using the horizontal velocity data from point measurements by 2-D INFINITY-EM loggers, and the line is an analytical approximation; (b) relationship between the mean ﬂow velocity at z = 1m and the turbulent stress. Figure 10. Bulk characteristics of the ice boundary layer: (a) bulk transfer coefﬁcient for momentum CZ as a function of distance from the IWI, where dots are values calculated using the horizontal velocity data from point measurements by 2-D INFINITY-EM loggers, and the line is an analytical approximation; (b) relationship between the mean ﬂow velocity at z = 1m and the turbulent stress. Figure 11. Daily averaged heat balance at (a) station S1 and (b) station S2. Figure 11. Daily averaged heat balance at (a) station S1 and (b) station S2. Figure 12. Buoyancy ﬂux at the ice–water interface B as a function of (a) TKE dissipation rate ε and (b) friction velocity u∗. the former suggests Qiw ∝ε1/3 and the latter agrees with the observed linear dependence Qiw ∝ε. Herewith, the re- sult discards the widely used bulk relationship Qiw ∝u∗and suggests that Qiw scales as friction velocity squared (Eq. 20) or, in terms of Eq. (19), CQ ∝u∗. The dependence Qiw ∝u2 ∗ is well supported by our data, though the scaling is less ap- parent at very low u∗due to the lack of values at low tur- bulence levels. Still, the data on Qiw(u∗) (Fig. 12b) clearly demonstrate the inappropriateness of the quasi-neutral scal- ing (Eq. 18). Instead, the ﬂux can be parameterized as Biw = 0.015u2 ∗NS, (25) (25) Biw = 0.015u2 ∗NS, Figure 12. Buoyancy ﬂux at the ice–water interface B as a function of (a) TKE dissipation rate ε and (b) friction velocity u∗. where NS is the quasi-constant buoyancy frequency in the boundary layer 0.4 < z < 10m. where NS is the quasi-constant buoyancy frequency in the boundary layer 0.4 < z < 10m. Here, ε is taken at the distance zcrit = 0.85m from the ice base, which corresponds to the boundary between the ice- adjacent sublayer and the linearly stratiﬁed boundary layer (see Sect. 4.2). 5 Discussion Our study presents the ﬁrst detailed assessment of mixing conditions under the ice cover of Lake Baikal and their ef- fect on the growth and melt of the ice cover. The seasonal ice cover is Lake Baikal’s inherent feature, whose role in the functioning of this unique ecosystem remains not fully un- The linear correlation between the ice–water heat ﬂux and ε supports the scaling (Eq. 20) in the stratiﬁed boundary layer under ice: from the two bulk relationships Eqs. (18) and (20), www.hydrol-earth-syst-sci.net/24/1691/2020/ 4.5 Heat budget at the ice base and relation of ice–water heat ﬂux to under-ice mixing The simple result has large potential for application in The good agreement of the measured velocity proﬁles with the logarithmic approximation at z < zcrit allowed estimation of the roughness of the ice bottom surface z0 from Eq. (5). The mean z0 amounted to 1.00 mm with a maximum of 3.5 mm and minimum of 0.2 mm. The roughness had a sig- niﬁcant (Pearson’s coefﬁcient of −0.52, p ≪0.01) negative correlation with the mean velocity as z0 ≈1.2 × 10−4U−1 mean. Our estimations of z0 and u∗yielded the following pa- rameters for the bulk formula Eq. (6): C1 m ≈3.4 × 10−3 and CZ = C1 mZ−1, where C1 m is the bulk transfer coef- ﬁcient for the momentum ﬂux at 1 m depth. The indepen- dent measurements of current velocities at four depths made by the single-point 2-D horizontal current loggers INFIN- ITY demonstrated good agreement with Eq. (6) when scaled against u∗from the HR-Aquadopp measurements (Fig. 10). An attempt to link the ice–water heat ﬂux with the mixing characteristics in the stratiﬁed boundary layer in the form of a bulk relationship (Eq. 19) provided a remarkable result: the heat ﬂux at the IWI and the dissipation rate of the TKE are linked linearly (Fig. 12a), or in terms of buoyancy ﬂux B: The simple result has large potential for application in modeling of the ice–water boundary layer at strong under-ice currents with a minimum of input information; care should be taken, however, regarding the thickness of the nearly log- arithmic layer and its dependence on the under-ice stratiﬁ- B = gαQiw = 0.065ε. (24) (24) B = gαQiw = 0.065ε. Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 www.hydrol-earth-syst-sci.net/24/1691/2020/ www.hydrol-earth-syst-sci.net/24/1691/2020/ G. Kirillin et al.: Turbulence under ice derstood. In this regard, the outcomes of the study underscore the importance of the lake-wide circulation for the ice cover duration and ice thickness. The applicability of the results extends, however, beyond Baikal-speciﬁc conditions. Lake Baikal shares the major features of seasonal ice cover with other lakes, as well as with inland and marginal seas, allow- ing extension of the results to other ice-covered waters. Fur- thermore, the ice–water boundary layer in Lake Baikal pos- sesses a remarkable feature relevant to fundamental problems of environmental ﬂuid mechanics: a strong boundary-layer ﬂow in the background of permanent stable density stratiﬁ- cation. In our study we successfully tested an alternative ap- proach to the traditional Monin–Obukhov similarity theory, based on the Dougherty–Ozmidov scaling. We also revealed several important facets of the turbulent energy budget in stratiﬁed boundary layers and established a relationship be- tween the shear turbulence under ice and the heat ﬂux at the ice–water boundary. ergy downwards, contributing to destruction of stratiﬁcation in the main SL δ < z < hS. The opposing trend is created by solar radiation, which increases the temperature of the con- vectively mixed layer Tm and thereby creates an upward ex- ponential decrease of temperature from Tm to the freezing point Tf . The observed quasi-linear stratiﬁcation in the SL is apparently a result of the two opposing forces leading to a nearly steady-state conditions characterized by a constant N within the layer δ < z < hS. A particular advance in estimation of the turbulent energy budget under ice was achieved by direct estimation of the TKE dissipation rate using the velocity structure method. By doing so, we (i) avoided applying Taylor’s “frozen turbu- lence” hypothesis, which remains questionable at relatively low current velocities under ice, and (ii) were able to trace the vertical distribution of ε across the boundary layer (at least a part of it, covering ∼2m). On the one hand, this ex- tended information allows better quantiﬁcation of the turbu- lent structure; on the other hand, it poses some fundamen- tal questions about the major forces behind under-ice mixing and heat transport, to be discussed below. The high values of water–ice heat ﬂuxes in Lake Baikal and their apparent relationship to the intensity of under-ice currents were previously noted by Aslamov et al. (2014a, 2017). G. Kirillin et al.: Turbulence under ice In the present study, the measured ﬂuxes reached up to 40 Wm−2 at their peaks, which is an order of magnitude higher than values reported from small Arctic lakes (Kirillin et al., 2018) and comparable to the highest reported values in alpine thermokarst ponds (Huang et al., 2019) and in the ocean (Gallaher et al., 2016; Peterson et al., 2017). Con- current registration of ﬂuxes, current velocities, and dissipa- tion rates of the TKE reveals the direct link between turbu- lence production by the velocity shear and ice growth (ab- lation). The ﬁnding contradicts the conventional assumption on the major role of convection produced by the solar ra- diation penetrating the ice in under-ice mixing of freshwa- ter lakes. While radiatively driven convection is a prominent feature of freshwater lakes (Farmer, 1975; Yang et al., 2017; Volkov et al., 2019) and effectively mixes the upper water column of Lake Baikal in winter (Granin et al., 2000; Jew- son et al., 2009), its effect on the boundary mixing and heat transport to the ice base appears to be restricted by the sta- ble stratiﬁcation in the relatively thin interfacial layer, with water temperature increasing from the melting point at the IWI to that of the convectively mixed layer (Kirillin et al., 2018). As a result, the energy of convection partially dissi- pates within the convective layer and is partially spent for entrainment of deeper waters at the base of the mixed layer (Mironov et al., 2002). The rate of the energy dissipation pro- duced by convection in small lakes (Volkov et al., 2019) is about 10−9–10−8 Wkg−1, which is roughly an order of mag- nitude lower than that measured in this study. Consequently, the turbulence budget in the boundary layer differs signiﬁ- cantly from that reported in previous lake studies. The TKE production averaged over the entire observation period pre- vails over dissipation at z < LN, while in the deeper part of the boundary layer ε slightly exceeds the production. Here- with, the boundary mixing continuously pumps turbulent en- p Close to the ice base, vertical proﬁles of the TKE dis- sipation rates decayed as ε ∝z−1, supporting the scaling of the turbulent mixing length with the distance from the solid boundary, similar to neutral or nearly neutral condi- tions. www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 1704 G. Kirillin et al.: Turbulence under ice One of the derivatives of the D–O boundary layer scaling is the relationship ε ∝u2 ∗N, which also implies that the length scale u∗N−1 can be used instead of LN without any basic changes in the model as- sumptions. This scaling was previously considered, for ex- ample, by Zilitinkevich and Mironov (1996) and is preferable for testing and reﬁning the model parameters if observations of u∗are available at high resolution. Figure 13. Formation of the vertical temperature proﬁle in a shear- driven ice boundary layer. Gray line is a no-shear original proﬁle; black line is the result of shear-driven turbulence on the background of stable stratiﬁcation. See text for notations. (density) distribution is formed by absorption of solar radia- tion and the upward heat ﬂux to the ice, producing the proﬁle typically observed in ice-covered lakes without strong cur- rents: a nearly homogeneous convectively mixed layer with a relatively thin stratiﬁed layer on top. Mixing by the veloc- ity shear reduces the density gradient. In the top layer, shear mixing is balanced by solar heating from above, so that the density gradient tends to the critical value between turbulent and non-turbulent states. In the upper part of the homoge- neous layer, a weaker nearly linear density gradient forms. In the larger part of this layer, at z > LN, the turbulence pro- duction is balanced by the stratiﬁcation, in accordance with z-less linear scaling, Eq. (13). Beneath the depth LN, the scaling suggests ∂U∂z−1 = const, which is also supported by the measured mean velocities (Fig. 8). The stratiﬁcation is nearly linear, suggesting also that ε is nearly constant across it. The thickness of the layer depends apparently on the scales of the under-ice ﬂow. The shape of the observed tempera- ture proﬁles slightly deviates from linearity, with a tendency to re-stratiﬁcation corresponding to weaker currents at S2 (Fig. 3b). Apparently, variations in the current speeds may affect the general tendency to linear stratiﬁcation, produc- ing changes in the heat content of the upper water column caused by horizontal advection of heat, or by re-stratiﬁcation during periods of low shear. We do not possess enough well- resolved data on temperature to investigate these effects. The presence of an ice-adjacent interfacial sublayer 0 < z < δ is another remarkable feature of the shear-dominated ice boundary layer. G. Kirillin et al.: Turbulence under ice 1705 Figure 13. Formation of the vertical temperature proﬁle in a shear- driven ice boundary layer. Gray line is a no-shear original proﬁle; black line is the result of shear-driven turbulence on the background of stable stratiﬁcation. See text for notations. production rate than the sum of the lost terms at z > LN. The imbalance can be tentatively attributed to downward ad- vection of the TKE. Furthermore, the balance was estimated under the assumption of constant buoyancy frequency N, ne- glecting a possibly stronger loss of the TKE closer to the ice, where N increases. We also neglected possible transport of turbulent energy produced by convection due to solar heat- ing. The latter can, however, be assumed to be small: within the stratiﬁed layer, the radiation levels under ice produced a destabilizing buoyancy ﬂux of only O(10−10) Wkg−1, and the convectively mixed layer was located several meters be- neath the deepest point covered by measurements. production rate than the sum of the lost terms at z > LN. The imbalance can be tentatively attributed to downward ad- vection of the TKE. Furthermore, the balance was estimated under the assumption of constant buoyancy frequency N, ne- glecting a possibly stronger loss of the TKE closer to the ice, where N increases. We also neglected possible transport of turbulent energy produced by convection due to solar heat- ing. The latter can, however, be assumed to be small: within the stratiﬁed layer, the radiation levels under ice produced a destabilizing buoyancy ﬂux of only O(10−10) Wkg−1, and the convectively mixed layer was located several meters be- neath the deepest point covered by measurements. The proposed scaling of the TKE budget differs from the conventional MOST approach by using the Dougherty– Ozmidov length scale instead of the Obukhov length scale, i.e., by replacing the surface buoyancy ﬂux with the mean stratiﬁcation as a major scaling variable. This alternative ap- proach is convenient for analysis of observational data: in contrast to the surface ﬂuxes, N is easily measurable in oceanic and lake studies. Noteworthily, these scaling ap- proaches have been shown to be interchangeable (Grachev et al., 2015). In the particular case of ice-covered waters, the D–O approach is also more physically sound than MOST; since the surface buoyancy ﬂux does not dominate the turbu- lent conditions under ice, it is rather a result of the upward heat transport from deeper waters. G. Kirillin et al.: Turbulence under ice This fact gives a solid background to estimation of the shear velocity u∗from the mean velocity proﬁles: the latter method is often uncertain, given that neither depth of the logarithmic layer nor ice roughness are known a priori. However, the thickness of the layer with ε ∝z−1 varies de- pending on the current speed and mixing intensity. At strong currents, dissipation followed the scaling across the entire depth of the high-resolution velocity measurements of 1.4 m; at ε ≪10−7, it reduced to several tens of centimeters. The maximum mixing length scales remarkably well against the Dougherty–Ozmidov length scale: using NS as the character- istic value for stratiﬁcation, LN = 1.2m at ε = 10−7 Wkg−1 and LN = 0.4m at ε = 10−8 Wkg−1. Qualitatively the result can be treated as follows: the shear at the ice base domi- nates in the turbulence production at distances from the ice base less than LN, while farther from the source of the shear the stratiﬁcation limits the size of the turbulent eddies. This structure is also supported by comparison of the friction ve- locities computed from the two mixing lengths, Eqs. (7) and (13): u∗tends to be overestimated by the Ozmidov scaling at high mixing rates close to the ice base, and it becomes lower than that produced by z scaling at larger distances from the ice. At z ≈LN both estimations provide equal results (see Fig. 8). In the TKE budget, the depth z > LN is also a turn- ing point, where Eq. (14) shows a close balance between tur- bulence production and damping. Generally, the simpliﬁed model of the TKE balance Eq. (14) was well supported by the data. The discrepancies included the prevalence of TKE production over the damp- ing terms closer to the ice cover and a slightly lower TKE www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 6 Conclusions We investigated the ﬁne vertical structure of turbulence char- acteristics in the boundary layer of Lake Baikal and proposed a model of stratiﬁed turbulent ice boundary layer based on the Dougherty–Ozmidov length scale of turbulence. In con- trast to small lakes, where solar radiation dominates ice– water heat exchange, the water–ice heat ﬂux in Lake Baikal was strongly affected by the mean ﬂow shear, similar to the ice boundary layer in the ocean. The shear-produced mixing was counteracted by the stable density stratiﬁcation beneath the ice cover. Absolute values of the water–ice ﬂuxes were an order of magnitude higher than those in no-shear conditions, ensuring basal ice melt even during cooling at the ice sur- face. The ultimate result consists in scaling of the water–ice heat ﬂux against the shear velocity squared. The result sug- gests large errors in heat ﬂux estimations when the traditional bulk approach is applied to stratiﬁed conditions with strong shear. It also implies that under-ice currents may have a much stronger effect on the ice melt than estimated by traditional models. y The main motivation of the study was seeking the rela- tionship between the shear mixing and stratiﬁcation on the one hand and the heat release from water to the ice cover on the other hand. In this regard, the scaling of the heat (buoy- ancy) ﬂux with the shear velocity squared and the buoyancy frequency under ice (Eq. 20) is our major ﬁnding, with impli- cations for a wide range of ice-related problems. The simple bulk approximation Eq. (19) is widely used in models of ice– water interaction, but its validity was never thoroughly tested before. The simple relationship Qiw ∝u∗1T , equivalent to Eq. (19), failed to describe the heat ﬂux dynamics in the ice boundary layer, replaced by the scaling Qiw ∝(gαT )−1u2 ∗N. The result suggests that the effect of the currents on the de- crease of the Arctic sea ice may appear much stronger than assumed by the present model projections, based on the bulk estimation Qiw ∝u∗. Further decline of the Arctic ice cover may result in both increase of the under-ice current speeds due to changed global circulation and increase of the strat- iﬁcation due to warming of the under-ice waters. Both fac- tors, according to our scaling will accelerate the vertical heat transport from water to ice, resulting in a positive feedback on the ice melt. G. Kirillin et al.: Turbulence under ice scale u∗f −1 based on the mean shear velocities is O(101)m. Herewith, the Coriolis force may have an effect on the thick- ness of the boundary layer. At weak stratiﬁcation, effects of both N and f on the boundary layer dynamics may ap- pear comparable, so that the model will perform better if the length scale LN (or its equivalent u∗N−1) is replaced by the combined length scale u∗(f N)−1/2. The latter scal- ing was proposed by Pollard et al. (1972). Zilitinkevich and Mironov (1996) suggested that the f N scaling has a rather limited area of application. However low ﬂow velocities and weak stratiﬁcation are typical for conditions under ice, and the Pollard et al. (1972) scaling may ﬁnd its application in ice-covered waters. More data are required here, in partic- ular, on the ﬁne density and ﬂow structure over the entire Ekman layer. scale u∗f −1 based on the mean shear velocities is O(101)m. Herewith, the Coriolis force may have an effect on the thick- ness of the boundary layer. At weak stratiﬁcation, effects of both N and f on the boundary layer dynamics may ap- pear comparable, so that the model will perform better if the length scale LN (or its equivalent u∗N−1) is replaced by the combined length scale u∗(f N)−1/2. The latter scal- ing was proposed by Pollard et al. (1972). Zilitinkevich and Mironov (1996) suggested that the f N scaling has a rather limited area of application. However low ﬂow velocities and weak stratiﬁcation are typical for conditions under ice, and the Pollard et al. (1972) scaling may ﬁnd its application in ice-covered waters. More data are required here, in partic- ular, on the ﬁne density and ﬂow structure over the entire Ekman layer. G. Kirillin et al.: Turbulence under ice The thickness of the layer (δ ≈0.2m) was close to the smallest estimate based on the solar radi- ation of the layer created between the ﬁxed temperature at the ice base and a convectively mixed homogeneous layer beneath. Our temperature measurements were too scarce to trace the evolution of its thickness at variable current veloci- ties. Some insight into the genesis of the layer can, however, be obtained by assuming the largest value of the Dougherty– Ozmidov length scale LN ≈0.85m as the maximal thickness of δ. In this case, the mean buoyancy frequency in the in- terfacial sublayer Nδ ≈0.02 s−1. That leads to the buoyancy Reynolds number Reb ≈16 and the gradient Richardson number Rig ≈0.4. Both values are close to the critical values of O(101) and 0.25, respectively. The layer 0 < z < δ may therefore be assumed to stay in a near-critical turbulence- free state. In the rest of the boundary layer, both Rig and Reb are far beyond the critical values, indicating developed turbulence. We did not consider the rotational effects on the bound- ary layer characteristics. A slight Ekman-like rotation of the mean current was observed under ice (Fig. 3), and the Ekman The following structure can be tentatively drawn from the above analysis (Fig. 13): the background vertical temperature www.hydrol-earth-syst-sci.net/24/1691/2020/ Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 1706 Hydrol. Earth Syst. Sci., 24, 1691–1708, 2020 References Hampton, S. E., Izmest’eva, L. R., Moore, M. V., Katz, S. L., Den- nis, B., and Silow, E. A.: Sixty years of environmental change in the world’s largest freshwater lake–Lake Baikal, Siberia, Global Change Biol., 14, 1947–1958, 2008. Aslamov, I., Kozlov, V., Kirillin, G., Mizandrontsev, I., Kucher, K., Makarov, M., Gornov, A. Y., and Granin, N.: Ice–water heat ex- change during ice growth in Lake Baikal, J. Great Lakes Res., 40, 599–607, 2014a. 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Kirillin et al.: Turbulence under ice retische und angewandte Limnologie: Verhandlungen, 27, 2812– 2814, 2000. Review statement. This paper was edited by Louise Slater and re- viewed by two anonymous referees. Hamblin, P. and Carmack, E.: On the rate of heat transfer between a lake and an ice sheet, Cold Reg. Sci. Technol., 18, 173–182, 1990. 6 Conclusions Hence, incorporation of the under-ice den- sity stratiﬁcation in the heat ﬂux parameterizations may sig- niﬁcantly improve the outcomes of the oceanic ice models, especially for the ice melt periods, when freshening of the under-ice boundary layer produces strong salinity stratiﬁca- tion. Data availability. Data are available from the authors by request. Data availability. Data are available from the authors by request. Data availability. Data are available from the authors by request. Author contributions. GK, IA, and NG conceived the study; IA, RZ, and GK designed and performed the ﬁeld experiments; GK developed the model; GK, IA, VK, and NG performed ﬁeld data analysis; GK wrote the manuscript with contributions from all co- authors. Author contributions. GK, IA, and NG conceived the study; IA, RZ, and GK designed and performed the ﬁeld experiments; GK developed the model; GK, IA, VK, and NG performed ﬁeld data analysis; GK wrote the manuscript with contributions from all co- authors. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. The study is a part of the research project “Ice- Bound” supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG Project KI-853/11-1, KI-853/11-2). Additional support by the DFG Project KI-853/13-1 is gratefully appreciated by Georgiy Kirillin. Field work and data analysis were carried out within the framework of LIN SB RAS State Task No. 0345-2019-0008 and additionally supported by project ISDCT SB RAS No. 0348-2020-0012. In contrast to the surface heat ﬂux, the bulk formulation Eq. (6) worked well for the momentum ﬂux thanks to the nearly constant shear conditions close to the ice base. The result is of practical use in simple models of ice-covered seas and lakes, where u∗can be directly approximated from the mean current speeds at a certain distance from the IWI (Fig. 10). The speeds should, however, be known at distances from the ice z < LN; otherwise, the stratiﬁcation effects on turbulence production make the bulk formula unrepresenta- tive. Financial support. This research has been supported by the Deutsche Forschungsgemeinschaft (DFG grant nos. KI-853/11-1, KI-853/11-2, and KI-853/13-1). The publication of this article was funded by the Open Access Fund of the Leibniz Association. 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https://openalex.org/W2796780143	https://europepmc.org/articles/pmc5793437?pdf=render	English	null	Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells	Biological procedures online	2,018	cc-by	6,517	Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells Alysia G. Buckley1, Kevin Looi2, Thomas Iosifidis2,3, Kak-Ming Ling4, Erika N. Sutanto4,5, Kelly M. Martinovich4, Elizabeth Kicic-Starcevich4, Luke W. Garratt2, Nicole C. Shaw4, Francis J. Lannigan6, Alexander N. Larcombe4, Graeme Zosky4,7, Darryl A. Knight8,9,10, Paul J. Rigby1, Anthony Kicic2,3,4,5,11,12* and Stephen M. Stick2,3,4,5 Buckley et al. Biological Procedures Online (2018) 20:3 DOI 10.1186/s12575-018-0070-0 Buckley et al. Biological Procedures Online (2018) 20:3 DOI 10.1186/s12575-018-0070-0 Open Access Open Access Abstract Background: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. Methods: Here, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. Results: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. Conclusions: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases. Keywords: Tight junctions, Confocal microscopy, Fixation, Airway epithelial cells, Air liquid interface Keywords: Tight junctions, Confocal microscopy, Fixation, Airway epithelial cells, Air liquid interfa * Correspondence: anthony.kicic@telethonkids.org.au Alysia G. Buckley and Kevin Looi are co-first authors. Anthony Kicic and Stephen M. Stick are co-senior authors. * Correspondence: anthony.kicic@telethonkids.org.au Alysia G. Buckley and Kevin Looi are co-first authors. Anthony Kicic and Stephen M. Stick are co-senior authors. 2School of Paediatrics and Child Health, The University of Western Australia, Nedlands, Western Australia 6009, Australia 3Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, The University of Western Australia, Nedlands, Western Australia 6009, Australia Full list of author information is available at the end of the article Methods R g The culture reagents Modified Eagle’s Medium (MEM), Penicillin/Streptomycin, L-Glutamine, Foetal Calf Serum (FCS) and Normal Goat Serum (NGS) were purchased from Life Technologies (CA, USA). Triton X-100, trizma base, sodium chloride, bovine serum albumin (BSA) and fibronectin were purchased from Sigma Aldrich (MO, USA). Collagen IV was purchased from BD Biosciences (New Jersey, USA). Abstract 2School of Paediatrics and Child Health, The University of Western Australia, Nedlands, Western Australia 6009, Australia 3Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, The University of Western Australia, Nedlands, Western Australia 6009, Australia Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Buckley et al. Biological Procedures Online (2018) 20:3 Page 2 of 9 Background initially in a representative airway epithelial cell line (16HBE14o-), followed by corroboration in primary airway epithelial cultures grown at air-liquid interface (ALI). g The airway epithelial layer remains the frontline of defence against pathogens, aeroallergens and noxious gases by establishing and maintaining a physical barrier. The integ- rity of this layer is typically maintained by the presence of a range of junctional complexes including: tight junctions; adherens junctions; and desmosomes [1–4]. Apically lo- cated tight junctions perform a fundamental role in regu- lating solute transport across the airway epithelium [5] by restricting macromolecule migration through paracellular spaces [6–9]. Several families of proteins have been identi- fied to form tight junctions between adjacent cells includ- ing the occludin and claudin families. These proteins contain four transmembrane domains with two extracellu- lar loops, where the extracellular loops fuse with their counterpart on adjacent cells [10] resulting in a belt-like structure around the apical surface of airway epithelial cells [4, 5, 11, 12]. In association with the transmembrane tight junction proteins is the intracellular protein zona occludens-1 (ZO-1) [10] which act by anchoring the tight junction proteins to the cytoskeleton [13]. Antibodies For immunocytochemistry, the following antibodies were used: Claudin-1 (polyclonal), Occludin (monoclonal, clone OC-3F10), ZO-1 (monoclonal, clone ZO1-1A12, and polyclonal), AlexaFluor 488 (Goat anti-Mouse and Goat anti-Rabbit) and AlexaFluor 568 (Goat anti-Mouse and Goat anti-Rabbit). These antibodies were purchased from Life Technologies (CA, USA). Hoechst 33,342 was purchased from Sigma Aldrich (MO, USA). Studies have observed that decreases in ZO-1, claudin-1 and occludin organisation within the cell membrane leads to disruption of barrier function in epithelial cells, thereby allowing entry of bacteria and other pathogens into the host [6–9, 14, 15]. Some evidence also suggests that alter- ation of adherens junctions can also facilitate the entry of pathogens into the host [6, 14]. Recent investigations sug- gest that epithelial barrier integrity may be dysfunctional in airway diseases such as asthma, where decreased tight junctional complexes and increased layer permeability have been observed [3, 16, 17]. Tight junction proteins ZO-1 and occludin have also been shown to have lower expression and a disorganised structure in asthmatic epi- thelium, when compared to non-asthmatic epithelium, resulting in reduced barrier function [15, 16]. Cell Culture and Maintenance 16HBE14o- cells, a SV-40 transformed bronchial epithe- lial cell line, were kindly provided by Dr. Dieter Gruenet (University of California, San Francisco, USA). Cells were cultured in MEM containing 10% (v/v) FCS, 100 U/mL (v/v) Penicillin/Streptomycin and 1% (v/v) L- Glutamine in a 37 °C, 5% CO2 incubator. For experi- ments, cells were seeded at a density of 10,000 cells/ coverslip on glass coverslips previously coated with 10 μg/mL fibronectin, 30 μg/mL collagen I and 100 μg/ mL BSA. Cells were maintained under standard culture conditions until 100% confluency over the coverslips was achieved. Cultures were then continued for a further 3 days before being fixed for subsequent immunocyto- chemical analysis to ensure complete generation of tight junction proteins. g Tight junction integrity has typically been assessed using Transepithelial Electrical Resistance (TEER) [18]. Higher resistance measurements are typically observed in conflu- ent polarised cultures with intact junctional complexes since ions cannot pass across the epithelial cellular layer into basal compartments [19]. Conversely, low TEER values are a consequence of increased ion transport across the epithelial layer, indicative of increased permeability result- ing from incomplete tight junctions [15, 19, 20]. Despite these measurements providing insight into the global changes, they fail to provide insight into localised changes that may be occurring between cells. Thus, confocal microscopy provides a valuable tool for the visualisation and assessment of local protein changes and interactions, and may also be used to corroborate TEER measurements. Here, we optimised and established a methodology for epithelial tissue fixation for the immunocytochemical ana- lysis of tight junctions (ZO-1, claudin-1 and occludin), Methanol, Acetone, Methanol: Acetone Primary antibodies were incubated on cells for 1 h at RT, followed by washing with 1× TBS at RT every 10 min for 1 h. Secondary antibody incubation and wash was performed as per the primary antibody incubation step. Cells were also incubated with Hoechst (2.5 μg/ mL) for 5 min at RT during the final wash step to stain for nuclei. All coverslips were mounted with mounting medium containing 19 mM polyvinyl alcohol (PVA, Sigma Aldrich, MO, USA), 45 mM Trizma Base (Sigma Aldrich, MO, USA), 45 mM NaH2PO4.2H2O, 27% (v/v) glycerol (Sigma Aldrich, MO, USA), and 4.9 mM chloro- butanol (Sigma Aldrich, MO, USA). Negative control samples were included to determine the level of non- specific binding of secondary antibodies to the tissue. Fixation with coagulant fixatives was performed using either ice cold 100% methanol, acetone or 1:1 methanol: acetone. Cells were fixed at −20 °C for 10 min, followed by washing with TBS for 30 min at RT, replacing wash TBS every 5 min. Cells were then stored in TBS at 4 °C until required. Blocking Solution To minimise non-specific binding of primary and second- ary antibodies in samples, blocking solution containing 10% (v/v) NGS, 10% (v/v) FCS and 1% (v/v) BSA in 1× TBS was incubated on cells for 30 min at RT. For parafor- maldehyde fixed samples, 0.2% (v/v) Triton X-100 was in- cluded in the blocking solution. In addition, all antibodies were diluted in the blocking solution outlined above. Paraformaldehyde Cells were fixed using 4% (v/v) paraformaldehyde in 71 mM Tris Buffered Saline (TBS), pH 7.4, at room temperature (RT) for 15 min, followed by washing with TBS for 30 min at RT, replacing wash TBS every 5 min. Cells were then stored in TBS at 4 °C until required. Permeabilization Following fixation, permeabilization was performed on a number of samples. Here, cells were treated with 0.1% (v/v) Saponin in 1× TBS and incubated at RT for 10 min. Cells were then washed with 1× TBS for 30 min at RT, replacing wash TBS every 5 min. Cells were then stored in TBS at 4 °C until required. Establishment of ALI Cultures Primary airway epithelial cells (AECs) were obtained from children admitted for elective surgery for non-respiratory related conditions [21–23] and de-identified prior to down- stream analysis. Primary AECs were then grown on 6.5- mm Transwell-Clear inserts 0.4 μm pore size (Corning, NY, USA) pre-coated with 30 μg/mL human placental collagen type I, which has been previously demonstrated to support AEC growth [24]. Cells were grown under submerged conditions in Bronchial-Air Liquid Interface (B-ALI™, Lonza, MD, USA) growth media until confluent. To differentiate into ciliated pseudostratified AECs, media Buckley et al. Biological Procedures Online (2018) 20:3 Page 3 of 9 either methanol or paraformaldehyde as described above. Cells were then stored in TBS at 4 °C until required. was removed from the apical side and this was considered Day 0 of ALI culture and the start of the experimental period. Cells were then grown in B-ALI™differentiation media, added to the basolateral side every alternate day and the apical side washed with tissue-culture sterile 1X PBS weekly. Cultures were grown for 28 days at ALI to ensure maximal differentiation as assessed by the presence of beat- ing cilia as well as mucus production, as evident by mucus build-up on the apical side of the cultures. Fixation This study sought to investigate various fixation methods suitable for the reproducible staining of epithelial airway cells. All treatments were repeated in triplicate. All fixation combinations can be found in Table 1. Triton X-100 Pre-extraction Cells were incubated with 0.2% (v/v) Triton X-100 in 1× TBS on ice for 10 min, followed by gentle washing with TBS for 30 min at RT, replacing wash TBS every 5 min. Fixation following pre-extraction was performed with Table 1 Fixative combinations used in this study. All fixative combinations were performed in triplicate on 16HBE14o- cultured cells. Immunocytochemistry was performed on cells as d il d cultured cells. Immunocytochemistry was performed on cells as detailed Pre-extraction Fixation Permeabilization – 4% Paraformaldehyde – 0.2% Triton X-100 4% Paraformaldehyde – – 4% Paraformaldehyde 0.1% Saponin – 4% Paraformaldehyde + Acetone – – 100% Methanol – 0.2% Triton X-100 100% Methanol – – 100% Methanol 0.1% Saponin – 1:1 Methanol:Acetone – – 100% Acetone – Discussion Positive staining was observed following methanol fixation for ZO-1 and claudin-1 tight Table 2 Qualitative assessment of fluorescent staining of tight junction antibodies, where: - indicates negative staining; + indicates weak staining with no consistent structure; ++ indicates moderate staining of tight junctions, with some structure present; +++ indicates strong staining, with consistent structures present Fixation Claudin-1 Occludin ZO-1 4% Paraformaldehyde – – – 0.2% Triton Pre-extraction + 4% Paraformaldehyde – – – 4% Paraformaldehyde + 0.1% Saponin – – + 4% Paraformaldehyde + Acetone – – – Methanol +++ – +++ 0.2% Triton Pre-extraction + Methanol – – – Methanol + 0.1% Saponin + – + Methanol + Acetone – +++ +++ Acetone – – – Table 2 Qualitative assessment of fluorescent staining of tight junction antibodies, where: - indicates negative staining; + indicates weak staining with no consistent structure; ++ indicates moderate staining of tight junctions, with some structure present; +++ indicates strong staining, with consistent structures present Fixation Claudin-1 Occludin ZO-1 4% Paraformaldehyde – – – 0.2% Triton Pre-extraction + 4% Paraformaldehyde – – – 4% Paraformaldehyde + 0.1% Saponin – – + 4% Paraformaldehyde + Acetone – – – Methanol +++ – +++ 0.2% Triton Pre-extraction + Methanol – – – Methanol + 0.1% Saponin + – + Methanol + Acetone – +++ +++ Acetone – – – Table 2 Qualitative assessment of fluorescent staining of tight junction antibodies, where: - indicates negative staining; + indicates weak staining with no consistent structure; ++ indicates moderate staining of tight junctions, with some structure present; +++ indicates strong staining, with consistent structures present Methodologies to perform reproducible immunocyto- chemistry for tight junction proteins (ZO-1, occludin and claudin-1) in epithelial derived cells to date are inconsist- ent and, at times, conflicting [6, 26, 27]. Particularly for epithelial cells, the fixation method must be carefully chosen to ensure optimal staining for the antigens of interest [28, 29]. Routine histological fixatives are often used without thought as to why one fixative may be better suited for a particular antigenic epitope than another. The most common types of fixatives used in immuno- cytochemistry fall into two categories: (1) non-coagulative or cross-linking and (2) coagulative fixatives. The cross- linking family include formaldehyde and glutaraldehyde [29]. These fixatives transform the cytosol into an insol- uble gel by the formation of methylene bridges between proteins, which halt autolysis and harden tissue [30, 31]. Confocal Microscopy Treated and control samples were imaged using a Nikon A1 inverted confocal microscope (Nikon, Japan), with a Nikon Plan Apo VC 60× NA 1.4 oil immersion objective (Nikon, Japan) and NIS-AR Elements software (v4.2.22, Nikon, Japan). Individual channels were captured sequen- tially, where a 405 nm laser was used for Hoechst 33,342 with collection through a 450/50 bandpass filter, AF488 excited using a 488 nm laser with collection through 525/ 50, and AF568 excited with a 561 nm laser and collected through a 585/50 bandpass filter. Z-stack images with step size of 0.5 μm were collected with a pinhole of 35.8 μm (1.2 A.U. for 488 nm laser), where the top and bottom of the stacks were determined visually. Buckley et al. Biological Procedures Online (2018) 20:3 Page 4 of 9 Page 4 of 9 Results junction proteins, but no staining was detected for occlu- din (Fig. 2). Fixation with acetone failed to expose any positive staining for ZO-1, occludin or claudin-1 in the samples (data not shown). Permeabilization with saponin following methanol fixation was unsuccessful in produ- cing tight junction staining for any of the antibodies assessed (data not shown). Combinatorial coagulative fix- ation produced consistent positive staining for both ZO-1 and occludin. However, no claudin-1 staining was ob- served (Fig. 3). No fluorescence was observed in negative controls for all fixation methods, where positive sample settings were used (data not shown). To determine the extent of tight junction formation in epithelial cells we examined the effect of various fixatives on the epithelial cell line 16HBE14o- (Table 2). Initial ex- periments did not produce staining for any fixation combi- nations where fibronectin/collagen coating of coverslips was omitted (data not shown). Coverslip coating was used following these preliminary experiments to aid adherence and cell growth [25]. Image analysis of paraformaldehyde fixed cells, with a Triton X-100 permeabilization step, showed no specific staining of tight junction complexes ZO-1, occludin or claudin-1 (Fig. 1). Saponin was used fol- lowing paraformaldehyde fixation as an alternative permeabilization agent to Triton X-100. Data generated showed that use of saponin slightly increased junctional staining post paraformaldehyde fixation for ZO-1, but positive staining was highly variable within samples (data not shown). Occludin and claudin-1 staining was absent following paraformaldehyde-saponin fix- ation and permeabilization. Pre-extraction with 0.2% Triton X-100 on ice, followed by fixation, was also tested. However, following the pre-extraction treat- ment, all cells lost attachment to the coverslip and immunocytochemistry was not performed (data not shown). Using a combinatorial approach of parafor- maldehyde fixation, followed by acetone fixation to increase permeabilization, also failed to yield positive staining for ZO-1, occludin or claudin-1 (data not shown). Following optimisation of the staining protocol, stain- ing on primary cells cultured to ALI was performed to verify compatibility between the transformed cell line and primary cells. Fixation of ALI culture with methanol showed positive staining for both claudin-1 and ZO-1, as seen with the 16HBE14o- cell line, whilst fixation with 1:1 methanol: acetone produced positive staining for occludin and ZO-1 (Fig. 4). Discussion In the current study, we found that fixation of 16HBE14o- cells for the tight junction proteins ZO-1, claudin-1 and occludin require different fixation proto- cols for reliable staining patterns. For consistent staining of claudin-1, fixation with ice cold methanol was re- quired, whilst occludin needed a combinatorial approach of methanol: acetone. We found that staining for ZO-1 could be positively identified using both fixation ap- proaches, and as such, could be used as a counter stain for both claudin-1 and occludin. Furthermore, we found that staining patterns in cell line 16HEB14o- was con- gruent with primary airway epithelial cells grown in ALI. This consistency in staining pattern reinforces their use- fulness as a substitute for protocol optimization, as ac- cess to paediatric primary epithelial cells is often limited. Coagulative fixation methods were also tested to deter- mine epitope accessibility. Discussion Fixation via this method may alter some of the tertiary Page 5 of 9 Buckley et al. Biological Procedures Online (2018) 20:3 Fig. 1 Paraformaldehyde fixation (4%) of 16HBE14o- cells in culture. The top row of panels show absence of specific staining for claudin-1 (Green) and ZO-1 (Red). The bottom row of panels show co-staining of occludin with ZO-1. Merged images showed nuclei staining with Hoechst (blue) Fig. 1 Paraformaldehyde fixation (4%) of 16HBE14o- cells in culture. The top row of panels show absence of specific staining for claudin-1 (Green) and ZO-1 (Red). The bottom row of panels show co-staining of occludin with ZO-1. Merged images showed nuclei staining with Hoechst (blue) may also be attributable to the restricted epitope access due to cross-linked adjacent proteins, or steric hindrance. protein structure within the tissue, but generally maintains secondary protein structures [29, 31–33]. Absence of claudin-1 and occludin antibody labelling using this fix- ation method in our laboratory may be due to the location of the proteins within the cell membrane, preventing suffi- cient access of the antibody to the epitope. Whilst the ZO-1 protein is not located within the cellular membrane, the negative staining following paraformaldehyde fixation As the cross-linking fixatives change cytoplasm into an insoluble gel, permeabilization steps may be required for immunocytochemical analysis of intracellular com- ponents [32, 34, 35]. Surfactants and non-ionic deter- gents, such as saponin and Triton X-100 respectively, are commonly used in immunocytochemistry for the Fig. 2 Methanol fixation of 16HBE14o- cells in culture. The top row of panels show claudin-1 co-stained with ZO-1. The bottom row of panels show the destruction of occludin staining using methanol as the fixative. Merged images showed nuclei staining with Hoechst (blue) Fig. 2 Methanol fixation of 16HBE14o- cells in culture. The top row of panels show claudin-1 co-stained with ZO-1. The bottom row of panels show the destruction of occludin staining using methanol as the fixative. Merged images showed nuclei staining with Hoechst (blue) Page 6 of 9 Buckley et al. Biological Procedures Online (2018) 20:3 Fig. 3 Methanol-acetone (1:1) fixation of 16HBE14o- cells in culture. The top row of panels show the absence of claudin-1 staining, whilst ZO-1 staining is clearly visible. The bottom row of panels show co-staining of occludin with ZO-1 using methanol-acetone as the fixative. Merged im- ages showed nuclei staining with Hoechst (blue) Fig. Discussion 3 Methanol-acetone (1:1) fixation of 16HBE14o- cells in culture. The top row of panels show the absence of claudin-1 staining, whilst ZO-1 staining is clearly visible. The bottom row of panels show co-staining of occludin with ZO-1 using methanol-acetone as the fixative. Merged im- ages showed nuclei staining with Hoechst (blue) purpose of increasing cellular permeability [35–38]. Solu- bilisation of lipid components non-specifically by Triton X-100, or specific cholesterol removal by saponin, facili- tates antibody access to intracellular compartments and epitopes without changing the cells’ ultrastructural integ- rity [28]. Saponin might be expected to increase epitope exposure for ZO-1, occludin or claudin-1. However, in our samples, permeabilization with saponin did not alter antibody staining of occludin or claudin-1 tight junction proteins, with occasional variable staining for ZO-1. This negligible staining following the use of surfactants may be due to the epitope for claudin-1 and occludin being located in a position that is not altered by the removal of, nor coupled to, lipids or cholesterol. Fig. 4 Methanol-acetone fixation of primary airway epithelial cells (AEC) grown at air liquid interface (ALI). The top row of panels show positive staining for both claudin-1 and ZO-1. The bottom row of panels show positive co-staining of occludin and ZO-1 in primary AECs grown at ALI. Merged images showed nuclei staining with Hoechst (blue) Fig. 4 Methanol-acetone fixation of primary airway epithelial cells (AEC) grown at air liquid interface (ALI). The top row of panels show positive staining for both claudin-1 and ZO-1. The bottom row of panels show positive co-staining of occludin and ZO-1 in primary AECs grown at ALI. Merged images showed nuclei staining with Hoechst (blue) Buckley et al. Biological Procedures Online (2018) 20:3 Page 7 of 9 Fig. 5 Schematic representation of the workflow required for the visualization of tight junctional complexes in airway epithelial cells. * denotes key points within the workflow which requires specialized optimization Fig. 5 Schematic representation of the workflow required for the visualization of tight junctional complexes in airway ep key points within the workflow which requires specialized optimization Pre-extraction with Triton X-100, followed by fixation with paraformaldehyde, has been suggested to remove some background staining in tissues, as some soluble components within the cell are removed prior to fixation [29, 34]. As such, this should provide greater access for antibodies to bind to epitopes of interest, as the lipids are removed in a non-selective manner. Discussion However, following exposure of confluent 16HBE14o- cells to Triton X-100, all cells appeared to lose attachment to the extracellular matrix (ECM). There are also suggestions that paraformal- dehyde is unable to sufficiently cross-link proteins in situ [39], although other studies suggest that formaldehyde is only released from tissues following years of washing tis- sues in water, and cross-linking bonds cannot be broken by urea [40]. In our study, it is likely that the epitopes of interest are insufficiently exposed via the cross-linking fix- ation and permeabilization methods commonly employed. In our samples, fixation with acetone failed to produce positive staining for any tight junction proteins. As acetone is a stronger organic solvent than alcohol, cell membrane loss can be observed following cellular fixation [31, 43]. To change the epitope exposure, without complete loss of cel- lular membranes, a fixative of 1:1 methanol: acetone was performed. Staining following dual fixation showed positive fluorescence for ZO-1 and occludin proteins, however claudin-1 staining was destroyed. It is plausible that the extra denaturation required for occludin antibody access results in masking of the claudin-1 epitope. The optimized fixation protocol was then repeated on primary airway epithelial cell culture samples derived from healthy participants, where cells had successfully reached a differentiated state when grown under ALI conditions. Fixation of the cultures with methanol yielded positive staining with claudin-1 and ZO-1, whilst methanol: acet- one fixation yielded positive staining for occludin and ZO- 1, reproducing the findings seen with the 16HBE14o−cul- tures. However, it should be noted that there were subtle differences in the staining intensity as well as the pattern of staining, suggesting that the final interpretation of staining should be restricted to primary cultures and not with the surrogate optimisation model. p y p y Coagulant fixatives are also commonly used to fix tissue for immunocytochemistry. This family includes alcohols such as ethanol and methanol, as well as acetone [33]. Fix- ation of our samples with coagulant fixatives produced varied results. The use of methanol fixation revealed posi- tive staining for ZO-1, but occludin staining was absent. Alcohol fixatives simultaneously fix and permeabilize cells, by extracting phospholipids and precipitating proteins in tissue [41]. They are frequently used for observing cellular cytoskeletal elements, as shown with the positive ZO-1 tight junction staining. The coagulant fixatives displace water molecules from proteinaceous materials, thereby breaking hydrogen bonds [42]. Discussion Alterations of hydrogen bonds can change the tertiary structure of proteins but does not alter the amino acid sequence of the epitope [42]. This can result in exposure of epitopes which were previously buried within the protein, thereby allowing antibody access and binding [42]. This alteration of pro- tein tertiary structure protein may not have been sufficient to unmask the occludin epitope, and as such, further in- vestigation was required. Funding Thi k This work was supported by grants from the National Health and Medical Research Council of Australia (1026494 & 1048910). Stephen M. Stick is a NHMRC Practitioner Fellow. 4. Schneeberger EE, Lynch RD. The tight junction: a mulitfunctional complex. Am J Physiol Cell Physiol. 2004;286:C121328. 5. Kojima T, Go M, Takano K, et al. Regulation of tight junctions in upper airway epithelium. Biomed Res Int. 2013; https://doi.org/10.1155/2013/947072. Availability of Data and Materials 6. Yeo N-K, Jang YJ. Rhinovirus infection-induced alteration of tight junction and adherens junction components in human nasal epithelial cells. Laryngoscope. 2010;120:34652. All data generated or analysed during this study are included in this published article and are available from the corresponding author on reasonable request. 7. Comstock AT, Ganesan S, Chattoraj A, et al. Rhinovirus-induced barrier dysfunction in polarized airway epithelial cells is mediated by NADPH oxidase 1. J Virol. 2011;85(13):6795–808. Acknowledgements Received: 25 July 2017 Accepted: 22 January 2018 The authors acknowledge the facilities, and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, a facility funded by the University, State and Commonwealth Governments. We would like to thank the contribution and assistance of all the respiratory fellows, anesthetists, nurses and hospital staff at Princess Margaret Hospital and St John of God. Finally, we would also like to thank the families and children participating in this study. Competing Interests Competing Interests The authors declare that they have no competing interests. Competing Interests Competing Interests The authors declare that they have no competing interests. 18. Stewart CE, Torr EE, Mohd Jamili NH, et al. Evaluation of differentiated human bronchial epithelial cell culture sytems for asthma research. J Allergy. 2012; https://doi.org/10.1155/2012/943982. Abbreviations AEC: Airway epithelial cell; ALI: Air-liquid interface; B-ALI: Bronchial Air-Liquid Interface; BSA: Bovine Serum Albumin; FCS: Foetal Calf Serum; MEM: Minimum Essential Medium; NGS: Normal Goat Serum; pAECs: Primary airway epithelial cells; PBS: Phosphate Buffered Saline; PVA: Polyvinyl Alcohol; RT: Room Temperature; TBS: Tris Buffered Saline; TEER: Transepithelial Electrical Resistance; ZO-1: Zona Occludens-1 Publisher’s Note 19. DeBenedetto A, Rafaels NM, McGirt LY, et al. Tight junctions defects in in patients with atopic dermatitis. J Allergy Clin Immunol. 2011;127:773–86. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 20. Rezaee F, Meednu N, Emo JA, et al. Polyinosinic-polycytidylic acid induces protein kinase d-dependent disassembly of apical junctions and barrier dysfunction in airway epithelial cells. J Allergy Clin Immunol. 2011;128:1216–24. Conclusions In conclusion, this study successfully established a meth- odological workflow (Fig. 5) using confocal microscopy to compare and evaluate staining expression levels of mul- tiple tight junction complexes in the human airway. Via the workflow, we established that there was no universal methodological approach appropriate for staining and visualising all tight junction proteins investigated. How- ever, we identified key points within the methodological workflow which after specialised optimisation lead to sub- sequent visualisation of each tight junction protein. Finally, we successfully demonstrated the reproducibility and translation of the workflow in primary AEC ALI cul- tures, indicating the adaptability of this method in other cell types. Of significance, this workflow can now be used Acetone is another coagulative fixative with strong lipid removal activity, particularly triglycerides and sterols [43]. Page 8 of 9 Page 8 of 9 Page 8 of 9 Buckley et al. Biological Procedures Online (2018) 20:3 Page 8 of 9 to visualise epithelial tight junctions and assess barrier in- tegrity in established cell cultures derived from chronic airway diseases including cystic fibrosis, chronic obstruct- ive pulmonary disorder and asthma. Centre for Health Research, The University of Western Australia, Crawley, Western Australia 6009, Australia. 5Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth, Western Australia 6001, Australia. 6School of Medicine, Notre Dame University, Fremantle, Western Australia 6160, Australia. 7School of Medicine, Faculty of Health, University of Tasmania, Hobart, Tasmania 7000, Australia. 8School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, New South Wales, Australia. 9Priority Research Centre for Asthma and Respiratory Disease, Hunter Medical Research Institute, Newcastle, New South Wales, Australia. 10Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada. 11School of Public Health, Curtin University, Bentley, Western Australia 6102, Australia. 12Telethon Kids Institute, Subiaco, Perth, Western Australia 6008, Australia. References F H 1. 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Authors’ Contributions AGB performed all the optimisation experiments in the cell line, conducted all experiments in the primary cell cultures and drafted the manuscript. KL, assisted with initial sample processing, cell culture establishment, maintenance of the primary cell cultures grown at air liquid interface and critically revised the manuscript. KML, ENS, KMM, EKS, LWG, NCS, all assisted with the human sample recruitment, sample processing, cell culture establishment and maintenance. FL performed the airway sampling and critically revised the manuscript. ANL, GZ, DAK, PJR and SMS were involved in the concept design and coordination of the study and critically revised the manuscript. AK optimized and established the protocols for cell culture, assisted with the concept and design of the study, assisted in sample collection and processing and critically revised the manuscript. All authors have read and approved the manuscript. 8. Sajjan U, Wang Q, Zhao Y, et al. 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Biological electron microscopy: theory, techniques, and troubleshooting. New York: Springer-Verlag; 1992. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W2211683832	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/CAB4EA86B65C7615436B4656F09E65BB/S1479262115000374a.pdf/div-class-title-longitudinal-analysis-of-maize-diversity-in-yucatan-mexico-influence-of-agro-ecological-factors-on-landraces-conservation-and-modern-variety-introduction-div.pdf	English	null	Longitudinal analysis of maize diversity in Yucatan, Mexico: influence of agro-ecological factors on landraces conservation and modern variety introduction	Plant genetic resources	2,015	cc-by	10,575	(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. ISSN 1479-2621 (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. ISSN 1479-2621 Abstract Transformations that farmers bring to their traditional farming systems and their impacts on the conservation and evolution of maize varieties over a 12-year period are investigated using a longitudinal analysis. Despite the increased introduction and supply of improved maize variety seeds in the Yucatan Peninsula, Mexico, over the last 12 years farmers continue to maintain a substantial amount of traditional maize variety diversity. Even with the increased availability of hybrid seeds, farmers in the community of Yaxcaba on average plant more than three quarters of their milpa ﬁelds to traditional maize varieties, with the latter one fourth predominately planted to a locally improved variety Nal Xoy, a farm cross of a traditional variety and an improved variety. We observed a signiﬁcant reduction in yellow – x-Nuuk nal, a long-cycle traditional landrace, paralleled by an increase in short- and intermediate-cycle locally adapted improved maize varieties. We found great differences in the distribution of maize varieties by soil type, with modern varieties being targeted for the rarer, deeper and ﬁne-grained soils, while traditional varieties predominate on the more prevalent stony and thin soils. Our results provide a picture in which most traditional maize varieties in Yaxcaba continue to be main- tained by farmers, coexisting with locally adapted improved varieties on the same landscape, and allowing the continued evolution of maize populations. Keywords: crop evolution; crop genetic diversity; milpa; traditional varieties * Corresponding author. E-mail: d.jarvis@cgiar.org Longitudinal analysis of maize diversity in Yucatan, Mexico: inﬂuence of agro-ecological factors on landraces conservation and modern variety introduction Marianna Fenzi1, Devra I. Jarvis2*, Luis Manuel Arias Reyes3, Luis Latournerie Moreno4 and John Tuxill5 1Centre Alexandre-Koyre´ – E´cole des hautes e´tudes en sciences sociales (EHESS), Paris, France, 2Bioversity International, Maccarese, Rome Italy, 3CINVESTAV-IPN Unidad Me´rida, Merida, Yucatan, Mexico, 4Instituto Tecnolo´gico de Conkal, Division de Estudios de Posgrado e Investigacio´n, Conkal, Yucatan, Mexico and 5Fairhaven College of Interdisciplinary Studies, Western Washington University, Bellingham, WA, USA Received 15 December 2014; Revised 10 April 2015; Accepted 3 July 2015 – First published online 2 September 2015 ceived 15 December 2014; Revised 10 April 2015; Accepted 3 July 2015 – First published online Study site Agro-morphological studies (Ortega Paczka and Dzib, 1992; Arias, 1995; Arias et al., 2002) have shown that the local maize populations planted in Yaxcaba corre- spond to the Mesoamerican and precolombian races Nal t’eel and Tuxpen˜o (known in Yucatan as x-Nuuk nal) and the subrace Ts’ı´it bakal (within the race Olotillo), which represent 75% of the maize diversity collected in the whole Yucatan Peninsula (Ortega Paczka and Dzib, 1992; Arias, 1995; Arias et al., 2002). From the data obtained in 1999 and 2000 surveys, four genetic groups have been identiﬁed in Yaxcaba municipality based on the female ear maturation time and cob charac- teristics. Accordingly, we followed planting trends of an early maturing group of 62–72 days (local varieties Nal t’eel, x-T’uup nal and x-Mejen nal), and a late group of 83–90 days (Ts’ı´it bakal and x-Nuuk nal varieties) within the Tuxpen˜o race and intermediate groups. All of the above varieties occur in yellow-, white- and blue-seeded populations (or seed lots), and farmers usually do not mix seed colours within a given popu- lation. We also documented trends in creolized varieties that are based on advanced generations of hybrids and open-pollinated varieties released by regional breeding Yaxcaba is in the middle of the zona maicera (maize- producing zone) that stretches across central and eastern Yucatan state, where small-scale milpa agriculture retains an important presence on the landscape and in the local economy. Approximately 9970 ha of milpa were culti- vated in 2011 in Yaxcaba municipality with a mean production of 0.69 ton/ha of maize (SIAP, 2011). The principal rainfed milpa crops in Yaxcaba are maize, beans (Phaseolus vulgaris and Phaseolus lunatus), squash (Cucurbita moschata and Cucurbita argyros- perma) and chile peppers (Capsicum annuum and Cap- sicum chinense). The rural population is predominantly Yucatec Maya in ethnicity and culture, and the zona maicera is considered as one of the most ‘traditional’ or Mayero regions of the Yucatan peninsula (Re Cruz, 1996). In the 1990s, several pioneering research projects on maize diversity management were launched in Mexico, which paved the way to a deeper understanding of ‘gen- etic erosion’ and maize diversity management strategies. However, little focus was placed on understanding how traditional crop genetic diversity is affected by the dynamics of the introduction of new technologies, in par- ticular the technological packages, capital investment, and hired labour needed to introduce the HYV in traditional farming systems. Materials and methods isolated (Frankel, 1973) gave way to more complex studies focused on a more interconnected view of crop genetic diversity (Zimmerer, 1996; Brush, 2004; Jarvis et al., 2011; Leclerc and Coppens, 2012). isolated (Frankel, 1973) gave way to more complex studies focused on a more interconnected view of crop genetic diversity (Zimmerer, 1996; Brush, 2004; Jarvis et al., 2011; Leclerc and Coppens, 2012). Introduction since the late 1960s (Frankel, 1967; Frankel and Bennett, 1970; Wilkes and Wilkes, 1972; Harlan, 1975). The 1980s and 1990s saw the launch of projects to investigate the complex association between genetic erosion and changes in traditional agriculture (Tuxill and Nabhan, 2001; Brush, 2004). The study of the replacement of indi- genous crop varieties with modern ones and the idea that traditional agricultural systems are static and relatively Genetic erosion linked to the loss of local crop varieties has been a major concern within scientiﬁc community https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press M. Fenzi et al. 52 https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press Study site Mexico is one of the world’s centres of crop diversity and contains the centre of origin of maize (Zea mays) with 62 races out of the 350 documented in all of Latin America (Kato et al., 2009). Mexico has also been a cen- tral actor in the Green Revolution, since the 1940s through the Rockefeller Foundation’s programme, and the 1960s through CIMMYT’s maize improvement programmes (Cotter, 2003). Mexican agricultural devel- opment has created a regime of coexistence of high yielding maize seed varieties (HYV) and landraces (Ortega Paczka, 2003). Trade liberalization, launched in 1994, reinforced the role of transnational agribusiness, which supported the spread of technological packages (improved seeds, herbicides and chemical fertilizers) through subsidies or campaigns of new production models (Fox and Haight, 2010). The municipality of Yaxcaba is located in the central maize-growing zone of Yucatan state at 2083205200 North and 8884904000 West longitude, encompassing approxi- mately 147,400 ha and lying 108 km east-southeast of Merida, the principal urban centre of the Yucatan peninsula. The region has a seasonally dry tropical climate of sub- type Awo with a mean annual temperature of 268C and a mean annual precipitation of 1024 mm. Temperature and altitudinal gradients are homogenous throughout the municipality of Yaxcaba. The major source of agroe- cological heterogeneity relates to soil differences. cological heterogeneity relates to soil differences. Yaxcaba is in the middle of the zona maicera (maize- producing zone) that stretches across central and eastern Yucatan state, where small-scale milpa agriculture retains an important presence on the landscape and in the local economy. Approximately 9970 ha of milpa were culti- vated in 2011 in Yaxcaba municipality with a mean production of 0.69 ton/ha of maize (SIAP, 2011). The principal rainfed milpa crops in Yaxcaba are maize, beans (Phaseolus vulgaris and Phaseolus lunatus), squash (Cucurbita moschata and Cucurbita argyros- perma) and chile peppers (Capsicum annuum and Cap- sicum chinense). The rural population is predominantly Yucatec Maya in ethnicity and culture, and the zona maicera is considered as one of the most ‘traditional’ or Mayero regions of the Yucatan peninsula (Re Cruz, 1996). Study site The management of genetic resources of maize is the product of social processes (Anderson, 1946; Hernandez, 1985) and farmer percep- tions of local and introduced varieties (Louette et al., 1997; Arias et al., 2000; Bello´n and Hellin, 2011). These local dynamics, together with ecological restrictions, shape the traditional agricultural practices that support landrace conservation (Perales et al., 2003b; Brush and Perales, 2007). Agro-morphological studies (Ortega Paczka and Dzib, 1992; Arias, 1995; Arias et al., 2002) have shown that the local maize populations planted in Yaxcaba corre- spond to the Mesoamerican and precolombian races Nal t’eel and Tuxpen˜o (known in Yucatan as x-Nuuk nal) and the subrace Ts’ı´it bakal (within the race Olotillo), which represent 75% of the maize diversity collected in the whole Yucatan Peninsula (Ortega Paczka and Dzib, 1992; Arias, 1995; Arias et al., 2002). From the data obtained in 1999 and 2000 surveys, four genetic groups have been identiﬁed in Yaxcaba municipality based on the female ear maturation time and cob charac- teristics. Accordingly, we followed planting trends of an early maturing group of 62–72 days (local varieties Nal t’eel, x-T’uup nal and x-Mejen nal), and a late group of 83–90 days (Ts’ı´it bakal and x-Nuuk nal varieties) within the Tuxpen˜o race and intermediate groups. All of the above varieties occur in yellow-, white- and blue-seeded populations (or seed lots), and farmers usually do not mix seed colours within a given popu- lation. We also documented trends in creolized varieties that are based on advanced generations of hybrids and open-pollinated varieties released by regional breeding The aim of this study is to understand what happens when modern and local varieties compete, using a longitudinal analysis based on two different surveys per- formed in 1999 and 2011 in the municipality of Yaxcaba, Yucatan, Mexico. Longitudinally based case studies of farmers’ management of crop varieties have recently been identiﬁed as a key research priority for genetic resources conservation (Dyer et al., 2014, 2015; Brush et al., 2015). We investigate why Yaxcaba farmers have continued to plant and manage maize landraces when modern varieties and commercial maize hybrids have become widely distributed and relatively easily accessed within the community and region. We also investigate transformations that farmers bring to their traditional farming systems and their impacts on the conservation and evolution of maize varieties over a 12-year period. Data collection Surveys of farmers were conducted in two different periods: 1999 and 2011 in Yaxcaba during the primary growing season (April to September). Sixty-one farmers in 1999 and 71 farmers in 2011 who had milpas in Yaxcaba were sampled, corresponding to approximately 10% of ejidatarios – ejido members with usufruct rights on common land in the community. The ejido is a legal form of common lands established by Mexico’s Land Reform during the 20th Century (1915–1992), in which members have usufruct rights on land that is owned by the community and managed for public beneﬁts, primar- ily agriculture. Accordingly to what was reported by farmers in informal discussions during interviews, only about two hundred of the ejidatarios in Yaxcaba are actu- ally making milpa. In addition, in 2011 more than 100 farmers not having formal ejidatario status made milpa on the ejido’s lands, therefore not disposing of govern- mental agricultural subsidy payments. The entire area of milpa cultivated annually in the ejido is estimated at approximately 1800 ha. The sampled farmers were initially selected in 1999 through a random selection of households stratiﬁed by geographic quadrants within Yaxcaba town (Jarvis et al., 2008; Tuxill et al., 2010). Thirty farmers interviewed in 1999 were included in the same group of farmers surveyed in 2011. In addition to speciﬁc information about seed lots, basic demographic information was collected for each farm household interviewed. Farmers also were asked about other jobs and activities (such as beekeeping; hunt- ing; livestock production; and local food processing, preparation and consumption) and other household income sources, including government subsidy and household support programs (e.g. PROCAMPO and DIC- ONSA). The interviews were conducted in either the farmers’ homes or at their milpas, depending on the pre- ference of the farmer. The interviews were conducted in either Spanish or Yucatec Maya depending on the language preference of the farmer; a local interpreter assisted with Maya translations. To supplement the infor- mation gained from interviews, a subset of farmers was also visited on separate occasions to observe their milpas and other household production sites and activi- ties. Beekeeping, for instance, is a major income-generat- ing activity that many farm households combine with milpa in rural Yucatan. Study site 53 Longitudinal analysis on maize varieties in Yucatan a single unit by one farmer during a single population generation (i.e. one cropping cycle). Farmers were asked about the quantity of maize seed that they used in the previous year; where and how they obtained their seed; the frequency with which they change seeds; their seed storage strategies; and the qualities and traits they valued in their maize varieties. Information from the farmers about other varieties not currently planted but that they had grown in the previous 10 years was collected separately. programmes that have been present in Yaxcaba since at least the 1980s (mejorado), and commercial varieties, mainly hybrids and a few open-pollinated varieties that have been recently (1–2 years) bought in a formal market and planted by farmers (hibrido). Data analysis Processed data were analysed by using descriptive statistics to score scale responses, frequency distribu- tions and mean comparisons. Statistical analyses were performed using the GraphPad Prism software Inc., (La Jolla, California, USA). Total area planted to each maize variety, both local and modern, was calculated based on GPS measurements and farmers’ diagrams and descriptions of their plots using the methods described in Jarvis and Campilan (2006). Standard diversity indices for crop varietal diversity (Jarvis et al., 2008), including richness (number) of maize varieties grown, and even- ness estimated as a complement of D (1 2 D), where D is the Simpson measure of dominance, were calculated and transformed logarithmically 1/(1 2 LN) (Magurran, 2003; Jarvis et al., 2008). Percentage divergence (i.e. the partition of diversity between and within farms) was cal- culated as the difference between community and farm index values divided by the community Simpson index. The average number of maize varieties per household and mean household Simpson Index was calculated for https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press Interviews Semi-structured interviews and participant observation were carried out in 2011 based on a questionnaire derived from the 1999 survey (see online Supplementary materials for complete survey). The variables quantiﬁed in the interviews in 1999 and 2011 included: the fre- quency and area to which farmers planted each different varieties of maize; the size and location of each milpa planted; the agroecological conditions under which the milpa is grown (association of cultivars, soil types and duration of cultivation); number of years each variety was grown; quantity of yield obtained in the last harvest, i.e. previous year (the latter expressed both using a quali- tative scale and with quantitative estimates). To analyse information on seed ﬂows, the concept of seed lots was used (following Louette et al., 1997) where a seed lot is an identiﬁable variety managed as M. Fenzi et al. M. Fenzi et al. 54 Table 1. Demographics and socio-economic characteristics of study participants. Median age ^ standard deviation is indicated in years. For other characteristics, numbers or percentages (in brackets) of farmers are presented Year Number Median age (range) Buying maize Selling maize Sons making milpa Paying for milpa Honey 1999 61 54 (30–86) 45 (63%) 19 (31%) 35 (57%) 26 (43%) 18 (30%) 2011 71 62 (26–90) 54 (76%) 10 (14%) 35 (49%) 33 (47%) 21 (30%) the two years surveyed (1999 and 2011). The total maize variety richness was calculated by summing the number of distinct maize varieties found in Yaxcaba. Groups were compared by using a non-parametric Mann– Whitney U-test. Spearman’s rank test was used to determine correlations. P values above 0.05 were consi- dered not statistically signiﬁcant. the two years surveyed (1999 and 2011). The total maize variety richness was calculated by summing the number of distinct maize varieties found in Yaxcaba. Groups were compared by using a non-parametric Mann– Whitney U-test. Spearman’s rank test was used to determine correlations. P values above 0.05 were consi- dered not statistically signiﬁcant. both time periods. Analysis of evenness at the household level for maize revealed similar low values (0.27 ^ 0.24 in 1999 versus 0.33 ^ 0.25 in 2011, P ¼ 0.2221; Fig. 1(a) right panel), which suggests that most farmers’ ﬁelds were and still are dominated by one maize variety. Results 5 (a) (b) (c) 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 0.3588 0.2221 0.0881 0.0451 4 3 Richness Evenness Evenness 1.0 0.8 0.6 0.4 0.2 0.0 0 2 4 Richness 6 8 Evenness Richness 2 1 0 8 6 4 2 0 1999 2011 1999 2011 1999 2011 1999 r=0.8641 P<0.0001 2011 r=0.8484 P<0.0001 1999 2011 Fig. 1. Comparison of diversity estimates in 1999 and 2011 surveys. (a) Mean richness (left panel) and evenness (right panel) in 1999 and 2011. (b) Longitudinal analysis for single farmer’s richness (left panel) and evenness (right panel) in 1999 and 2011. (c) Relationship between farm evenness and richness in 1999 (left panel) and 2011 (right panel). 5 (a) 0.3588 4 3 Richness 2 1 0 1999 2011 0.8 0.6 0.4 0.2 0.0 0.2221 Evenness 1999 2011 Demographics and socio-economic characteristics of study participants Demographic and socio-economic characteristics of the 61 farmers in 1999 and 71 farmers in 2011 interviewed are summarized in Table 1. Median age of farmers increased from 54 (range 30–86) years in 1999 to 62 (range 26–90) years in 2011 (P ¼ 0.0018) reﬂecting that many of the original farmers interviewed in 1999 still were included in the 2011 survey. Slightly higher pro- portions of farmers buying maize were observed in 2011 (76%) compared with 1999 (63%; P ¼ 0.0921), while we observed a reduction in the proportions of farmers selling maize from 1999 (31%) to 2011 (14%; P ¼ 0.0214). The proportion of farmers who have sons helping them to make milpa was 57% in 1999 and 49% in 2011 (P ¼ 0.3855). A slight increase was observed in the proportions of farmers paying for milpa between 1999 and 2011 (43 and 47% respectively; P ¼ 0.7266). Finally, the proportion of farmers producing honey as a part of their household activities was the same in 1999 (30%) and 2011 (30%; P ¼ 1). (b) 1.0 0.8 0.6 0.4 0.2 0.0 0.0881 0.0451 Evenness Richness 8 6 4 2 0 1999 2011 1999 2011 Evenness (c) 1.0 0.8 0.6 0.4 0.2 0.0 0 2 4 Richness 6 8 Evenness 1999 r=0.8641 P<0.0001 2011 r=0.8484 P<0.0001 Interviews When longitudinal analysis was restricted to those farmers (n ¼ 30) who were present in both the 1999 and the 2011 surveys, a trend increasing towards richness not reaching https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press Maize richness and evenness stability between 1999 and 2011 in Yaxcaba A comparison of average richness and evenness between 1999 and 2011 was performed in order to determine trends in maize varietal diversity conservation on farm. We failed to detect any signiﬁcant difference in average household richness for maize between the data obtained in 1999 (2.21 ^ 1.08) and 2011 (2.42 ^ 1.24) (P ¼ 0.3588; Fig. 1(a) left panel and Table 2) indicating that similar num- bers of maize varieties were cultivated by households at Richness Fig. 1. Comparison of diversity estimates in 1999 and 2011 surveys. (a) Mean richness (left panel) and evenness (right panel) in 1999 and 2011. (b) Longitudinal analysis for single farmer’s richness (left panel) and evenness (right panel) in 1999 and 2011. (c) Relationship between farm evenness and richness in 1999 (left panel) and 2011 (right panel). Fig. 1. Comparison of diversity estimates in 1999 and 2011 surveys. (a) Mean richness (left panel) and evenness (right panel) in 1999 and 2011. (b) Longitudinal analysis for single farmer’s richness (left panel) and evenness (right panel) in 1999 and 2011. (c) Relationship between farm evenness and richness in 1999 (left panel) and 2011 (right panel). https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press Longitudinal analysis on maize varieties in Yucatan 55 Table 2. Community and household area statistics and estimates of diversity for maize varieties in Yaxcaba Table 2. Community and household area statistics and estimates of diversity for maize varieties in Yaxcaba 1999 2011 Non-parametric Mann–Whitney U-test Mean HH area planted to Maize (ha) 3.79 ^ 1.86 2.95 ^ 1.41 P ¼ 0.0103 Mean HH area (ha) and mean percent (%) HH area planted to modern varietiesa 0.27 þ 0.84 (7%) 0.21 ^ 0.41 (9%) NS Mean HH area (ha) and percent (%) HH area of locally adapted improved varieties (locally bred landrace £ modern cross) 0.33 ^ 0.90 (2%) 0.476 ^ 0.657 (12%) P ¼ 0.0006 Mean HH area (ha) and percent (%) HH area planted to traditional varieties 3.46 þ 1.83 (91%) 2.47 ^ 1.55 (79%) P ¼ 0.002 Mean HH richness 2.21 ^ 1.08 2.42 ^ 1.24 P ¼ 0.3588 Mean HH evenness 0.27 ^ 0.24 0.33 ^ 0.25 P ¼ 0.2221 Community richness 13 13 N/A Number of modern varieties 2 2 N/A Number of locally adapted improved varieties 1 1 N/A Community evenness 0.71 0.80 N/A Divergence 0.61 0.59 N/A NS, not signiﬁcant. Maize richness and evenness stability between 1999 and 2011 in Yaxcaba When we analysed the areas cultivated with other traditional maize varieties, including – x-E´ek’ jub, white and yellow Ts’ı´it bakal, white and yellow x-T’uup nal, white and yellow x-Mejen nal and Nal t’eel, we failed to detect any signiﬁcant difference in both the area and proportions of cultivated areas between 1999 and 2011 (Fig. 2(a) and (b)). We next looked at the areas cultivated with locally adapted improved maize varieties. As shown in Fig. 2(a), no difference between 1999 and 2011 was observed in mean area cultivated with either Maı´z mejorado or Maı´z hı´brido. In contrast, a signiﬁcant increase was observed when proportions of land allocated to Maı´z hı´brido cultivation were analysed (from 2.63 ^ 1.08% in 1999 to 7.27 ^ 1.99% in 2011; P ¼ 0.019) (Fig. 2(b)). Interestingly, the most notable changes were observed when we considered areas allocated to the variety Nal Xoy, a locally adapted cross established by Ruﬁno Chi, a Mayan farmer from the village of Xoy, between the improved maize variety PR-7822 and the traditional var- iety Nal t’eel. We found a signiﬁcant increase in surfaces allocated to Nal Xoy cultivation from 1999, when Nal Xoy cultivation represented only 2.21 ^ 1.16% of the maize acreage, to 2011 when 12.27 ^ 2.98% of the maize cultivation area was constituted by Nal Xoy (P ¼ 0.0042) (Fig. 2(a) and (b)). Collectively, these results show that between 1999 and 2011 a decrease in the areas statistical signiﬁcance (P ¼ 0.0582) and a signiﬁcant increase in evenness (P ¼ 0.0451) were observed (Fig. 1(b)). The analysis of the relationship between the two measures of diversity, richness and evenness, at house- hold level was highly correlated in both 1999 and 2011 (Fig. 1(c)). At the community level, the number of varieties remained the same (community richness ¼ 13 varieties; Table 2). Community evenness increased slightly, indicat- ing a more even distribution of the area planted to the 13 varieties in the community of Yaxcaba in 2011 than in 1999, which can be seen in Table 2. The divergence or the difference between values of richness and evenness among households in the Yaxcaba community decreased in 2011 (Table 2). Maize richness and evenness stability between 1999 and 2011 in Yaxcaba a Modern varieties include improved (advanced generations of commercial varieties) and hybrid maize. (‘yellow maize’) represented in 1999 the most widely cultivated varieties, covering 32.71 ^ 5.05% and 42.12 ^ 5.35% respectively of milpa surface (Fig. 2(a) and (b)). Similar proportions of white x-Nuuk nal culti- vated areas were still planted in 2011, while a signiﬁcant decrease in the proportion of milpa areas allocated to yellow x-Nuuk nal was observed (25.4 ^ 4.35% in 2011; P ¼ 0.0188; Fig. 2(b)). When we analysed the areas cultivated with other traditional maize varieties, including – x-E´ek’ jub, white and yellow Ts’ı´it bakal, white and yellow x-T’uup nal, white and yellow x-Mejen nal and Nal t’eel, we failed to detect any signiﬁcant difference in both the area and proportions of cultivated areas between 1999 and 2011 (Fig. 2(a) and (b)). We next looked at the areas cultivated with locally adapted improved maize varieties. As shown in Fig. 2(a), no difference between 1999 and 2011 was observed in mean area cultivated with either Maı´z mejorado or Maı´z hı´brido. In contrast, a signiﬁcant increase was observed when proportions of land allocated to Maı´z hı´brido cultivation were analysed (from 2.63 ^ 1.08% in 1999 to 7.27 ^ 1.99% in 2011; P ¼ 0.019) (Fig. 2(b)). Interestingly, the most notable changes were observed when we considered areas allocated to the variety Nal Xoy, a locally adapted cross established by Ruﬁno Chi, a Mayan farmer from the village of Xoy, between the improved maize variety PR-7822 and the traditional var- iety Nal t’eel. We found a signiﬁcant increase in surfaces allocated to Nal Xoy cultivation from 1999, when Nal Xoy cultivation represented only 2.21 ^ 1.16% of the maize acreage, to 2011 when 12.27 ^ 2.98% of the maize cultivation area was constituted by Nal Xoy (P ¼ 0.0042) (Fig. 2(a) and (b)). Collectively, these results show that between 1999 and 2011 a decrease in the areas (‘yellow maize’) represented in 1999 the most widely cultivated varieties, covering 32.71 ^ 5.05% and 42.12 ^ 5.35% respectively of milpa surface (Fig. 2(a) and (b)). Similar proportions of white x-Nuuk nal culti- vated areas were still planted in 2011, while a signiﬁcant decrease in the proportion of milpa areas allocated to yellow x-Nuuk nal was observed (25.4 ^ 4.35% in 2011; P ¼ 0.0188; Fig. 2(b)). https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press Maize variety cultivation and land use 56 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Surface (ha) White x-nuuk nal Yellow x-nuuk nal x-Éek’ jub White Ts’iit bakal Yellow Ts’iit bakal White x-T’uup nal Yellow x-T’uup nal White x-Mejen nal Yellow x-Mejen nal Nal Xoy Nal t’eel Improved Hybrid 1999 2011 (a) 0.0119 0.0188 0.0078 M 0 White x-nuuk nal Yellow x-nuuk nal x-Éek’ jub White Ts’iit bakal Yellow Ts’iit bakal White x-T’uup nal Yellow x-T’uup nal White x-Mejen nal Yellow x-Mejen nal Nal Xoy Nal t’eel Improved Hybrid 5 10 15 20 25 30 35 40 45 50 Surface (%) 1999 2011 (b) 0.0188 0.0042 0.0190 Fig. 2. Cultivated maize varieties in 1999 and 2011. Hectares (a) or proportions (b) of surfaces cultivated with the indicated maize varieties in 1999 and 2011. See online Supplementary materials for detailed values of the graphs. Fig. 2. Cultivated maize varieties in 1999 and 2011. Hectares (a) or proportions (b) of surfaces cultivated with the indicated maize varieties in 1999 and 2011. See online Supplementary materials for detailed values of the graphs. with particular traits. We observed a signiﬁcant reduction in yellow x-Nuuk nal, a long-cycle traditional landrace, paralleled by an increase in short- and intermediate- cycle locally adapted improved maize varieties and Nal Xoy in particular. We found a signiﬁcant decrease in pro- portions of areas cultivated with long-cycle traditional varieties, namely yellow and white x-Nuuk nal, x-E´ek’ jub and yellow and white Ts’ı´it bakal from 1999 (86.33 ^ 2.89%) to 2011 (71.47 ^ 4.28%) (P ¼ 0.0078). We detected a parallel increase in the proportions of areas cultivated with either traditional (yellow and white x-T’uup nal, yellow and white x-Mejen nal, allocated to traditional varieties paralleled by an increase in the areas devoted to locally adapted improved maize varieties was observed in Yaxcaba milpas. Interestingly, the changes in locally adapted improved maize varieties were mainly linked to a strong increase in Nal Xoy cultivation. Maize variety cultivation and land use The mean area per household devoted to maize cultiva- tion decreased signiﬁcantly from 3.79 ^ 1.86 ha in 1999 to 2.95 ^ 1.41 ha in 2011 (P ¼ 0.0103; Table 2). This reduction appears to result from a decrease in the culti- vated area allocated to traditional varieties, from 3.46 ^ 1.83 ha or 91% of the total household area devoted to maize in 1999 to 2.47 ^ 1.55 ha (79%) in 2011 (P ¼ 0.002), while the area devoted to locally adapted improved maize varieties signiﬁcantly increased from 0.33 ^ 0.90 ha (2%) in 1999 to 0.48 ^ 0.66 ha (12%) in 2011 (P ¼ 0.0006). To better determine the reasons for this change, we next studied the relative amount of cultivated area allo- cated to each maize variety in 1999 and 2011 (Fig. 2; see also online Supplementary materials). X-Nuuk nal (Tuxpen˜o) either Sak nal (‘white maize’) or K’an nal 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Surface (ha) 0 White x-nuuk nal Yellow x-nuuk nal x-Éek’ jub White Ts’iit bakal Yellow Ts’iit bakal White x-T’uup nal Yellow x-T’uup nal White x-Mejen nal Yellow x-Mejen nal Nal Xoy Nal t’eel Improved Hybrid White x-nuuk nal Yellow x-nuuk nal x-Éek’ jub White Ts’iit bakal Yellow Ts’iit bakal White x-T’uup nal Yellow x-T’uup nal White x-Mejen nal Yellow x-Mejen nal Nal Xoy Nal t’eel Improved Hybrid 5 10 15 20 25 30 35 40 45 50 Surface (%) 1999 2011 1999 2011 (a) (b) 0.0119 0.0188 0.0078 0.0188 0.0042 0.0190 Fig. 2. Cultivated maize varieties in 1999 and 2011. Hectares (a) or proportions (b) of surfaces cultivated with the indicated maize varieties in 1999 and 2011. See online Supplementary materials for detailed values of the graphs. M. Fenzi et al 56 M. Fenzi et al. https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press Discussion In the central part of Yucatan state, where the municipal- ity of Yaxcaba is located, the agricultural system is still largely traditional and centered on milpa cultivation due to extremely stony lands and because of strong com- munity cohesion in continuing to manage ejido lands as common property, with individual farm households having usufruct rights to milpa, beekeeping and other productive activities. Nal t’eel) or locally adapted improved short-cycle varieties (Nal Xoy, Mejorado, Hibrido) from 1999 (13.67 ^ 2.89%) to 2011 (28.53 ^ 4.28%) (P ¼ 0.0078). Trend towards shorter-cycle varieties (a) 55% Tsek’el lu’um Púus lu’um K’áankaab 34% 11% Long cycle Short cycle (b) Tsek’el lu’um Púus lu’um K’áankaab 93% 7% 43% 57% 30% 70% (b) Púus lu’um Púus lu’um Tsek’el lu’um Tsek’el lu’um K’áankaab Short cycle Traditional Modern (c) Tsek’el lu’um Púus lu’um K’áankaab 5% 28% 50% 95% 72% 50% (c) aditional Modern Púus lu’um K’áankaab 28% 50% 72% 50% (c) Tsek’el lu’um (c) Tsek’el lu’um 72% Modern Traditional Traditional Fig. 3. Distribution of maize varieties depending on soil types. (a) Pie chart representing the proportions of different soil types: Tsek’el lu’um (light grey part), Pu´us lu’um (dark grey part) and K’a´ankaab (black part). (b) Pie chart repre- senting the proportions of lands cultivated with long-cycle (dark grey parts) or short-cycle (light grey parts) varieties. (c) Pie chart representing the proportions of lands cultivated with traditional (dark grey part) or modern (light grey part) varieties. https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press Trend towards shorter-cycle varieties We next asked whether the observed changes in culti- vated varieties were linked to any trend of varieties https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press Longitudinal analysis on maize varieties in Yucatan 57 Long cycle Short cycle Traditional Modern (c) (b) (a) 55% Tsek’el lu’um Púus lu’um K’áankaab Tsek’el lu’um Púus lu’um K’áankaab Tsek’el lu’um Púus lu’um K’áankaab 34% 11% 93% 7% 43% 57% 30% 70% 5% 28% 50% 95% 72% 50% Fig. 3. Distribution of maize varieties depending on soil types. (a) Pie chart representing the proportions of different soil types: Tsek’el lu’um (light grey part), Pu´us lu’um (dark grey part) and K’a´ankaab (black part). (b) Pie chart repre- senting the proportions of lands cultivated with long-cycle (dark grey parts) or short-cycle (light grey parts) varieties. (c) Pie chart representing the proportions of lands cultivated with traditional (dark grey part) or modern (light grey part) varieties. g y (a) which represented 55.34 ^ 4.70% of cultivated land in 2011. A second, stony, dark-coloured soil type called Pu´us lu’um (Redzina) of variable depth, which presents a relatively high organic matter content, represented 10.58 ^ 2.51% of cultivated areas. Finally, K’a´ankaab (Cambisol), called kankabal in Spanish, is a deep, well- drained, reddish or reddish-black coloured, ﬁne-grained soil type, and represented 34.09 ^ 4.42% of maize culti- vated areas. We then looked at long- and short-cycle varieties’ distribution depending on soil type. The great majority of Tsek’el lu’um under cultivation was allocated to long-cycle varieties; the areas cultivated with short- cycle varieties represented only 6.9% of these soils (Fig. 3(b)). Interestingly, short-cycle varieties covered 42.7% of Pu´us lu’um areas and reached 70.3% in the case of K’a´ankaab plots (P , 0.0001; Fig. 3(b)). We next looked at the proportions of lands cultivated with traditional or modern varieties depending on soil type. Tsek’el lu’um and Pu´us lu’um lands were mainly cultivated with traditional varieties (95.4 and 72.4%, respectively), while half of the K’a´ankaab areas (50.2%) were allocated to the cultivation of modern varieties (P , 0.0001; Fig. 3(c)). These results reveal great differ- ences in the distribution of maize varieties depending on soil type, with short-cycle and modern varieties being more represented especially on the deeper and ﬁne-grained K’a´ankaab soils. Agroecological distribution of maize varieties depending on soil types Our results show that more farmers in 2011 failed to satisfy their household consumption needs with their own maize production compared with in 1999, and therefore needed to buy maize from off-farm sources. Accordingly, fewer farmers had sufﬁcient surplus maize production to commercialize for sale in 2011 when com- pared with in 1999. These observations indicate that maize cultivation in Yaxcaba continues to be mainly aimed at household consumption. Interestingly, farmers who still commercialize their maize production expressed a growing interest in the maize seed trade, considering it a better way to add value to their pro- duction and their knowledge of seed selection. Despite a signiﬁcant increase in the proportions of areas allocated to cultivating short-cycle maize varieties, the great majority of milpa lands are still devoted to long- cycle traditional varieties. Results of analysing the agroe- cological distribution of maize varieties in Yaxcaba according to soil types in 2011 are shown in Fig. 3(a). Yaxcaba lands devoted to maize cultivation are domi- nated by three main types of soil, which we identify here using both their local (Yucatec Mayan) and scientiﬁc taxonomy. Tsek’el lu’um (Lithosol), called pedregoso in Spanish, is a young, extremely stony, shallow, thin soil mainly present in the form of calcareous outcrops, By performing a longitudinal analysis on a farmer population, our study shows that in 2011 the number of 58 M. Fenzi et al. farmers paying other farmers to work in their milpa increased when compared with 1999. Such a result indicates an increase in household ﬁnancial resources deployed for growing milpa. Agriculture-related activities such as beekeeping for honey production, already represented in 1999, are an important source of household income but did not increase over time. We can hypothesize that other, not agriculture-related, sources of economic support increased in latter years, with increasing numbers of farmers performing seasonal jobs or migrating for short periods to earn money. In many cases, wage employment and intermittent migration out of Yaxcaba provide a funding system that allows the household to pay other younger farmers for the most laborious activities involved in the process of making milpa. Whether such changes in economic resources for milpa could affect maize varietal diversity maintenance and conservation by farmers needs further investigation. Agroecological distribution of maize varieties depending on soil types Soil taxonomy and land quality affect both the distri- bution of maize varieties across the landscape and the probability of farmers’ adoption of improved varieties (Latournerie et al., 2006; Bello´n and Taylor, 1993). Agriculture in the state of Yucatan has been shaped by signiﬁcant environmental constraints, including the pre- dominance of shallow stony soils, periodic shortfalls of rainfall during the growing season and the risk of major disturbance from cyclones (Duch, 1991). Certain maize varieties are speciﬁcally targeted for different soil types based on their agronomic competitiveness (Bello´n and Taylor, 1993). The permanence of shifting cultivation systems in extremely stony lands probably plays an important role as well in local varieties’ adoption. The management of varieties depending on soil quality can help to explain the low adoption of improved varieties. In Yaxcaba, locally adapted improved maize varieties generally fail to outperform traditional varieties on low- quality soils. Farmers plant inaccessible and stony plots with landraces in order to not be dependent on exogen- ous factors requiring high monetary investment and best care. In contrast, K’a´ankaab soils that are easily readily accessible to farm households require relatively high quantities of inputs and more efforts to control for weeds compared with milpa plots managed under longer fallow periods in the ‘monte’ (woods). Kankabales are the areas where locally adapted improved maize is mainly represented and where a logic prevails of intensi- ﬁed production, with shortened fallow periods (often approaching continuous cultivation) and a high depen- dency on the use of commercial NPK fertilizer and herbi- cides (Tuxill, 2005). Farmers consider their traditional maize varieties to be better adapted to the conditions of the ejido outside of the kankabales sites. This study high- lights the importance of maize landraces for Yaxcaba farmers in capitalizing on all soils in the ejido, as kanka- bales represent only about one third of the area available for milpa agriculture in Yaxcaba. Even with the increased availability of improved and hybrid seeds over the last 12 years, farmers in Yaxcaba on average plant more than three quarters of their milpa ﬁelds to traditional maize varieties, and plant the latter remaining quarter predominately to a locally improved variety Nal Xoy. Collectively, these results demonstrate stability in the average household maize diversity cultivated in Yaxcaba between 1999 and 2011. Analysis of changes in the proportions of cultivated var- ieties in Yaxcaba shows that all improved maize varieties are not equivalent. https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press Agroecological distribution of maize varieties depending on soil types In contrast, the majority of studies measuring the amount of crop gen- etic diversity held by smallholder farmers in traditional agricultural communities worldwide, and in Mexico in particular, show that the number of varieties captured by surveying a limited number of farmers within a com- munity is generally not representative of the pool of var- ieties available to farmers at the community level (Jarvis et al., 2008; Bajracharya et al., 2010; Mulumba, 2012; Brush et al., 2015). Our longitudinal survey monitored varieties grown by 61 individual interviewees of the Yax- caba community in 1999 and 71 in 2011, and we did not ﬁnd any signiﬁcant genetic erosion at the community level, even with substantial ﬂuctuation in the relative abundance of some varieties. A second methodological difference with our study is that the ENHRUM survey addressed maize varieties within a very extensive questionnaire that was not focused primarily on maize diversity. It is our opinion that this is not an optimal way to obtain high-quality information on maize diversity. In our study, particular attention was given to gather maximum information about seed lots, seed distri- bution and seed ﬂows, through speciﬁc and repeated questions on each variety, both in local Spanish and Yuca- tec Maya names. These deep discussions allowed conﬁr- mation of the amount and distribution of hybrid maize varieties in the community, a methodology widely used to better understand the number of varieties accessed Despite the increased introduction and supply of improved maize varieties in the Yucatan Peninsula over the last 12 years, farmers continue to maintain a substan- tial amount of traditional maize variety diversity. Accord- ingly, less than 25% of maize seeds in Mexico were purchased from formal sectors in 1999 (Ortega Paczka et al., 2000). Farmers choose among a portfolio of maize varieties: traditional, locally improved, nationally improved and hybrid. Over the 12 years of this study, the richness (number of varieties) and their evenness in terms of area planted by Yaxcaba farm households remained the same. Agroecological distribution of maize varieties depending on soil types (2014) conclude that previous community-based case studies have failed to ﬁnd genetic erosion in maize populations due to a preference or bias for atypically high-diversity sites on the part of researchers. farmers have modiﬁed through selection (Vigouroux et al., 2011). The existence in Yaxcaba of two racial complexes (long cycle and short cycle) can be explained by the need to avoid having maize plants that are ﬂowering or ﬁlling out ears when periodic short-term interaestival drought conditions (known locally as la canicula) occur (Tuxill et al., 2010). Environmental factors such as recent changes in rainfall regime or hurricanes may inﬂuence farmers’ decision-making, potentially leading to changes in two ways. The ﬁrst is the change in milpa planting months that has moved from May/June to June/July (Canul Ku, 2009). The second is, as observed in this study, a reduction of long-cycle traditional maize varieties paralleled by an increase in short-cycle locally improved varieties. This change may mean that producers are moving these two complexes toward a ‘variety balance’ as an additional way to ensure production in a context of increased climate uncertainty. Our research in Yaxcaba addresses one of the principle critiques of previous case studies on maize diversity raised by Dyer et al. (2014, 2015), in that we utilize matched longitudinal data that required following a survey sample of farmers over an extended period of time, in our case 12 years. Although our study area, Yaxcaba, was not included in the ENHRUM national survey sample, our study time period overlaps entirely with that of Dyer et al. (2014). We suspect our conclusions differ predominantly because of the sampling method- ology of the ENHRUM survey on which Dyer et al. (2014)’s conclusions are based. The ENHRUM survey strategy was to sample many communities but relatively few households per community, i.e. 68 maize sowing communities were covered (Dyer et al., 2014), but only a total of 606 households sowing maize were interviewed (Dyer and Lo´pez-Feldman, 2013), resulting in a variable and probably too low number of households sampled in each community covered in the survey. Agroecological distribution of maize varieties depending on soil types The area of locally adapted improved varieties signiﬁcantly increased primarily due to the adop- tion of the Nal Xoy variety, while the nationally released and hybrid varieties did not change signiﬁcantly in their extent of adoption from 1999 to 2011. Nal Xoy’s history and diffusion pattern suggest that this new variety devel- oped by farmers for agricultural households is efﬁciently adapted to local needs. The title of Ruﬁno Chi Canul’s communication (2002) is very eloquent: ‘Nalxoy, maize for traditional milpa’. In 1983, upon the introduction of the improved variety named PR-7822 in the village of Xoy, people realized that, although providing good yields, this variety presented some drawbacks in its sus- ceptibility to pest attacks during storage, and in its culin- ary qualities. To solve these problems the new variety was crossed by farmers in Xoy with a population of Nal t’eel. As Ruﬁno Chi reported, people ‘could store in “trojes”, maize that could be intercropped with common beans and lima beans, maize that would be easy to degrain by hand, and maize that would be good for ’tortillas, pozole, and other foods’ (Chi Canul, 2002, p. 37). Because of the interest that developed for what has thereafter been named Nal Xoy variety and with some institutional help, Nal Xoy rapidly spread among farmers all over the Yucatan Peninsula. The wide panel of varieties accessed creates a mosaic of traditional and improved maize varieties within and among the Yaxcaba agricultural landscape. Such a mosaic pro- vides the opportunity for continuous gene ﬂow among maize varieties, sustaining the on-going evolution of the crop (Bello´n and Brush, 1994; Jarvis and Hodgkin, 1999; Louette and Smale, 2000; Bello´n and Risopoulos, 2001). However, management practices employed by farmers for selection of features to be maintained in the next gen- eration may limit gene ﬂow between varieties. Farmers’ selection of agro-morphological characteristics may reﬂect farmers’ reactions to new genetic diversity introduction in their crop populations, accepting or promoting desired traits from improved varieties or conserving favourable traits from old ones (Perales et al., 2003b). The cycle of maturation is probably one of the characteristics that 59 Longitudinal analysis on maize varieties in Yucatan from random household interviews in Mexico show a notable drop in richness between 2002 and 2007 of 1.43 to 1.22 varieties at the household level. From these results, Dyer et al. https://doi.org/10.1017/S1479262115000374 Published online by Cambridge University Press Agroecological distribution of maize varieties depending on soil types Several previous case studies at the community level in Mexico (Perales et al., 2000; Perales et al., 2005; Latourn- erie-Moreno et al., 2006; Brush and Perales, 2007; Bello´n and Hellin, 2011; Tuxill et al., 2010) and elsewhere (Brush, 2004; Guzman et al., 2005; Bisht et al., 2007; Rana et al., 2007; Jarvis et al., 2008; Bezanc¸on et al., 2009; FAO, 2010; Jackson et al., 2010; Mulumba et al., 2012; Zimmerer, 2013) have demonstrated the persistence of landraces for climatic, ecological, cultural or social reasons. This trend is reafﬁrmed by the data from Mexico’s national effort for maize diversity recollection (CONABIO, 2014). Comparing race richness from this last collection and of historical collections started in the late 1940s, Perales and Golicher, (2014) conclude that maize diversity in Mexico is relatively stable although not evenly distributed within their 11 designated biogeo- graphic regions. In contrast, in a recent study employing matched longitudinal data from the Mexico National Household Survey (ENHRUM), Dyer et al. (2014) argue that the data obtained from case studies, which collec- tively suggest maize diversity is maintained in Mexico, are overshadowed by a widespread loss of maize diversity at a national scale. Dyer et al. (2014)’s measurements of richness of maize varieties, elaborated M. Fenzi et al. 60 within a community (Louette et al., 1997; Pautasso et al., 2012). 2011; Thomas et al., 2011; Jarvis et al., 2016). Our ﬁndings conﬁrm that in areas with limiting climatic and soil conditions, which constitute a large part of Mexico, farmers have continued to maintain their traditional var- ieties or the creole varieties that contain the germplasm of their traditional varieties crossed with commercial seed. We note that the ﬁrst wave of traditional variety replacement has already passed in Mexico. Longitudinal case studies have supplied important insights on farmers’ adjustments after this ﬁrst wave and the roles traditional varieties continue to play in ensuring agricultural pro- duction and cultural identity for smallholder farmers in the Yucatan. Dyer et al. (2014) warn against the continuing decline in average number of maize varieties per household, focusing on the ﬁrst decade of the 21st century. In fact, the maize varietal landscape in Mexico began to undergo substantial change much earlier than 2002. Mexico is one of the ﬁrst countries to have developed hybrid maize varieties (or to introduce them from USA) through the Rockefeller Foundation’s programmes in the mid-1940s (Cotter, 2003). Acknowledgements The authors thank the Swiss Agency for Development and Cooperation (SDC) for providing ﬁnancial support to this study, and the International Development Research Centre (IDRC), Canada for funding the 1999 survey. Marianna Fenzi was supported by a PhD scholar- ship from the Centre Alexandre-Koyre´, E´cole des hautes e´tudes en sciences sociales (EHESS). The authors thank Dr. Luis Dzib Aguilar for helpful discussions. The 1999 Yaxcaba survey was carried out by Jose Vidal Cob Uicab, Jaime Canul Ku, Luis Burgos May and Teresa Quinones. They also thank them for their data collection, and thank all Yaxcaba farmers for generously sharing their experience and knowledge. They thank Efraim Moo Ake for his crucial help in carrying out the ﬁeld work. Our longitudinal analysis on a time frame that overlaps with that studied by Dyer et al., found no evidence of genetic erosion in Yaxcaba, a community representative of much of southern and south-east Mexico in terms of maize diversity. In Mexico, as elsewhere in the world, many farmers continue to keep their traditional varieties for pragmatic reasons: as a means for improving agri- cultural production and productivity in low input conditions; as an insurance to maintain productivity in heterogeneous environments, or under changing climates; for the sustained local consumer demand for diverse food products, and because of the concerns and interests of the farmers and communities themselves who wish to retain control over their production systems (Perales et al., 2003a; Edmeades et al., 2006; Salazar et al., 2007; Giuliani, 2007; Bello´n et al., 2009; Bocci and Chable, 2009; Kontoleon et al., 2009; Practical Action, Agroecological distribution of maize varieties depending on soil types Commercial varieties have been spread and tested in a wide range of environments in rural Mexico since 1950. In the USA and France, hybrid maize varieties largely replaced local materials in less than a decade during the 1950s for USA and 1960s for France (Bonneuil and Thomas, 2009). There is no reason to think that farmers in the marginal zones of Mexico waited for 60 years to access and adopt the new seeds when fertilizer, and later herbicides, were inte- grated widely by farmers in the same zone. The replace- ment phenomenon was not evenly distributed: in regions of Mexico where farming of new varieties gave a more important commercial advantage, local varieties have been replaced since the 1950s, whereas in regions where landraces displayed a competitive advantage thanks to their local adaptation, today we still ﬁnd con- siderable diversity and gene ﬂow. Observations by Dyer et al. in the North and West of Mexico reﬂect this dynamic in explaining the origins of the current low levels of maize diversity there as well as the low replacement rates indicative of a stabilized situation. 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https://openalex.org/W2964122881	http://economiclaw.kiev.ua/index.php/economiclaw/article/download/847/824	Ukrainian	null	PRINCIPLES OF ENERGY LEGISLATION	Ekonomìka ta pravo	2,019	cc-by	5,961	ERROR: type should be string, got "https://doi.org/10.15407/econlaw.2019.02.047\nУДК 346.5 М.М. ДУТОВ, канд. юрид. наук, старш. наук. співроб. Інститут економіко-правових досліджень НАН України, м. Київ, Україна\n orcid.org/0000-0002-4661-2833 М.М. ДУТОВ, канд. юрид. наук, старш. наук. співроб. Інститут економіко-правових досліджень НАН України, м. Київ, Україна\n orcid.org/0000-0002-4661-2833 В.В. СИДОРЕНКО, канд. юрид. наук, старш. наук. співроб. Інститут економіко-правових досліджень НАН України, м. Київ, Україна\n orcid.org/0000-0002-2787-9102 ISSN 1681-6277. Економіка та право. № 2 (53), 2019\nродне енергетичне\nміжнародних норм\n© М.М. ДУТОВ, \n В.В. СИДОРЕНКО, 2019 © М.М. ДУТОВ, \n В.В. СИДОРЕНКО, 2019 https://doi.org/10.15407/econlaw.2019.02.047 https://doi.org/10.15407/econlaw.2019.02.047\nУДК 346.5 ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА Проаналізовано основоположні принципи енергетичного законодавства, на яких \nґрунтується сучасне європейське та міжнародне енергетичне право. Шляхом ана-\nлізу положень європейських і міжнародних нормативно-правових документів ви-\nявлено сім основних принципів енергетичного законодавства, що мали вирішальний \nвплив на формування енергетичного права та є підґрунтям для фор мування май-\nбутніх норм енергетичного права Європейського Союзу та України. Ключові слова: енергетичне \nправо, енергетичне зако но-\nдавство, принципи законо-\nдавства, ресурсний сувере-\nнітет, енергетичні послуги, \nенергетична справедливість, \nклімат, природні ресурси. Ключові слова: енергетичне \nправо, енергетичне зако но-\nдавство, принципи законо-\nдавства, ресурсний сувере-\nнітет, енергетичні послуги, \nенергетична справедливість, \nклімат, природні ресурси. Вступ. Наукове визначення «енергетичного законодавства» та його \nзасадничих принципів, було сформовано в науковій літературі біль-\nше 20 років тому [1]. Але за минулий час енергетичне законодавство \nздолало чималий шлях у своєму еволюційному розвитку, зазнавши \nбагато змін і модернізацій, головним чином через приватизацію та \nлібералізацію енергетичних ринків у всьому світі, а також через ін-\nтернаціоналізацію та відповідні зміни на енергетичних ринках. Однак на відміну від таких інститутів, як, наприклад, законодав-\nство про захист навколишнього середовища, основні принципи енер-\nгетичного законодавства на міжнародному та європейському рівні \nдосі ще виділені науковцями. Відповідно, це призводить до відсут-\nності однакової законодавчої техніки під час створення та модерніза-\nції міжнародного, європейського та національного законодавства. Ураховуючи, що згідно з Угодою про асоціацію Україна до кінця \n2025 р. має наблизити своє законодавство до законодавства ЄС та \nімплементувати (транспонувати) у своє законодавство положення \nбільш ніж півсотні директив, регламентів і рішень, ґрунтовне знан-\nня принципів, на яких базується енергетичне законодавство Євро-\nпейського Союзу (що, у свою чергу, безпосередньо пов’язане з між-\nнародним енергетичним законодавством), є ключовим моментом \nдля розроблення та удосконалення концепції енергетичного зако-\nнодавства України, створення відповідного законодавства з ураху-\nванням необхідності наближення до законодавства ЄС. Метою статті є аналіз основоположних принципів енергетичного \nзаконодавства, на яких ґрунтується сучасне європейське та міжна-\nродне енергетичне право шляхом аналізу положень європейських і \nміжнародних нормативно-правових документів, які є підґрунтям для 47 М.М. ДУТОВ, В.В. СИДОРЕНКО формування майбутніх норм енергетичного \nправа ЄС та України. формування майбутніх норм енергетичного \nправа ЄС та України. постколоніальних державах і, за замовчуван-\nням, доходи урядів шляхом регулювання ви-\nробництва. Це поставило серйозні обмеження \nнаціонального суверенітету та доступності цих \nенергетичних ресурсів для відповідних країн. Результати дослідження. На еволюцію зако-\nнодавства у сфері енергетики впливає декілька \nчинників, одним із яких є «енергетична спра-\nведливість». ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА Енергетична справедливість як \nпоняття має міждисциплінарний характер і \nнамагається дати відповіді на питання «що є \nправо?» і «яким воно має бути?». Це створює \nнеобхідність дослідити та визначити, якими \nмають бути керівні принципи енергетичного \nзаконодавства і роль права у розвитку кон-\nцепції енергетичної справедливості. Існує без-\nліч причин для визначення керівних прин ци-\nпів певної галузі знань. Для науковців у сфері \nенергетичного законодавства потреба у визна-\nченні керівних принципів є чіткою та зрозумі-\nлою. Оскільки це допоможе краще осмислити \nпроцес розробки та розвитку правового поля, \nщо формувалося поступово, у відповідь на різ-\nні геополітичні обставини і зростання еколо-\nгічності та обізнаності про витрати, що впли-\nвають на глобальне суспільство. Після Другої Світової війни, яка відзначала-\nся зростанням націоналізму в постколоніаль-\nному світі, багато колоніальних країн почали \nвимагати змін. Так само почало зростати втру-\nчання уряду в діяльність у сфері енергетики \nшляхом регулювання. Ця епоха відзначається \nстворенням Організації країн-експортерів наф-\nти (ОПЕК) та ухваленням кількох важливих \nрезолюцій ООН щодо постійного сувереніте ту \nнад природними ресурсами. Резолюція Гене-\nральної Асамблеї ООН 1962 р. визнала «не-\nвід’ємне право всіх держав вільно розпоряд-\nжатися своїми природними багатствами та \nресурсами відповідно до своїх національних \nінтересів» [2]. Резолюція Генеральної Асамблеї \nООН 3281 (XXIX) 1974 передбачає «Повний \nпостійний суверенітет кожної держави над її \nприродними ресурсами і всією економічною \nдіяльністю. З метою збереження цих ресурсів \nкожна держава має право здійснювати ефек-\nтивний контроль над ними та їх експлуатацію \nзасобами, придатними для її власної ситуації, \nвключаючи право на націоналізацію або пе ре-\nдачу власності своїм громадянам, це право є \nвираженням повного постійного суверенітету \nдержави. Жодна держава не може бути піддана \nекономічному, політичному чи будь-якому ін-\nшому примусу, щоб запобігти вільному і пов-\nному виконанню цього невід’ємного права». Попри досить тривале існування енерге-\nтичного законодавства, юридична наука ще не \nроз робила чіткої та обґрунтованої системи \nпринципів енергетичного законодавства. Для \nусунення цього слід виділити сім взаємо по-\nв’язаних керівних принципів, розроблених на \nпідставі аналізу практики та законодавства, \nнаведених і детально розглянутих нижче. Принципи енергетичного законодавства. Принципи енергетичного законодавства. 1. Принцип національного ресурсного су-\nверенітету. 2. Принцип доступу до сучасних енерге-\nтичних послуг. Крім того, принцип національного сувере-\nнітету був узгоджений як специфічний прин-\nцип у Стокгольмській та Ріо деклараціях 1972 \nта 1992 років відповідно (принцип № 21) [3]. На сьогодні постійний національний сувере-\nнітет над ресурсами визнається міжнародним \nправом, а його реалізація закріплюється у на-\nціональних конституціях. 3. Принцип енергетичної справедливості. 4. Принцип розумного, раціонального і ста-\nлого використання природних ресурсів. 5. ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА Принцип захисту довкілля, здоров’я лю-\nдини та боротьби зі зміною клімату. 6. Енергетична безпека та принцип надійності. 7. Принцип стійкості. 6. Енергетична безпека та принцип надійності. ISSN 1681 6277 Economics and Law №2 (53) 2019\nПринцип доступу до сучасних енергетичних \nпослуг. Лише декілька десятиліть тому, зі знач-\nним запізненням, було визнано, що для забез-\nпечення сталого розвитку в суспільствах країн, \nщо розвиваються, звичайне населення має от-\nримати доступ до сучасної системи енергопос-\nтачання. Уперше це важливе питання було по-\nрушено у 1986 р., у Доповіді Міжнародної комі-\nсії з довкілля та розвитку (Доповідь Брундтланд) \n[4]. Широкого розголосу це питання набуло Принцип національного ресурсного суверені-\nтету. Принцип постійного суверенітету над \nприродними ресурсами тісно пов’язаний із \nенергетичними ресурсами. Обговорення суве-\nренітету над природними ресурсами, зокрема \nнафтою, виникло після закінчення колоніаль-\nного періоду. До цього міжнародні нафтові \nкомпанії контролювали розвідку і видобуток \nнафтових ресурсів у багатьох колоніальних і ISSN 1681-6277. Economics and Law. № 2 (53), 2019 48 Принципи енергетичного законодавства 2000 р. завдяки спільній доповіді Програми \nрозвитку ООН (ПРООН), Департаменту з еко-\nномічних та соціальних питань ООН (ДЕСП \nООН), Світової енергетичної ради, під назвою \n«Оцінка стану світової енергетики: енергопос-\nтачання та виклик стабільності». Доповідь за-\nкликала розпочати дії глобального масштабу \nзадля забезпечення загального доступу до сис-\nтеми енергопостачання та обґрунтувала існу-\nвання зв’язку між енергетикою та бідністю. Текст Доповіді 2000 р. був оновлений та до-\nповнений Доповіддю ПРООН 2004 р.[5]. Важливе значення цієї теми підтверджує і \nтой факт, що згідно з останньою міжнародною \nдоповіддю з цієї тематики — Доповіддю про \nреалізацію ЦСР 2016 р. — 40 % населення сві-\nту, яке проживає в країнах, що розвиваються, \nвсе ще використовує горючі корисні копа-\nлини, що забруднюють довкілля та завдають \nшкоди здоров’ю, для приготування їжі та га-\nзозабезпечення. Більшість цих людей живуть \nв Африці та Південній Азії. На сьогодні по-\nнад 65 % населення Субсахарської Африки не \nмають доступу до електроенергії [8]. Водночас у правовій площині світ продов-\nжує жваве обговорення чотирьох тем, по в’я-\nзаних між собою: «Енергопостачання» є необхіднішим за на-\nявність «традиційних джерел енергії», адже \nсуспільство потребує не існування самої енер-\nгетики (вона не має справжньої цінності), а \nскоріше зміни того стилю життя, що може за-\nбезпечити сучасне енергопостачання. Воно \nвиникає за умов поєднання експлуатації пер-\nвинних джерел енергії, технологій, пов’язаних \nіз енергетичною сферою, робочої сили, сиро-\nвини та інфраструктури. ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА № 2 (53), 2019 М.М. ДУТОВ, В.В. СИДОРЕНКО турна приналежність, а також забезпеченні то-\nлерантності та поваги до певних груп і місць. Сучасні прояви цієї проблеми полягають у не-\nповазі ділових кіл, зацікавлених в альтерна-\nтивній енергетиці, до місцевих фермерських \nгруп, що виступають проти вітрових електро-\nгенераторів, і у зневазі, ігноруванні та прини-\nженні ролі і значення противників атомних \nелектростанцій. Справедливість визнання має \nна меті забезпечити однакові умови участі усіх \nзацікавлених сторін в ухваленні рішень щодо \nенергетичних ініціатив. країн), і національних питань (таких як забез-\nпечення доступу бідних верств населення до \nсистеми енергопостачання або оголошення \nнезаконною насильницької ліквідації домівок \nі селищ з метою реалізації нових широко-\nмасштабних гідроенергетичних проектів). країн), і національних питань (таких як забез-\nпечення доступу бідних верств населення до \nсистеми енергопостачання або оголошення \nнезаконною насильницької ліквідації домівок \nі селищ з метою реалізації нових широко-\nмасштабних гідроенергетичних проектів). В основі зародження і діяльності руху енер-\nгетичної справедливості лежить таке ж філо-\nсофське підґрунтя, як і в більшості питань \nекологічної, атмосферної та кліматичної спра-\nведливості. Література з цієї тематики тради-\nційно виокремлює три ключові теми у сфері \nенергетичної справедливості: справедливість \nрозподілу, справедливість процедури та спра-\nведливість визнання [10]. Принцип розумного, раціонального і сталого \nвикористання природних ресурсів. Декларація \nРіо щодо навколишнього середовища та роз-\nвитку 1992 р. [12] містить такі поняття як «збе-\nреження», «стале управління», «найкраще, \nефективне та раціональне використання» або \n«зменшення і викоренення нераціональних \nсхем виробництва та споживання», викладені \nу Принципі 8. Проте ще до Стокгольмської \nдекларації 1972 р. [13] було включено принцип \n(№ 5), за яким невідновлювані природні ре-\nсурси мають використовуватися таким чином, \nщоб запобігти їх виснаженню в майбутньому і \nзабезпечити доступ усієї світової спільноти до \nздобутків від такого використання. Справедливість розподілу має на меті пере-\nконатися, що не лише знедолене та бідне на-\nселення страждає найбільше від реалізації \nенергетичних проектів на місцях, і що уряди \nдержав та судові органи влади всесторонньо \nрозглядають скарги та заперечення стосовно \nреалізації нових енергетичних проектів і вино-\nсять рішення, не допускаючи надмірного тиску \nз боку забудовників. Раніше заперечення спра-\nведливості розподілу траплялося у випадках, \nколи уряд ухвалював рішення щодо розмі-\nщення вітрових електрогенераторів, затверд-\nження проектів вугільних чи атомних електро-\nстанцій. Це є доказом того, що абсолютно усі \nверстви суспільства мають отримувати користь \nі нести однакові збитки від таких рішень. ISSN 1681-6277. Economics and Law. № 2 (53), 2019\nПринцип сталого використання також за-\nкріплено в меті Рамкової конвенції ООН про \nзміну клімату (РКЗК ООН) у ст. ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА Послуги традиційних \nджерел енергії застосовуються лише для добу-\nвання вогню, шляхом спалення біомаси у фор-\nмі деревини, або призначені тільки для приго-\nтування їжі, обігріву або обслуговування тва-\nрин, що перевозять вантажі. На противагу їм, \nсучасне енергопостачання є необхідним для \nзабезпечення освітлення, охолодження, замо-\nрожування, екологічно чистого приготування \nстрав та обслуговування транспорту. • чи існує право доступу до енергопоста-\nчання в праві прав людини? • Якщо такого права не існує, то чи є інші \nстратегії в міжнародному праві, покликані за-\nбезпечити загальний доступ до енергопоста-\nчання? • Яку роль відіграє національне право дер-\nжави у просуванні та / або забезпеченні за-\nгального доступу до енергопостачання? • Чи може судова система вплинути на ви-\nрішення цього питання? Принцип енергетичної справедливості.Енер-\nгетична справедливість є моральним, філо-\nсофським та етнічним рухом, який виник у \nкінці ХХ — на початку ХХІ ст. і продовжує з \nкожним днем набувати більшої популярності. У публікаціях існують, наприклад, такі визна-\nчення енергетичної справедливості: це ство-\nрення системи глобального енергопостачан-\nня, що справедливо розподілятиме переваги \nта збитки енергопостачання, тобто такої сис-\nтеми, що сприятиме більшій участі держав і \nнеупередженості в ухваленні рішень стосов-\nно енергопостачання [9]. Порівняння Цілей розвитку тисячоліття Ге-\nнеральної Асамблеї ООН (ЦРТ), проголоше-\nних Тисячолітньою Декларацією 2000 р. [6], та \nЦілей сталого розвитку (ЦСР), також прого-\nлошених Генеральною Асамблеєю ООН у до-\nповіді «Перетворення нашого світу: Порядок \nденний у сфері сталого розвитку до 2030 року» \n[7], наочно демонструє, що світова спільнота з \nкожним днем усе більше визнає важливість за-\nгального доступу до системи енергопостачан-\nня. У першому випадку жодна з цілей не вио-\nкремлює важливість енергетичної сфери. На \nпротивагу цьому, ціль № 7 із переліку ЦСР \nповністю присвячена енергетичній тематиці: \n«За безпечити доступ всіх людей до прийнят-\nних за ціною, надійних, сталих і сучасних дже-\nрел енергії». Кожна ціль із переліку ЦСР міс-\nтить ряд завдань. Завдання 7.1. передбачає: \n«До 2030 року забезпечити загальний доступ \nдо недорогого, надійного та сучасного енерго-\nпостачання». Ця тема зі сфери соціальної справедливості \nвиходить далеко за межі впливу традиційного \nуряду та промислових ризиків, пов’язаних із \nенергобезпекою, економічним розвитком та \nтехнологіями, і має на меті розгляд питання \nморальності в ухваленні рішень. Вона стосу-\nється і міжнародних питань (таких як право \nлюдей із країн, що розвиваються, уникнути \n«капкану бідності», отримавши загальний до-\nступ до енергопостачання, та запобігти шкоді \nдовкіллю у результаті діяльності транснаціо-\nнальних енергетичних корпорацій або зне-\nшкодження ядерних відходів із розвинутих 49 ISSN 1681-6277. Економіка та право. ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА 2: «дозволити \nекосистемам здійснити природну адаптацію до \nзмін клімату, гарантувати безпеку вироб ниц-\nтва продовольства і сприяти забезпеченню по-\nдальшого економічного розвитку на сталій \nоснові», у ст. 3.4. «сприяти сталому розвитку… \nзахищати кліматичну систему від антропоген-\nних змін» і більш чітко в ст. 4 (г), де викладені \nзобов’язання Сторін «заохочувати збалансо-\nване управління, сприяти і співпрацювати за-\nдля збереження та покращення якості погли-\nначів та накопичувачів,… включаючи біома су, \nліси, океани та інші наземні, прибережні і \nморські екосистеми». Стаття 2 Кіотського \nпротоколу (1997) також передбачає, що для \nзменшення шкідливого впливу викидів в ат-\nмосферу система раціонального використан-\nня енергоресурсів прагне забезпечити необ-\nхідне просування принципів енергоефек тив-\nності, енергозбереження та використання \nвід новлюваних джерел енергії. Подібним чи-\nном Паризька кліматична угода (2015) ви знає \nпотребу просування ідей загального доступу Справедливість процедури передбачає од-\nнакову здатність усіх соціальних груп брати \nучасть у процесах ухвалення рішень щодо пи-\nтань, пов’язаних із певними енергетичними \nініціативами. Подібна практика вже існує у \nміжнародному екологічному праві та закріп-\nлена Орхуською конвенцією 1998 р. [11], але її \nзастосування подеколи відбувається з певни-\nми порушеннями. Це означає, що рішення \nможуть бути ухвалені без повного надання ін-\nформації постраждалим сторонам щодо від-\nповідних питань, а упередженість та політич-\nний тиск із боку впливових ділових кіл можуть \nсправляти вирішальний вплив під час розгля-\nду запропонованих енергетичних ініціатив. Нерівноцінний розподіл пільг на різні види \nенергоносіїв може також призвести до непра-\nвомірних рішень. Справедливість визнання полягає у розгляді \nвідмінних думок і точок зору суспільства на \nоснові таких критеріїв як стать, расова та куль- ISSN 1681-6277. Economics and Law. № 2 (53), 2019 50 Принципи енергетичного законодавства до системи сталої енергетики та використан-\nня альтернативних джерел енергії в країнах, \nщо розвиваються. ти нераціонального використання природних \nресурсів є чутливі питання життєвих циклів та \nподальшої долі планети. У 2015 р. Генеральна Асамблея ООН додала \nдо Цілей сталого розвитку 17-ту ціль, чим \nуперше з 2000 р. розширила перелік Цілей роз-\nвитку тисячоліття і зосередила увагу на пи-\nтаннях енергетики та безпосередньому вико-\nристанні природних ресурсів. Стале використання природних ресурсів ви-\nзнається вирішальним та життєво необхідним \nдля майбутнього економічного розвитку і вже \nперетворилося на головну тему в одній із семи \nпровідних ініціатив стратегії «Європа-2020» \n[15]. Ця Стратегія спрямована на підтримку \nзміни на користь ресурсоощадної та низько-\nвуглецевої економіки та зменшення залеж-\nності економічного розвитку від використан-\nня ресурсів та енергії. ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА Стратегія наголошує, \nщо стале використання ресурсів здатне запо-\nбігти погіршенню стану навколишнього се-\nредовища, втраті біорізноманіття та нераціо-\nнальному використанню ресурсів. Мета, до якої повинне прагнути кожне сус-\nпільство та громада, полягає у доступній і чис-\nтій енергетиці (№ 7). Інші додаткові цілі, такі \nяк сталий розвиток міст та громад (№ 11), \nвідповідальне споживання та виробництво \n(№ 13) також прямо стосуються сталого вико-\nристання природних багатств. На рівні ЄС стале використання природ-\nних ресурсів вітають та упроваджують. Стат-\nтя 11 Договору про функціонування ЄС на го-\nло шує: «Вимоги щодо охорони довкілля по-\nвинні бути невід’ємною частиною визначення \nта реалізації політик та дій Союзу, зокрема з \nогляду на сприяння сталому розвитку». А еко-\nлогічна політика ЄС має сприяти прагненню \nзберігати, охороняти та покращувати зважене \nта раціональне використання природних ре-\nсурсів і подолати зміну клімату. Мета енерге-\nтичної політики ЄС, проголошена у ст. 194, \nполягає у «сприянні ефективному викорис-\nтанню енергії та енергозбереженню, а також \nрозвитку нових та відновлюваних форм енер-\nгії». Протягом багатьох років за допомогою \nнизки директив вдалося перетворити ці дек-\nларативні політичні цілі на реальні зобов’я-\nзання усіх держав сприяти ефективному вико-\nристанню сміття, водних ресурсів, відновлю-\nваних джерел енергії та енергоефективності. Деякі держави, як і ЄС, також ухвалили кон-\nституційні положення чи акти, що вимагають \nсталого використання природного середови-\nща та його ресурсів. Надалі усі зазначені міжнародні угоди та ре-\nзолюції визнають існування допустимих меж \nвикористання земельних, прісно вод них, мор-\nських та природних ресурсів, як що світове сус-\nпільство прагне уникнути непоправної шкоди. Твердження про те, що міжнародне право на-\nкладає на держави зви чаєвий обов’язок стало-\nго використання природніх ре сурсів, може зда-\nтися досить сумнівним, проте очевидним є те, \nщо воно проголошено головною метою усього \nсвітового співтовариства, важливість якого ви-\nзнають усе більше країн через право договорів. На болюче питання про розподіл та спільне \nвикористання деяких ресурсів було знайдено \nвідповідь: уведено практику заохочення існу-\nвання загального обо в’яз ку гарантувати збе-\nреження та стале ви ко рис тання відкритих мо-\nрів, глибоководної частини морів, Антаркти-\nди та Місяця [16], і визнано, що всі ці ресурси \nцілковито належать усьому людству. На болюче питання про розподіл та спільне \nвикористання деяких ресурсів було знайдено \nвідповідь: уведено практику заохочення існу-\nвання загального обо в’яз ку гарантувати збе-\nреження та стале ви ко рис тання відкритих мо-\nрів, глибоководної частини морів, Антаркти-\nди та Місяця [16], і визнано, що всі ці ресурси \nцілковито належать усьому людству. ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА Органи контро-\nлю більше не можуть спиратися на їхню здат-\nність упроваджувати нормативні положення \nза фактом розгляду і вирішення конкретної \nпоточної проблеми, наприклад витоку нафти \nчи аварії на атомній електростанції. Натомість \nнормативні положення, орієнтовані на май-\nбутнє та на попередження можливих загроз, є \nнеобхідними для зменшення викидів вугле-\nкислого газу та спроб зупинити зміну клімату. Оскільки існує нагальна потреба, гострі пи-\nтання у відносинах між енергетикою та нав-\nколишнім середовищем мають бути ретельно \nвивчені та розглянуті, і що найважливіше — \nвирішені. Історично так склалося, що стриж-\nнем концепції традиційної енергетики є пи-\nтання економічного розвитку та зростання. Дешева, загальнодоступна та надійна енерге-\nтика була і залишається основною складовою \nчастиною будь-якої країни з розвинутою еко-\nномікою. Окрім доступної енергії на сьогодні \nенергетика, національна безпека та захист \nдов кілля є основними визначальними факто-\nрами будь-якої сучасної енергетичної політи-\nки. Усі форми виробництва енергії чинять \nшкідливий вплив: чи на навколишнє середо-\nвище, чи на стан здоров’я людини, чи на енер-\nгетичну безпеку та економіку. Ключовим мо-\nментом є те, що потрібно постійно аналізувати \nвплив повного «життєвого циклу» кожного \nджерела енергії. Проте у кожного правила \nможе бути виняток: горючі енергоносії мають \nочевидніші, численніші, тяжчі та довготри-\nваліші ризики за більшість альтернативних \nджерел енергії. Більше того, перехід із вуг ле-\nцевої економіки на низьковуглецеву є не ли-\nше питанням захисту навколишнього середо-\nвища, а й потребою вижити в майбутньому. Органи контролю енергетичної сфери по-\nвинні зрозуміти, що майбутнє енергетики та \nдовкілля передбачає наявність складних проб-\nлем, що мають міждисциплінарний харак тер, \nпов’язаний також із поглядами і досвідом різ-\nних поколінь та юрисдикціями, вони сповнені \nнаукової, технологічної, економічної і соці-\nальної невизначеності. До того ж зміна кліма-\nту не є черговою буденною проблемою, а ста-\nвить власні завдання для вирішення, такі як: \nбрак часу для віднайдення рішення; неспро-\nможність влади жодної держави вирішити цю \nпроблему самостійно; відкладання вирішення \nцієї проблеми зараз означатиме дедалі більшу \nвитрату ресурсів на це в майбутньому. Тим не \nменш, майбутнє потребує особливої уваги сьо-\nгодні, тому що зволікання вартуватиме силу \nгрошей і ресурсів. Центральним для розгляду \nпроблеми зміни клімату є питання визнання \nвзаємозв’язку між енергетикою та навколиш-\nнім середовищем і руйнівного впливу людини \nяк наслідку ігнорування цього зв’язку. З нерозривного зв’язку енергетики та нав-\nколишнього середовища випливає декілька \nважливих висновків. Перший полягає у тому, \nщо розробити політику використання чистої \nенергетики цілком можливо. ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА Шоста Програма дій з охорони навколиш-\nнього середовища [14] демонструє конкретні \nприклади реалізації політики ЄС, де стале ви-\nкористання та управління ресурсами вважа-\nються однією з пріоритетних галузей. Вони \nпокликані підготувати «головну стратегію ста-\nлого використання та управління ресурсами…» \nі сприяти вживанню практичних заходів задля \nзменшення негативного впливу експлуатації \nприродних ресурсів на довкілля. Принцип захисту довкілля, здоров’я людини \nта боротьби зі зміною клімату. З точки зору \nфізики, енергетика та довкілля є складовими \nприродного паливного циклу. Екологічні на-\nслідки стають відчутними вже на початково-\nму етапі розвідування та видобування, потім \nна етапі обробки та транспортування, і наре-\nшті на етапах розповсюдження, споживання \nприродних ресурсів, що використовуються \nдля виробництва енергії, та на стадії утилізації \nїхніх залишкових продуктів. Таким чином, за- ISSN 1681-6277. Економіка та право. № 2 (53), 2019\nПриродні ресурси визначають як сукупність \nусіх сировинних ресурсів, включаючи енергію \nбіомас, гідро-, вітро-, геотермальну, сонячну \nенергетику та енергію припливів та відпливів. Особливо важливими темами у боротьбі про- 51 М.М. ДУТОВ, В.В. СИДОРЕНКО конодавство та політика у сфері енергетики та \nекологічне законодавство і політика не можуть \nвважатися окремими царинами регулювання. Особливо важливим є те, що феномен зміни \nклімату ускладнив проблеми навколишнього \nсередовища, пов’язані з енергетичною сфе-\nрою — сектор енергетики залишається го-\nловним забруднювачем довкілля вуглекислим \nгазом. До цих екологічних викликів належать \nпогіршення природних умов середовища та \nзростання кількості ризикових ситуацій та ка-\nтастроф, що загрожують населенню планети. комплексу. Другий і важливіший висновок \nполягає у тому, що сучасна та майбутня енер-\nгетична політики піддаються суттєвому впли-\nву зміни клімату та нерозривно пов’язані з \nнею. У той же час надзвичайно просто стверд-\nжувати, що використання чистої енергії в \nмайбутньому вже є найбільшою цінністю для \nсвітової спільноти; майбутнє з чистою енер-\nгетикою часто об’єднують та пов’язують із ви-\nрішенням питань змін клімату. Особливо важливим є те, що феномен зміни \nклімату ускладнив проблеми навколишнього \nсередовища, пов’язані з енергетичною сфе-\nрою — сектор енергетики залишається го-\nловним забруднювачем довкілля вуглекислим \nгазом. До цих екологічних викликів належать \nпогіршення природних умов середовища та \nзростання кількості ризикових ситуацій та ка-\nтастроф, що загрожують населенню планети. Зміна клімату має такі структурні характе-\nристики, за якими цю проблему було визнано \nнадзвичайно небезпечним викликом, що на-\nразі потребує нової форми регулювання; такої \nформи, що поєднуватиме законодавство та \nполітику у сфері енергетики з екологічними \nзаконодавством та політикою. ISSN 1681-6277. Economics and Law. № 2 (53), 2019 ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА Політика вико-\nристання традиційної енергетики, що спира-\nлась на експлуатацію дешевих і загальнодос-\nтупних горючих копалин, тепер є несумісною \nз вимогами, що диктує нова система. Це озна-\nчає, що відновлювані, низьковуглецеві ре сур-\nси та енергоефективність набувають дедалі \nбільшого значення у структурі енергетичного Енергетична безпека та принцип надійності. Енергетична безпека є стрижнем будь-якої \nенергетичної політики і закріплена у низці за-\nконів та нормативних актів певної держави ISSN 1681-6277. Economics and Law. № 2 (53), 2019 52 Принципи енергетичного законодавства Значна відмінність між цими підсекторами \nполягає у тому, що кожен із них має особливі \nфізичні па раметри. Наприклад, паливо для \nтранспорту легко впізнати та зберігати. Елект-\nроенергію отримують різними способами, а її \nзберіган ня в найкращому випадку можливе \nпротягом нетривалого часу. Важливішим є те, \nщо система постачання електроенергії має бу-\nти весь час стабільною та доступною для всіх. Проте, незалежно від цих відмінностей, енер-\nгетика для двох підсекторів повинна бути на-\nдійною та доступною. Крім того, обидві сис-\nтеми повинні бути стійкими. Ураховуючи той \nфакт, що паливо для транспорту легко збері-\nгати та перевозити територією країни, тран-\nспортна система є відносно стійкою, на відмі-\nну від системи електропостачання. Фактично, \nна сьо годні стійкість є головним викликом \nпідсектору електропостачання, адже неспри-\nятливі та складні погодні умови зупиняють \nпостачання електроенергії, що призводить до \nзначних економічних витрат. Таким чином, \nмайбутнє енергетичної сфери залежить від за-\nбезпечення її стійкості. щодо енергетичної сфери. Її значення по в’я-\nзане із загальною важливістю енергетики для \nсуспільства. Концепція стосується двох зовсім \nрізних, проте пов’язаних між собою, цілей \nенергетичної політики. Вона звертає увагу на \nнадійність пропозиції, під якою зазвичай ро-\nзуміють довготривалу доступність енергії за \nприйнятною ціною. Більшість сучасних енер-\nгетичних політик держав додають до цього ви-\nзначення витрати на соціальне забезпечення \nта захист довкілля. Концепція стосується і на-\nдійності попиту, під яким розуміють довготри-\nвалу потребу в енергетичних ресурсах, що ви-\nробляє певна країна. Традиційно це стосується \nкраїн із великими запасами вуглеводневих ре-\nсурсів, але до цієї групи також належать і краї-\nни, що виробляють енергію за допомогою аль-\nтернативних джерел, наприклад, гідроенергія. Надійність пропозиції є певним чином уні-\nверсальною метою енергетичної політики, але \nїї практичне закріплення та втілення на рівні \nзаконодавства та політики певної держави без-\nпосередньо залежить від національних особ-\nливостей. Для країни чи регіону, що імпортує \nенергетичні ресурси, надійність пропозиції \nозначає, передовсім, надійність імпорту. ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА шкоди, запобігання руйнуванню, уникнення \nабо полегшення наслідків несприятливої си-\nтуації; розроблення варіантів для продовжен-\nня функціонування системи електропостачан-\nня під час настання несприятливої події; за-\nбезпечення швидкого повернення до звичного \nстану роботи після дестабілізації системи елек-\nтропостачання. Іншими словами, заходи щодо \nзабезпечення стійкості стосуються як можли-\nвого впливу на надійність такої системи, так і \nвпливу на її здатність швидше відновлювати \nфункціонування. шкоди, запобігання руйнуванню, уникнення \nабо полегшення наслідків несприятливої си-\nтуації; розроблення варіантів для продовжен-\nня функціонування системи електропостачан-\nня під час настання несприятливої події; за-\nбезпечення швидкого повернення до звичного \nстану роботи після дестабілізації системи елек-\nтропостачання. Іншими словами, заходи щодо \nзабезпечення стійкості стосуються як можли-\nвого впливу на надійність такої системи, так і \nвпливу на її здатність швидше відновлювати \nфункціонування. Для України, з огляду на початковий стан \nфор мування енергетичного законодавства, роз-\nроб ка системи принципів енергетичного за-\nконодавства та їх використання науков цями \nта практиками мають велике значення. Також \nце дасть змогу глибше зрозуміти цілі, завдання \nта принципи законодавства ЄС у сфері енер-\nгетики, що також зараз формується, і надасть \nможливість ефективніше провести гармоніза-\nцію за конодавства України з правовою сис-\nтемою ЄС з урахуванням недавно ух валених \nзмін до Конституції України, що закріплюють \nстратегічний курс України на членство в ЄС та \nНАТО [21]. Висновки. Виконаний аналіз принципів енер-\nгетичного законодавства дає підстави для вис-\nновку, що його гармонійний розвиток на між-\nнародному, європейському та національному \nрівні неможливий без ґрунтування такого зако-\nнодавства на чітко визначених, науково дове-\nдених і перевірених практикою принципів, що \nвстановлюють його системотвірні положення. Практика створення нормативно-правових \nактів у сфері енергетики, а також правозасто- СПИСОК ЛІТЕРАТУРИ / REFERENCES 1. Adrian J. Bradbrook. Energy Law as an Academic Discipline. Journal of Energy & Natural Resources Law. 1996. Vol. 14. Iss. 2. P. 193-217. https://doi.org/10.1080/02646811.1996.11433062. 1. Adrian J. Bradbrook. Energy Law as an Academic Discipline. Journal of Energy & Natural Resources Law. 1996. Vol. 14. Iss. 2. P. 193-217. https://doi.org/10.1080/02646811.1996.11433062. p //\ng/\n/\n 2. General Assembly resolution 1803 (XVII) of 14 December 1962, \"Permanent sovereignty over natural resources\". URL: https://www.ohchr.org/Documents/ProfessionalInterest/resources.pdf [last accessed 10.02.2019]. p //\ng/\n/\n 2. General Assembly resolution 1803 (XVII) of 14 December 1962, \"Permanent sovereignty over natural resources\". URL: https://www.ohchr.org/Documents/ProfessionalInterest/resources.pdf [last accessed 10.02.2019]. 3. Declaration of the UN Conference on the Human Environment 1972 and Declaration of the UN Conference on the \nEnvironment and Development 1992. URL: http://legal.un.org/avl/pdf/ha/dunche/dunche_e.pdf [last accessed \n10.02.2019]. 3. Declaration of the UN Conference on the Human Environment 1972 and Declaration of the UN Conference on the \nEnvironment and Development 1992. URL: http://legal.un.org/avl/pdf/ha/dunche/dunche_e.pdf [last accessed \n10.02.2019]. ]\n 4. Report of the World Commission on Environment and Development \"Our Common Future\", UN Doc A/42/427 \n(1987). URL: https://digitallibrary.un.org/record/139811/files/A_42_427-EN.pdf [last accessed 10.02.2019]. ]\n 4. Report of the World Commission on Environment and Development \"Our Common Future\", UN Doc A/42/427 \n(1987). URL: https://digitallibrary.un.org/record/139811/files/A_42_427-EN.pdf [last accessed 10.02.2019]. //\n/\n/\n/\n/\n 5. World Energy Assessment Overview: 2004 Update / United Nations Development Programme. 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Transforming our world: the 2030 Agenda for Sustainable Development. Resolution adopted by the General Assem-\nbly on 25 September 2015. URL: http://www.un.org/en/development/desa/population/migration/generalassembly/\ndocs/globalcompact/A_RES_70_1_E.pdf [last accessed 15.02.2019]. 7. Transforming our world: the 2030 Agenda for Sustainable Development. Resolution adopted by the General Assem-\nbly on 25 September 2015. URL: http://www.un.org/en/development/desa/population/migration/generalassembly/\ndocs/globalcompact/A_RES_70_1_E.pdf [last accessed 15.02.2019]. /g\np\n/ _\n_\n_ _\np\n[\n]\n 8. Goal 7: Ensure access to affordable, reliable, sustainable and modern energy for all. URL: http://unstats.un.org/\nsdgs/report/2016/goal-07/ [last accessed 15.02.2019]. g /\np\n/\n/g\n/ [\n]\n 9. Benjamin K. Sovacool, Raphael J. Heffron, Darren McCauley & Andreas Goldthau. ПРИНЦИПИ ЕНЕРГЕТИЧНОГО ЗАКОНОДАВСТВА Дер-\nжава чи регіон прагнуть досягти цього стану \nшляхом диверсифікації імпорту — за рахунок \nурізноманітнення джерел постачання, тран-\nспортних шляхів, необхідного і доцільного \nфінансування проектів імпортної інфраструк-\nтури, зобов’язань щодо зберігання ресурсів \nтощо. Це також призводить до виникнення за-\nцікавленості в альтернативних джерелах енер-\nгії, включаючи як нетрадиційні джерела нафти \nта газу, так і джерела відновлюваної енергії. Для країни, що виробляє енергетичні ресурси, \nнадійність пропозиції може мати різні прояви. У випадках, коли держава має змогу (хоча б \nчастково) самостійно забезпечити себе енер-\nгетичними ресурсами, надійність політики \nпропозиції не завжди базується на потребі \nмати стабільні джерела імпортних ресурсів, \nпроте спрямована на запобігання надмірному \nекспорту. Держава може досягти цього шляхом \nупровадження політики мінімізації експорту \nпаливно-енергетичних ресурсів [18, c. 527]. Національна академія наук США визначила \nстійкість як «здатність реагувати за планом, \nзберігати, відновлюватися від та успішно адап-\nтуватися до несприятливих подій» [19, c. 114]. Надзвичайно сильний шторм Сенді, ураган \nКатріна, навіть горіння гілки дерева були в \nзмозі зупинити постачання електроенергії, \nвиявивши таким чином уразливість системи \nелектропостачання. Наприклад, збитки від \nвідключення електроенергії на Східному узбе-\nрежжі США в 2003 р. обійшлися державі в \n4—10 млрд дол. [20]. Складні погодні умови \nвиникають дедалі частіше, що зазвичай по-\nв’язують із глобальним потеплінням. Це під-\nвищує важливість розробки такої політики в \nсфері енергетики, що здатна розуміти та ре а-\nгувати на виклики зміни клімату. Окрім теми зміни клімату, питання стійкос-\nті не оминули і протидію загрозам у сфері кі-\nбербезпеки. Наприклад, Міністерство енерге-\nтики США визнає, що система електропоста-\nчання перебуває в очевидній небезпеці через \nкібератаки, які фактично стаються дедалі час-\nтішими та вигадливішими [20]. Принцип стійкості. Енергетичний сектор \nекономіки можна умовно поділити на дві час-\nтини: транспортування ресурсів і вироб ництво \nелектроенергії, хоча горючі корисні копалини \nшироко використовують в обох підсекторах. Залежно від причини, якою можуть бути по-\nгодні катаклізми чи кібератака, працівники, \nщо обслуговують систему електропостачання, \nмають забезпечити її стійкість для мінімізації 53 ISSN 1681-6277. Економіка та право. № 2 (53), 2019 М.М. ДУТОВ, В.В. СИДОРЕНКО совна практика демонструють, що деякі прин-\nципи енергетичного законодавства вже роз-\nроблені та досить широко використовуються. Однак для формування ефективнішого зако-\nнодавства потрібно здійснити класифікацію \nтаких принципів на науковій основі з одно-\nчасним обґрунтуванням необхідності їх існу-\nвання. Саме цьому присвячено дану статтю. ISSN 1681-6277. Economics and Law. № 2 (53), 2019 СПИСОК ЛІТЕРАТУРИ / REFERENCES Energy decisions reframed as \njustice and ethical concerns. 2016. URL: http://sro.sussex.ac.uk/61327/ [last accessed 15.02.2019]. //\n/\n/\n10. McCauley, Darren & Heffron, Raphael & Stephan, Hannes & Jenkins, Kirsten. Advancing Energy Justice: The tri-\numvirate of tenets. International Energy Law Review. 2013. Vol. 32. P. 107-110. g\n11. Convention on Access to Information, Public Participation in Decision-Making and Access to Justice in Environ-\nmental Matters (Aarhus, Denmark, 25 June 1998). URL: https://treaties.un.org/doc/Treaties/1998/06/19980625%20\n08-35%20AM/Ch_XXVII_13p.pdf [last accessed 15.02.2019]. /\n_\n_\np p\n[\n]\n12. Rio Declaration on Environment and Development, adopted 14 June 1992. URL: http://www.unesco.org/\neducation/pdf/RIO_E.PDF [last accessed 15.02.2019]. 13. Declaration of the UN Conference on the Human Environment adopted in Stockholm, 16 June 1972. URL: http://\nwww.un.org/ga/search/view_doc.asp?symbol=A/CONF.48/14/REV.1 [last accessed 15.02.2019]. g/g /\n/\n_\np\ny\n/\n/\n/\n[\n]\n14. Decision No 1600/2002/EC of the European Parliament and of the Council of 22 July 2002 laying down the Sixth \nCommunity Environment Action Programme. Official Journal. 2002. L 242. P. 0001-0015. 14. Decision No 1600/2002/EC of the European Parliament and of the Council of 22 July \nCommunity Environment Action Programme. Official Journal. 2002. L 242. P. 0001-001 54 Принципи енергетичного законодавства 15. EUROPE 2020. A strategy for smart, sustainable and inclusive growth: Communication from the Commission. URL: https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:52010DC2020&from=EN [last ac-\ncessed 15.02.2019]. ]\n16. Benjamin J. Richardson. Sustainable Development in International and National Law. Review of European, Com-\nparative & International Environmental Law. 2009. Vol.18. Iss. 1. P. 105-107. https://doi.org/10.1111/j.1467-9388. 2009.625_2.x. _\n17. Eduardo Pereira, Kim Talus. National Petroleum Supply Reservations: Background and Comparison. Journal of \nWorld Energy Law and Business. 2014. Vol. 7. Iss. 6. P. 527—537. https://doi.org/10.1093/jwelb/jwu023. 18. National Research Council. 2012. Disaster Resilience: A National Imperative. Washington, DC: The National Acad-\nemies Press. https://doi.org/10.17226/13457. 19. US-Canada Power System Outage Task Force, Final Report on the August 14, 2003 Blackout in the USA and Cana-\nda: Causes and Recommendations. 2014. URL: https://www.energy.gov/sites/prod/files/oeprod/Documentsand-\nMedia/BlackoutFinal-Web.pdf [last accessed 15.02.2019]. 20. US Department of Energy, Transforming the Nation’s Electricity System: The Second Installment of the Quad-\nrennial Energy Review. 2017. URL: https://www.energy.gov/sites/prod/files/2017/02/f34/Quadrennial%20Energy%\n20Review--Second%20Installment%20%28Full%20Report%29.pdf [last accessed 15.02.2019]. 21. On Amendments to the Constitution of Ukraine (Regarding the Strategic Course of the State on Acquiring Full-\nfledged Membership of Ukraine in the European Union and the North Atlantic Treaty Organization): Law of Uk-\nraine dated 02.07.2019 No. 2680-VIII. Holos Ukrainy. 2019. No. М.М. Dutov М.М. Dutov\nInstitute of Economic and Legal Researches of the NAS of Ukraine, Kyiv, Ukraine\norcid.org/0000-0002-4661-2833 Institute of Economic and Legal Researches of the NAS of Ukraine, Kyiv, Ukraine\norcid.org/0000-0002-2787-9102 СПИСОК ЛІТЕРАТУРИ / REFERENCES 34 [Про внесення змін до Конституції України \n(щодо стратегічного курсу держави на набуття повноправного членства України в Європейському Союзі та \nв Організації Північноатлантичного договору): Закон України від 07.02.2019 № 2680-VIII. Голос України. 2019. № 34] [in Ukrainian]. Надійшла / Received17.02.2019 М.М. Дутов, В.В. Сидоренко ISSN 1681-6277. Економіка та право. № 2 (53), 2019 Keywords: Energy Law, energy legislation, principles of legislation, resource sovereignty, energy services, energy justice, \nclimate, natural resources. ПРИНЦИПЫ ЭНЕРГЕТИЧЕСКОГО ЗАКОНОДАТЕЛЬСТВА Проанализированы основные принципы энергетического законодательства, на которых основывается совре-\nменное европейское и международное энергетическое право. Путем анализа положений европейских и между-\nнародных нормативно-правовых документов выявлено семь основных принципов энергетического законода-\nтельства, которые имели решающее влияние на формирование энергетического права, а также составляют \nоснову формирования будущих норм энергетического права Европейского Союза и Украины. Ключевые слова: энергетическое право, энергетическое законодательство, принципы законодательства, ре-\nсурсный суверенитет, энергетические услуги, энергетическая справедливость, климат, природные ресурсы. PRINCIPLES OF ENERGY LEGISLATION It’s been over 20 years since the scholars could coin such term as Energy Law. The purpose of the present Article is to \nexamine what the nature of Energy Law is today, twenty years on. By means of analysis of the provisions of the regulatory \nand legal documents the seven core principles of the Energy Law were determined; they were crucial in the establishment \nof the Energy Law itself and also are the basis for development of its future principles. The purpose of this article is to analyze the fundamental principles of energy law, which are based on modern Euro-\npean and International Energy Law by analyzing the provisions of European and international regulatory documents, \nwhich are the basis for shaping the future norms of Energy Law of the European Union and Ukraine. The purpose of this article is to analyze the fundamental principles of energy law, which are based on modern Euro-\npean and International Energy Law by analyzing the provisions of European and international regulatory documents, \nwhich are the basis for shaping the future norms of Energy Law of the European Union and Ukraine. The two main targets to be pursued by the present article are intertwined and interdependent. The first is about develop-\nment of a concept for scholars and experts what the essence of Energy Law is and how it may be made comprehensible \nboth for the law specialists and specialists of other domains. The second target is to describe of the core principles frame-\nwork which influenced the establishment of the Energy Law and will play a pivotal role in its future development. It is \nsupposed the core principles framework should be the center for the Energy Law despite the absence of the unified legal \nregulatory document consolidating those principles. The present article will be definitely useful for the scholars and ex-\nperts engaged in the establishment of the Energy Law, its enhancement as well as its practical application and usage in the \ncourt practice. Keywords: Energy Law, energy legislation, principles of legislation, resource sovereignty, energy services, energy justice, \nclimate, natural resources. 55 ISSN 1681-6277. Економіка та право. № 2 (53), 2019"
https://openalex.org/W2966371480	https://europepmc.org/articles/pmc6683403?pdf=render	English	null	Diagnostic and grading accuracy of 18F-FDOPA PET and PET/CT in patients with gliomas: a systematic review and meta-analysis	BMC cancer	2,019	cc-by	6,953	Xiao et al. BMC Cancer (2019) 19:767 https://doi.org/10.1186/s12885-019-5938-0 Xiao et al. BMC Cancer (2019) 19:767 https://doi.org/10.1186/s12885-019-5938-0 Open Access © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Diagnostic and grading accuracy of 18F- FDOPA PET and PET/CT in patients with gliomas: a systematic review and meta- analysis Jiarui Xiao1,2†, Yizi Jin2†, Ji Nie1,4†, Fukun Chen3* and Xuelei Ma1* Jiarui Xiao1,2†, Yizi Jin2†, Ji Nie1,4†, Fukun Chen3* and Xuelei Ma1* Jiarui Xiao1,2†, Yizi Jin2†, Ji Nie1,4†, Fukun Chen3* and Xuelei Ma1* Abstract Background: Positron emission tomography (PET) and PET/computed tomography (PET/CT) imaging with 3,4-dihydroxy-6-[18F] fluoro-L-phenylalanine (18F-FDOPA) has been used in the evaluation of gliomas. We performed a meta-analysis to obtain the diagnostic and grading accuracy of 18F-FDOPA PET and PET/CT in patients with gliomas. Methods: PubMed, Embase, Cochrane Library and Web of Science were searched through 13 May 2019. We included studies reporting the diagnostic performance of 18F-FDOPA PET or PET/CT in glioma patients. Pooled sensitivity, specificity, and area under the summary receiver operating characteristic (SROC) curve were calculated from eligible studies on a per-lesion basis. Results: Eventually, 19 studies were included. Across 13 studies (370 patients) for glioma diagnosis, the pooled sensitivity and specificity of 18F-FDOPA PET and PET/CT were 0.90 (95%CI: 0.86–0.93) and 0.75 (95%CI: 0.65–0.83). Across 7 studies (219 patients) for glioma grading, 18F-FDOPA PET and PET/CT showed a pooled sensitivity of 0.88 (95%CI: 0.81–0.93) and a pooled specificity of 0.73 (95%CI: 0.64–0.81). Conclusions: 18F-FDOPA PET and PET/CT demonstrated good performance for diagnosing gliomas and differentiating high-grade gliomas (HGGs) from low-grade gliomas (LGGs). Further studies implementing standardized PET protocols and investigating the grading parameters are needed. Conclusions: 18F-FDOPA PET and PET/CT demonstrated good performance for diagnosing gliomas and differentiating high-grade gliomas (HGGs) from low-grade gliomas (LGGs). Further studies implementing standardized PET protocols and investigating the grading parameters are needed. Keywords: PET, 18F-FDOPA, Glioma, Meta-analysis Keywords: PET, 18F-FDOPA, Glioma, Meta-analysis (HGGs) [2]. The treatment of gliomas requires multidis- ciplinary care. Appropriate surgical or radiotherapy regi- men is highly dependent on the delineation and grade of tumors, and therefore imaging assessment is critical to the clinical management of affected patients. For the past few decades, conventional magnetic resonance imaging (MRI) has been the method of choice for glioma diagnosis. How- ever, it lacks sensitivity in non-enhancing gliomas and cannot reliably provide the differentiation between tumor recurrence and radiation-induced changes (e.g., pseudo- progression and radionecrosis) [3, 4]. Thus, more accurate imaging modalities need to be found. Background Glioma is the most common primary brain tumor, accounting for 81% of all malignant brain tumors with an annual incidence of 5.26 per 100,000 individuals [1]. According to the World Health Organization (WHO) 2007 classification, grade I and II tumors are together referred to as low-grade gliomas (LGGs), while grade III and IV tumors are categorized into high-grade gliomas * Correspondence: cfk007@126.com; drmaxuelei@gmail.com †Jiarui Xiao, Yizi Jin and Ji Nie, contributed equally to this work. 3Department of Nuclear Medicine, Yunnan Cancer Hospital, the Third Affiliated Hospital of Kunming Medical University, Kunming 650118, Yunnan, China 1Department of Biotherapy, Cancer Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center, No.37, Guoxue Alley, Chengdu 610041, Sichuan, China Full list of author information is available at the end of the article The Response Assessment in Neuro-Oncology (RANO) working group has recently recommended the use of posi- tron emission tomography (PET) imaging for gliomas Xiao et al. BMC Cancer (2019) 19:767 Page 2 of 11 Page 2 of 11 complementary to MRI [5]. Compared with MRI, PET provides additional insight into tumor metabolism and has been shown to improve tumor delineation and grading [6]. The glucose metabolic agent 18-fluoro-deoxyglucose (18F-FDG) has been the classic PET tracer used in tumor imaging, but it is not ideal in detecting gliomas due to the high physiologic uptake in normal brain tissue. Given the diagnostic limitations of 18F-FDG, amino acid tracers have been extensively investigated, such as 3,4-dihydroxy-6- [18F] fluoro-L-phenylalanine (18F-FDOPA). Unlike gado- linium, 18F-FDOPA is transported across the intact blood brain barrier (BBB) [7]. Hence the use of 18F-FDOPA PET enables the depiction of tumor components beyond con- trast enhancement in MRI [8]. Furthermore, the accuracy of 18F-FDOPA PET and PET/CT in detecting gliomas has been reported to be superior as compared with 18F-FDG PET and PET/CT in previous studies [9, 10]. However, existing studies are inconclusive because of relatively small sample sizes and heterogeneous designs. Also, previous studies have provided contradictory conclu- sions on whether there are significant differences in 18F-FDOPA PET and PET/CT between low-grade gli- omas (LGGs) and high-grade gliomas (HGGs) [11–13]. We performed this meta-analysis to systemically review all relevant publications in attempt to (1) evaluate the overall diagnostic performance of 18F-FDOPA PET and PET/CT in patients with gliomas and to (2) access the ability of 18F-FDOPA PET and PET/CT in discrimin- ating HGGs from LGGs. complementary to MRI [5]. Compared with MRI, PET provides additional insight into tumor metabolism and has been shown to improve tumor delineation and grading [6]. The glucose metabolic agent 18-fluoro-deoxyglucose (18F-FDG) has been the classic PET tracer used in tumor imaging, but it is not ideal in detecting gliomas due to the high physiologic uptake in normal brain tissue. Given the diagnostic limitations of 18F-FDG, amino acid tracers have been extensively investigated, such as 3,4-dihydroxy-6- [18F] fluoro-L-phenylalanine (18F-FDOPA). Unlike gado- linium, 18F-FDOPA is transported across the intact blood brain barrier (BBB) [7]. Hence the use of 18F-FDOPA PET enables the depiction of tumor components beyond con- trast enhancement in MRI [8]. Furthermore, the accuracy of 18F-FDOPA PET and PET/CT in detecting gliomas has been reported to be superior as compared with 18F-FDG PET and PET/CT in previous studies [9, 10]. However, existing studies are inconclusive because of relatively small sample sizes and heterogeneous designs. Statistical analysis Meta-DiSc software, version 1.4 (Clinical Biostatistics Unit, Ramón y Cajal Hospital, Madrid, Spain) was used to calculate pooled data including sensitivity, specificity, positive likelihood (LR+), negative likelihood (LR-), diag- nostic odds ratio (DOR) (with 95% confidence intervals (CI)), and construct summary receiver operating charac- teristic (SROC) curves [17]. The Area Under the Curve (AUC) was computed to measure overall performance of tests (AUC is positively correlated with diagnostic value, 0.5 ≤AUC < 0.7 representing a low accuracy; 0.7 ≤AUC < 0.9 indicating a moderate accuracy; AUC ≥0.9 indicating a high diagnostic value) [18, 19]. StataSE, version 12 (Stata- Corp, College Station, TX, USA) was employed to assess publication bias through Deeks’ Funnel Plot Asymmetry Test [20]. The Spearman’s rank correlation coefficient was calculated between logarithmic sensitivity and logarithmic (1-specificity), and a strong positive correlation indicates the existence of threshold effect [17]. We used Chi- square, Cochran-Q, and I-squared test to evaluate the heterogeneity between studies [17]. The Random-Effects Model would be applied, unless no significant heterogen- eity was detected between studies [21]. A systematic search of the PubMed, Embase, Cochrane Library and Web of Science databases was performed for English and non-English publications through 13 May 2019 using the following search: “(DOPA [all fields] OR FDOPA [all fields] OR fluorodihydroxyphenylalanine [all fields]) AND (positron emission tomography [all fields] OR PET [all fields]) AND (glioma [all fields] OR gliomas [all fields] OR brain tumor [all fields] OR brain tumors [all fields])”. References to retrieved articles and unpublished clinical trials were also checked for potential findings. Data extraction and quality assessment y Two reviewers (JX and YJ) independently went through all eligible studies and extracted essential information, including name of principal author, year of publication, study country, type of study design (retrospective or pro- spective), number of specimens, number of patients, mean or median age of patients, male to female ratio, index test, prior treatment, tumor occurrence (newly diagnosed or recurrent), reference standard, comparative imaging ap- proaches, parameters and threshold (if existed), and the number of TP, FP, FN, TN. Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) [16] was applied to evaluate the quality of all included studies in Review Manager 5.3 software (Cochrane Collaboration, Oxford, England). QUADAS-2 tool is composed of four aspects, of which each item can be defined as “yes”, “no” or “unclear”. The overall assess- ments of each aspect interpret the bias risk as low, high or unclear. Two reviewers (JX and YJ) independently assessed each article with disagreements resolved. Also, previous studies have provided contradictory conclu- sions on whether there are significant differences in 18F-FDOPA PET and PET/CT between low-grade gli- omas (LGGs) and high-grade gliomas (HGGs) [11–13]. We performed this meta-analysis to systemically review all relevant publications in attempt to (1) evaluate the overall diagnostic performance of 18F-FDOPA PET and PET/CT in patients with gliomas and to (2) access the ability of 18F-FDOPA PET and PET/CT in discrimin- ating HGGs from LGGs. data. Case reports, reviews, letters and in vitro studies were excluded. Meanwhile, studies involving diagnosing brain metastases were also excluded (unless single cases can be differentiated). When study populations over- lapped, we only included the most recent one to avoid data duplication [14, 15]. Eligible literatures were then classified into two different groups according to different aims of study: (1) Studies focused on the diagnosis of gliomas; (2) Studies focused on the grading of gliomas. Study selection The comprehensive literature search yielded 605 rele- vant records in total. After excluding duplicates and screening through titles and abstracts, 67 records went into full-text review, with 19 studies fulfilling the inclu- sion criteria for the final meta-analysis. Eligible studies were further classified for the intended subanalyses: 13 studies for glioma diagnosis and 7 studies for glioma grading. The details of the selection process are illus- trated in Fig. 1. y Spearman correlation coefficient was −0.18 (p-value = 0.56), displaying no threshold effect. Deeks’ Funnel Plot test demonstrated no publication bias (p-value = 0.93, Fig. 2a). Aim 2: Investigating the performance of 18F-FDOPA PET and PET/CT for grading gliomas. Spearman correlation coefficient was −0.18 (p-value = 0.56), displaying no threshold effect. Deeks’ Funnel Plot test demonstrated no publication bias (p-value = 0.93, Fig. 2a). Aim 2: Investigating the performance of 18F-FDOPA PET and PET/CT for grading gliomas. As for the meta-analysis of grading gliomas, 7 studies [12, 13, 24, 32–35] with 219 patients were involved in total, with male/female ratio of 122/97 (1.26). 193 (88%) patients were newly diagnosed, and 26 (12%) patients Study selection All records were screened independently for eligibility by 2 reviewers (JX and YJ), and discrepancies were resolved by consensus. The inclusion criteria were: (1) Original studies investigating the diagnostic or grading capacity of 18F-FDOPA PET or PET/CT in patients with gliomas; (2) Studies with histopathology and/or clinical and imaging follow-up as reference standards; (3) Cer- tain numbers of true-positive (TP), false-positive (FP), false-negative (FN) and true-negative (TN) results in diagnostic or grading tests can be derived from sufficient Xiao et al. BMC Cancer (2019) 19:767 Xiao et al. BMC Cancer (2019) 19:767 Page 3 of 11 Page 3 of 11 Aim 1: Investigating the accuracy of 18F-FDOPA PET and PET/CT for diagnosing gliomas. Considering the differences between primary and recur- rent tumors in clinical management and post-treatment changes, we divided all studies into two subgroups: detect- ing (1) newly diagnosed gliomas and (2) recurrent (includ- ing residual) gliomas. TP, FP, FN, TN were recalculated in studies investigating in a mixed population of primary and recurrent gliomas when feasible. (Significant level: two- tailed p-value< 0.05). Altogether, 13 studies (370 patients) [9, 10, 15, 22–31] were included in this meta-analysis. 4 of the 13 studies (31%) were retrospective studies, whereas the others (69%) were designed prospectively. Other than Morana et al. [26], which focused on the diagnostic performance of 18F-FDOPA PET on pediatric gliomas, all researches were carried out among adults. 8 of 13 studies focused only on diagnosing recurrent gliomas with previous treat- ment, while the rest studies were conducted jointly in newly diagnosed and pretreated patients, and all of them can be further stratified into corresponding subgroups. All TP, FP, FN and TN results extracted were based on visual analysis. Study characteristics The characteristics of the included studies are demon- strated in Table 1 and Table 2. Fig. 1 Flow chart of study selection. 19 studies are included eventually Page 4 of 11 Xiao et al. BMC Cancer (2019) 19:767 Table 1 Baseline information of included studies for glioma diagnosis Reference Year Country Design Specimens No. Patients No. Age, yr M/F Test Prior treatment Occurrence Gold Standard Meana Median Chen et al. [9] 2006 US Prospective 27 30 (27b) – – – PET With or without 7 New+ 20 Recur Histo+Radio+follow-up Tripathi et al. [10] 2009 India Prospective 15 15 28.4 ± 11.1 – 9/6 PET/CT With (Sx/CT/RT) or without 3 New+ 12 Recur Histo+Radio+follow-up Sellam et al. [22] 2010 India Prospective 30 30 – – – PET/CT Sx+/−RT Recur Histo+Radio+follow-up Jora et al. [23] 2011 India Prospective 23 23 43.25 ± 14.9 – – PET/CT 15 with (Sx + RT) + 8 without 8 New+ 15 Recur Histo+Radio+follow-up Karunanithi et al. [15] 2013 India Prospective 35 35 36.62 ± 0.86 – 28/7 PET/CT Sx + RT+/−CT Recur Histo+Radio+follow-up Pafundi et al. [24] 2013 US Prospective 23 10 40.8 ± 18.9 – 9/1 PET/CT With or without 8 New+ 2 Recur Histo Herrmann et al. [25] 2014 US Retrospective 110 110 51.7 ± 12.1 52.5 72/38 PET/CT Sx Recur Histo+Radio+follow-up Moran et al. [26] 2015 Italy Retrospective 27 27 10 10 15/12 PET 1 with (Sx + CT + RT) + 20 without 20 New+ 1 Recur Histo+Radio+follow-up Sharma et al. [27] 2016 India Prospective 12 11 34 – 6/5 PET/CT Sx Recur Histo+Radio+follow-up Paquet et al. [28] 2017 France Prospective 60 35 60 – – PET Sx + CT + RT Recur Histo+Radio Evangelista et al. [29] 2018 Italy Retrospective 13 13 – 60 – PET/CT Unclear Recur Radio+follow-up Youland et al. [30] 2018 US Prospective 37 13 – 40 9/4 PET Sx/CT/RT Recur Histo Evangelista et al. [31] 2019 Italy Retrospective 21 21 58 ± 11 – – PET/CT Sx + CT/RT/immunotherapy Recur Radio+follow-up M male, F female, New newly-diagnosed, Recur recurrent, Histo histopathology, Radio radiology, Sx surgery, CT chemotherapy, RT radiation therapy aMean age is expressed as mean ± standard deviation Page 5 of 11 Xiao et al. BMC Cancer (2019) 19:767 Table 2 Baseline information of included studies for glioma grading Reference Year Country Design Specimens No. Patients No. Study characteristics Age, yr M/F Test Prior treatment Occurrence Gold Standard Parameter Cut-off Meana Median Fueger et al. [12] 2010 US 17 Prospective+ 42 Retrospective 59 59 (22b) – 44.5 13/9 PET or PET/CT With (Sx + CT/RT) or without New Histo SUVmax 2.72 Nioche et al. [32] 2013 France Prospective 33 33 51 ± 16 51 28/5 PET/CT With (Sx + CT/RT) or without 20 New+ 13 Recur Histo SUVmean 2.2 Pafundi et al. [24] 2013 US Prospective 23 10 (9b) 42.9 ± 19.2 – 8/1 PET/CT With or without 7 New+ 2 Recur Histo SUVmax T/ SUVmean N 2.0 Janvier et al. [13] 2015 France Retrospective 31 31 36.8 ± 12.1 – 13/18 PET With (Sx/CT/RT) or without 25 New+ 6 Recur Histo+Radio+ follow-up SUVmean T/N 1.33 Bund et al. [33] 2017 France Prospective 53 53 38 – 23/30 PET/CT Without New Histo SUVmax T/N 2.16 Morana et al. [34] 2017 Italy Retrospective 26 26 10.2 ± 4.6 9.5 15/11 PET Without New Histo SUVmax T/S 0.90 Patel et al. [35] 2018 US Prospective 45 45 46.4 ± 16.2 – 22/23 PET Without New Histo SUVmax T/N 1.7 M, male; F, female; Sx, surgery; CT, chemotherapy; RT, radiation therapy; New, newly-diagnosed; Recur, recurrent; Histo, histopathology; SUV, standardized uptake value; Radio, radiology; T, tumor; N, normal; S, Striatum aMean age is expressed as mean ± standard deviation b Xiao et al. BMC Cancer (2019) 19:767 Page 6 of 11 Fig. 2 Publication bias assessment of included studies (a, b) and quality assessment of included studies (c-f). a, b: Deeks’ Funnel Plot shows no publication bias in both detecting (a) and grading (b) gliomas. c-f: The graphs show risk of bias and applicability concerns regarding each study. Quality assessment result for diagnosis (c, e); for grading (d, f) Fig. 2 Publication bias assessment of included studies (a, b) and quality assessment of included studies (c-f). a, b: Deeks’ Funnel Plot shows no publication bias in both detecting (a) and grading (b) gliomas. c-f: The graphs show risk of bias and applicability concerns regarding each study. Quality assessment result for diagnosis (c, e); for grading (d, f) Fig. 2 Publication bias assessment of included studies (a, b) and quality assessment of included studies (c-f). a, b: Deeks’ Funnel Plot shows no publication bias in both detecting (a) and grading (b) gliomas. Study characteristics c-f: The graphs show risk of bias and applicability concerns regarding each study. Quality assessment result for diagnosis (c, e); for grading (d, f) Xiao et al. BMC Cancer (2019) 19:767 Xiao et al. BMC Cancer (2019) 19:767 Xiao et al. BMC Cancer (2019) 19:767 Page 7 of 11 Page 7 of 11 Page 7 of 11 As illustrated in the Fig. 3(d-f), 18F-FDOPA PET and PET/CT presented a pooled sensitivity of 0.88 (95% CI: 0.81–0.93), specificity of 0.73 (95% CI: 0.64–0.81), LR+ of 2.90 (95% CI: 2.19–3.85), LR- of 0.16 (95% CI: 0.08– 0.36), DOR of 25.87 (95% CI: 10.53–63.54), and AUC of 0.89 (SE = 0.03) for differentiating HGGs from LGGs. were diagnosed with recurrent gliomas. All studies were performed at the patient level. The number of TN repre- sents the number of discriminating HGGs from LGGs. All the interpretations were based on quantitative analysis, and the cut-off value of each study was determined through ROC analysis. For those used various parameters, the most predictive index was included in this analysis. were diagnosed with recurrent gliomas. All studies were performed at the patient level. The number of TN repre- sents the number of discriminating HGGs from LGGs. All the interpretations were based on quantitative analysis, and the cut-off value of each study was determined through ROC analysis. For those used various parameters, the most predictive index was included in this analysis. g Other than sensitivity (p-value = 0.0045), no signifi- cant heterogeneity was found among studies for specificity (p-value = 0.13), LR+ (p-value = 0.42), LR- (p-value = 0.07) and DOR (p-value = 0.53). Other than sensitivity (p-value = 0.0045), no signifi- cant heterogeneity was found among studies for specificity (p-value = 0.13), LR+ (p-value = 0.42), LR- (p-value = 0.07) and DOR (p-value = 0.53). Neither threshold effect nor publication bias was detected through Spearman correlation coefficient (= 0.67, p-value = 0.10) and Deeks’ Funnel Plot test (p-value = 0.57, Fig. 2b) respectively. Quality assessment We used QUADAS-2 to estimate the quality of literature in Review Manager 5.3. The assessment results were shown in Fig. 2(c-f). Meta-analysis of Diagnostic/Grading Performance in Gliomas. Meta-analysis of Diagnostic/Grading Performance in Gliomas. Aim 1: Investigating the accuracy of 18F-FDOPA PET for diagnosing gliomas. Meta-analysis resulted in a pooled sensitivity of 0.90 (95% CI: 0.86–0.93), and a pooled specificity of 0.75 (95% CI: 0.65–0.83). The pooled LR+ was 2.84 (95% CI, 2.09– 3.85), and the pooled LR- was 0.15 (95% CI: 0.09–0.26). The DOR was 24.05 (95% CI: 12.62–45.85) (Fig. 3a/b). SROC curve was established based on pooled sensi- tivity and specificity, and the overall AUC was 0.85 [Standard Error (SE) = 0.05], indicating a moderately high overall diagnostic value of 18F-FDOPA PET and PET/CT on gliomas among individuals (Fig. 3c). The diagnostic accuracy of 18F-FDOPA PET for distin- guishing radiation necrosis from brain tumor recurrence has been previously investigated by Yu J et al. [36]. How- ever, the use of 18F-FDOPA PET or PET/CT in accessing newly-diagnosed gliomas has not been systematically studied before. Moreover, the recommendations for the use of 18F-FDOPA in glioma grading remained contro- versial [5]. Our findings confirmed there is a meaningful role of 18F-FDOPA PET and PET/CT for the evaluation of glioma patients. Only a significant heterogeneity for sensitivity was detected (Chi-square = 34.40, p-value = 0.0006, I-square = 65.1%) through Chi-square, Cochran-Q, and I-squared tests (Fig. 3a). Discussion This meta-analysis demonstrated a pooled sensitivity of 0.90 and specificity of 0.75 for 18F-FDOPA PET and PET/CT in diagnosing gliomas, and a pooled sensitivity of 0.88 and specificity of 0.73 in grading gliomas. Sub- group analysis showed that 18F-FDOPA PET and PET/ CT had good diagnostic performance in both newly- diagnosed and recurrent groups. The specificity for de- tecting recurrent gliomas was slightly lower, which may be explained by the increased false positive rate caused by treatment response such as edema and inflammatory tissue. However, it is worth mentioning that the limited number of studies in newly-diagnosed group may impair the reliability to some degree. Subgroup analysis Of increased interest is the value of 18F-FDOPA PET and PET/CT in differentiating tumor recurrence from radiation-induced changes. At present, MRI remains the standard imaging method for assessing treatment effects in glioma patients [37]. However, the utility of contrast enhancement MRI mainly relies on the disruption of blood-brain barrier (BBB), which impairs its specificity in differentiating radiation-induced changes from recur- rent or residual brain tumors, such as pseudoprogression and radionecrosis. New amino acid tracers including 18F-FDOPA PET and PET/CT have been investigated to address this problem. Several studies have directly com- pared the performance of contrast enhanced MRI with 18F-FDOPA PET or PET/CT in the identification of residual and recurrent gliomas. Jora et al. [23] reported 18F-FDOPA PET to have higher overall sensitivity and specificity over MRI, although their sample size was too limited to show any statistical significance. Karunanithi et al. [15] proved significantly higher specificity of 18F- No threshold effect was detected in two subgroups. Pooled data and SROC curve manifested a high diagnostic value of 18F-FDOPA PET and PET/CT in re- current subgroup, with sensitivity of 0.92 (95% CI: 0.88–0.95), specificity of 0.76 (95% CI: 0.66–0.85), LR+ of 2.87 (95% CI: 2.01–4.11), LR- of 0.13 (95% CI: 0.07– 0.23), DOR of 29.65 (95% CI: 13.09–67.15), and AUC of 0.90 (SE = 0.06). In the newly-diagnosed subgroup, only two studies were eligible for meta-analysis (in another three studies only sensitivity can be computed after stratification). In comparison with recurrent group, pooled data of newly-diagnosed group revealed a lower sensitivity of 0.71 (95% CI: 0.54–0.85) and a higher spe- cificity of 0.86 (95% CI: 0.42–1.00). The LR+, LR- and DOR were 3.71 (0.87–15.81), 0.36 (0.19–0.68) and 10.88 (1.57–75.31) respectively. Aim 2: Investigating the Performance of 18F-FDOPA PET and PET/CT for Grading Gliomas. Xiao et al. BMC Cancer (2019) 19:767 Page 8 of 11 Fig. 3 Forest plots of sensitivity and specificity, and summary receiver operating characteristic curve of diagnosing gliomas (a-c) and differentiating HGGs from LGGs (d-f) Fig. 3 Forest plots of sensitivity and specificity, and summary receiver operating characteristic curve of diagnosing gliomas (a-c) and differentiating HGGs from LGGs (d-f) FDOPA PET in diagnosing recurrence than contrast enhanced MRI overall (p-value = 0.002), and for HGGs (p-value = 0.006) and LGGs (p-value = 0.004) respect- ively. Subgroup analysis Besides the evidence presented above, our systema- tical analysis has also shown that 18F-FDOPA PET and PET/CT provide more reliable results in discriminating recurrent lesions from treatment related changes, with a pooled sensitivity and specificity of 0.92 and 0.76. imaging approaches is of great significance. This meta- analysis proved the high overall value of 18F-FDOPA PET and PET/CT in evaluating tumor grade, with the AUC of 0.89. However, studies with respect to grading of recurrent gliomas demonstrated controversial results. In Fueger et al. [12], 18F-FDOPA uptake manifested no significant correl- ation with different tumor grades in recurrent gliomas, whereas Nioche et al. [32] reported a threshold of 1.8 to identify HGGs in pre-treated patients, with a sensitivity and specificity of 1.0 and 0.80 respectively. The discrepancy between limited studies concerned with grading recurrent gliomas made it unachievable for subgroup analysis. It is p y p y Another advantage of 18F-FDOPA PET and PET/CT is the utility in the detection of HGGs. Due to the differences between LGGs and HGGs in therapeutic regimen and prognosis evaluation, initial grading for gliomas through Xiao et al. BMC Cancer (2019) 19:767 Xiao et al. BMC Cancer (2019) 19:767 Page 9 of 11 Page 9 of 11 also worth noting that in Fueger et al. [12], only the data of newly-diagnosed group is available for meta-analysis. The exclusion of recurrent group might therefore introduce bias. In addition, since most patients included in our study are with newly-diagnosed gliomas (90%), our synthetic re- sults are not representative enough for grading accuracy within the recurrent population, which still requires further investigation with larger sample size. led to data loss in stratification of subgroups when true negative case was less than one (specificity could not be calculated in this case). Besides, multiple specimens were obtained from one patient in three studies [24, 27, 30], which impeded our analysis on a per-patient basis. In addition, different imaging protocols and grading param- eters from separate studies limited the quantitative ana- lysis we performed for glioma grading. Finally, since most studies included in our analysis were published before the WHO 2016 classification system [45], the correlation of 18F-FDOPA uptake with glioma molecular characteristics cannot be analyzed. Evidence for comparisons of 18F-FDOPA with other PET tracers are available in several existing studies. Subgroup analysis As the most predominant PET tracer in oncological diagnostics so far, 18F-FDG shares the feature of being transported through integrate BBB. However, the high glucose metab- olism within normal brain parenchyma may considerably hamper the differentiation between brain tumor and non- neoplastic tissue [38]. 18F-FDOPA has demonstrated better contrast than 18F-FDG in tumor versus normal tissues surrounding, and a higher sensitivity for detecting brain tumors than 18F-FDG [5, 10, 23]. The superiority in sensitivity of 18F-FDOPA can be further proved in com- parison with previous meta-analysis of 18F-FDG (pooled sensitivity of 0.38, 95% CI: 0.27–0.50) [39]. The longest established amino acid tracer for brain tumor imaging is [11C]-methyl-L-methionine (11C-MET). In the only study with direct comparison between 18F-FDOPA PET and 11C-MET, 18F-FDOPA performed equally well as 11C- MET in the imaging of brain tumors [11]. However, 11C- MET is being replaced by tracers labelled with fluorine-18 (half-life of 109.8 min) due to its short half-life (20 min). The use of another amino acid tracer O-(2-[18F]-fluor- ethyl)-1-tyrosine (18F-FET) has rapidly grown in recent years. A previous meta-analysis of 5 studies including 180 patients reported a sensitivity and specificity of 0.82 and 0.76 of 18F-FET PET for the differential diagnosis of pri- mary brain tumor [40]. In later comparison studies re- vealed that 18F-DOPA and 18F-FET shared similar uptake pattern in primary and recurrent HGGs and had similar diagnostic accuracy in recurrent HGGs [31, 41]. However, compared with 18F-FET, the elevated physiological 18F- FDOPA uptake in striatum has been a concern which may limits its use to detect brain tumor with striatum involve- ment [5, 42, 43]. Therefore, Kratochwil et al. recommended 18F-FET to examine patients with possible involvement of basal ganglia irrespective of tumor grade. Similarly, Morana et al. [44] reported that the diagnostic ability of 18F-FDOPA PET/CT in dorsal striatum was concordant with its overall accuracy, while not in the ventral striatum, which required fused MRI to improve its performance. Conclusions In conclusion, this meta-analysis provides evidence that 18F-FDOPA PET and PET/CT have high diagnostic accur- acy for the detection of gliomas, especially the discrimin- ation between tumor recurrence and radiation-induced changes. 18F-FDOPA PET and PET/CT also demonstrate good grading performance for the distinction between HGGs and LGGs. However, the grading parameters needs further investigation with standardized imaging protocols in prospective studies. Abbreviation 11 11 11C-MET: [11C]-methyl-L-methionine; 18F-FDG: 18-fluoro-deoxyglucose; 18F- FDOPA: 3,4-dihydroxy-6-[18F] fluoro-L-phenylalanine; 18F-FET: O-(2-[18F]- fluorethyl)-1-tyrosine; AUC: Area under the curve; BBB: Blood brain barrier; CI: Confidence intervals; CT: Computed tomography; DOR: Diagnostic odds ratio; FN: False negative; FP: False positive; HGGs: High-grade gliomas; LGGs: Low-grade gliomas; LR-: Negative likelihood ratio; LR + : Positive likelihood ratio; MRI: Magnetic resonance imaging; PET: Positron emission tomography; QUADAS-2: Quality Assessment of Diagnostic Accuracy Studies 2; SE: Standard Error; SROC: Summary receiver operating characteristic; TN: True negative; TP: True positive; WHO: World Health Organization Availability of data and materials ll d d l d d Availability of data and materials All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Several study limitations also need to be addressed. Although 19 studies were included in this meta-analysis, the sample size of each study tended to be small, and the number of true negative cases was particularly lim- ited, which might possibly yield less replicable results, especially the pooled specificity. Limited sample size also Authors’ contributions JX, YJ, and JN worked together on the conception, data analysis and interpretation, and drafting of the manuscript. They contributed equally to our work. FC and XM supervised development of work, helped to evaluate the manuscript and acted as corresponding author. All authors read and approved the final manuscript. Ethics approval and consent to participate Not applicable. References J Nucl Med. 2018;59. 11. Becherer A, Karanikas G, Szabo M, Zettinig G, Asenbaum S, Marosi C, Henk C, Wunderbaldinger P, Czech T, Wadsak W, et al. 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Model selection in historical research using approximate Bayesian computation Citation for published version: Rubio-Campillo, X 2016, 'Model selection in historical research using approximate Bayesian computation', PLoS ONE, vol. 11, no. 1, e0146491. https://doi.org/10.1371/journal.pone.0146491 Citation for published version: Rubio-Campillo, X 2016, 'Model selection in historical research using approximate Bayesian computation', PLoS ONE, vol. 11, no. 1, e0146491. https://doi.org/10.1371/journal.pone.0146491 Citation for published version: Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 RESEARCH ARTICLE Editor: John P. Hart, New York State Museum, UNITED STATES Editor: John P. Hart, New York State Museum, UNITED STATES Received: May 25, 2015 Accepted: December 17, 2015 Published: January 5, 2016 Received: May 25, 2015 Accepted: December 17, 2015 Published: January 5, 2016 Xavier Rubio-Campillo* Computer Applications in Science & Engineering department, Barcelona Supercomputing Centre, Barcelona, Spain Computer Applications in Science & Engineering department, Barcelona Supercomputing Centre, Barcelona, Spain * xavier.rubio@bsc.es * xavier.rubio@bsc.es * xavier.rubio@bsc.es a1111 Case Study This work examines an alternate approach to this evaluation based on a Bayesian-inspired model selection method. The validity of the classical Lanchester’s laws of combat is exam- ined against a dataset comprising over a thousand battles spanning 300 years. Four varia- tions of the basic equations are discussed, including the three most common formulations (linear, squared, and logarithmic) and a new variant introducing fatigue. Approximate Bayesian Computation is then used to infer both parameter values and model selection via Bayes Factors. Copyright: © 2016 Xavier Rubio-Campillo. 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: Dataset is distributed under the terms of a Creative Commons Attribution- ShareAlike 4.0 International License and accessible at https://github.com/xrubio/lanchester Formal Models and History Computational models are increasingly being used to study historical dynamics. This new trend, which could be named Model-Based History, makes use of recently published data- sets and innovative quantitative methods to improve our understanding of past societies based on their written sources. The extensive use of formal models allows historians to re- evaluate hypotheses formulated decades ago and still subject to debate due to the lack of an adequate quantitative framework. The initiative has the potential to transform the disci- pline if it solves the challenges posed by the study of historical dynamics. These difficulties are based on the complexities of modelling social interaction, and the methodological issues raised by the evaluation of formal models against data with low sample size, high variance and strong fragmentation. OPEN ACCESS Citation: Rubio-Campillo X (2016) Model Selection in Historical Research Using Approximate Bayesian Computation. PLoS ONE 11(1): e0146491. doi:10.1371/journal.pone.0146491 Introduction The discipline of History presents its ideas as descriptive models expressed in natural language. Historians use the flexibility of this communication system to explain the complexity and diversity of human societies though their written records. The approach is different than the majority of scientific disciplines, which formulate their theories in formal languages such as mathematics. Formal languages are not as flexible as natural languages, but they are much bet- ter defining concepts and relations without ambiguities [1]. Hypotheses defined in formal lan- guage can then be falsified against empirical evidence, and quantitative methods can then be applied to compare predictions generated by a theory to observed patterns. As a consequence, an old theory can be replace by a new one when it has superior explanatory power. This evaluation of ideas does not happen in History. Quantitative methods cannot be used to falsify descriptive models or perform cross-temporal and cross-spatial comparison. These inabilities are central to the current methodological debates of the discipline [2–6]. There is a clear desire to identify both the common trajectories and the observed differences between case studies with diverse spatiotemporal coordinates. However, it is unclear how this could be achieved using the common methods of the discipline. A possible approach to tackle this challenge is to shift the discipline from descriptive to for- mal models [7]. This innovation could allow historians to know under what extent a working hypothesis explains a historical dynamic by quantifying the distance between the predictions of a model and the patterns observed in the evidence. This new approach has clear benefits, but it is not an easy task as it requires a) formal models, b) quantified datasets and c) methods to compare both components. These debates are intrinsically linked with the increasing number of available databases and historical research using formal models [8–10]. The rise of what we could define as Model- Based History is changing the way researchers study historical trajectories [2]. To date, this new approach to the past has been focused on three main topics: trade networks [11], sociocul- tural evolution [7] and warfare [12–14]). The increase in the number of works is diversifying the topics examined by Model-Based History, and now it includes fields such as knowledge exchange [15] or the evolution of religion [16]. Quantitative comparison between models and observations is one of the advantages of this new approach. Impact Funding: Funding for this work was provided by the SimulPast Consolider Ingenio project (CSD2010- 00034) of the former Ministry for Science and Innovation of the Spanish Government and the European Research Council Advanced Grant EPNet (340828). Results indicate decisive evidence favouring the new fatigue model. The interpretation of both parameter estimations and model selection provides new insights into the factors guid- ing the evolution of warfare. At a methodological level, the case study shows how model selection methods can be used to guide historical research through the comparison between existing hypotheses and empirical evidence. Competing Interests: The author has declared that no competing interests exist. 1 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Introduction Both of them fit the different models to the observed patterns using maximum likelihood methods, and then they calculate an index of information loss (i.e. low values indicate better models). A different solution is to use the Bayesian statistical framework. It is based on the idea that the knowledge of a given system with uncertainty can be gradually updated through new evi- dence. The process is achieved by computing the probability that a given hypothesis is correct, considering both existing knowledge and new data. The main advantage of this approach is that it seems better fitted to evaluate competing models under high levels of uncertainty and equifinality [25]. Despite its interest, scientific research did not start using the Bayesian frame- work until recent years, even if it was formulated 200 years ago [26]. The delay on the adoption was mainly caused by the mathematical complexities of applying Bayesian statistics to non- trivial problems. The development of new computational methods such as Markov Chain Monte-Carlo and Approximate Bayesian Computation (ABC) has mainly solved this limita- tion, thus explaining the current success of Bayesian inference. Historians constantly deal with competing explanations of uncertain datasets, so it seems that Bayesian model selection can be useful to the discipline. This potential can also be inferred from the fact that other historical disciplines such as biology and archaeology are part of this Bayesian renaissance. Biology is particularly active in using these methods in fields such as population genetics and ecology [27, 28]. Archaeology traditionally limited Bayesian inference to C14 dates [29], but model selection techniques are becoming popular beyond this applica- tion [30–33]. These examples suggest that Bayesian model selection can be applied to History, considering the similarities between the three disciplines. First, all these fields study temporal trajectories using data with high levels of uncertainty. Second, the analysis of these datasets implies that they need to evaluate the plausibility of multiple competing hypotheses. Finally, all of them want to identify patterns generated as an aggregate of individual behaviour. As a con- sequence, it seems clear that Bayesian model selection would have significant utility for historians. This paper presents the use of Bayesian inference to perform model selection in historical research. The utility of a Bayesian-inspired computational method known as Approximate Bayesian Computation is discussed. Introduction The most common statistical framework to perform this evaluation is Null Hypothesis Significance Testing. First, the problem to solve is defined as a clear research ques- tion and a working hypothesis H1. This hypothesis is a possible answer which could be falsified by existing evidence. The explanation provided by H1 will then compete against a null hypoth- esis H0. H0 is an alternative that does not take into account H1. H1 is translated into a formal model, usually a computer simulation of the dynamics encapsulated in the hypothesis. Model- Based History often prefers bottom-up techniques such as Agent-Based Models [17] or com- plex network analysis [18]. Classical equation-based models are also applied, but these innova- tive approaches seem better suited to the type of social processes examined by the discipline. The created model defines the system at a small-scale level (e.g. individual or groups), and it evolves through the interaction between these entities. The emergence of distinctive large-scale patterns generated by this set of interactions is then compared to empirical data. If the proba- bility of getting the observed patterns without H1 is less than a given confidence interval (i.e. the p-value) we can reject H0, thus accepting H1. Null Hypothesis Significance Testing is useful to prove that our model has higher predictive power than a random process. However, it is not designed to compare multiple potentially valid explanations. Model selection is a different approach designed to quantify by how much a model is better at explaining evidence than alternate models. Model selection is having 2 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC increasing popularity due to the current debates on the use of statistics analysis for scientific research [19–23]. It is worth mentioning that neither method seems better than the other one, and the choice will depend on the aim of the research: Null Hypothesis Significance Testing aims to know if the observed process could be explained without the working hypothesis, while model selection aims to choose which hypothesis is better at matching evidence. The model selection approach provides a set of new methods to evaluate models. Most of them quantify the loss of information from each model to the evidence using information crite- ria [24]. Two of the most widely used methods are Akaike Information Criterion and Bayesian Information Criterion. Materials and Methods Case study: the evolution of combat Introduction The use of ABC is then illustrated with a classical example of formal model used in History: the classical Lanchester’s laws of warfare. Next section pres- ents the case study, the model selection framework and the competing models. Third section shows the results of the method, both in terms of model selection and parameter estimation. The text then interprets these results and concludes with an evaluation of the approach in the context of Model-Based History. Case study: the evolution of combat Warfare is probably the first human activity ever explored with formal models. Their use began in early XIXth century in the form of boardgames such as kriegsspiel. They were used to train 3 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC officers on managing armies and fighting the enemy. These practices had a major impulse dur- ing Second World War with the creation of Operations Research. This new research field focused on developing formal models able to help commanders on decision-making [34]. The introduction of the first computers expedited the use of these quantitative methods during the Cold War, establishing them as a standard procedure for training and planning. In contrast, History is only now incorporating some of these techniques to the study of past conflicts [35]. Boardgames, mathematical models and computer simulations are proving their utility in the task of studying warfare understood as an unfortunate part of human culture [36]. The theoretical model formulated by F.W. Lanchester in 1916 is one of the most popular mathematical formulations used in the field [37]. Lanchester aimed to design the laws predict- ing the casualties of two enemy forces engaged in land battle. He proposed a system of coupled differential equations where casualties were dependent on two factors: a) force size and b) fighting value. The first factor takes into account the importance of sheer numbers on the out- come of military conflict, while the second factor encapsulates qualitative differences between individual fighting skills (e.g. morale, training, technology, etc.). Two models were initially pro- posed: the linear law and the square law. The linear law aimed to capture the dynamics of ancient battles, where the supremacy of hand-to-hand combat meant that each soldier could only attack an opponent at a given moment. The equations defining the rate of casualties in a battle between armies Blue and Red are defined in Eq 1: dB dt ¼ rBR dR dt ¼ bRB ð1Þ ð1Þ with B, R as the size of the forces and r, b as their ﬁghting value. The rate of casualties is propor- tional to both sizes, so even highly disproportionate odds would cause similar casualties to both opponents. The square law models warfare after the introduction of gunpowder-based weapons. This technological innovation increased the range, thus allowing each soldier to attack multiple ene- mies. Case study: the evolution of combat The squared law models the casualties of a force as the enemy’s force size multiplied by the fighting value of its individuals, as seen in Eq 2: dB dt ¼ rR dR dt ¼ bB ð2Þ ð2Þ The Lanchester’s laws generated a large amount of interest during the Cold War [38–42]. h d b d h l d f h l d l d d h f l g g g The debate was centred on the actual predictive power of the laws, and it included the formula- tion of alternate proposals such as the popular logarithmic model. It suggested that the casual- ties suffered by a force are not dependant on the enemy’s size, but on its own size as defined in Eq 3 [40]: dB dt ¼ rB dB dt ¼ bR ð3Þ ð3Þ Several works discussed the validity of the laws [42, 43]. Other contributions extended the original framework introducing concepts such as spatial structure or system dynamics [44, 45]. The utility of the model was also expanded beyond its initial purpose, and has been successfully applied to study competition dynamics in ecology [46–48], evolutionary biology [49] or eco- nomics [50]. Model selection were also applied to compare the plausibility of these models against histor- ical evidence. The most extensive effort was made by Charles D. Allen’s in his monograph [51]. The author tested the validity of different models to explain a dataset of 1080 land battles from the middle of XVIIth century to the beginnings of the XXth century. The analysis suggested 4 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC that the logarithmic model has higher explanatory power than the two classical models. How- ever, the coarse-grained results assumed that this power remained constant during the whole period, thus not examining the validity of the models for the different phases of warfare. Simi- lar works used Bayesian inference to evaluate the Lanchester’s laws in specific scenarios. They included biological case studies [48], daily casualties during Inchon-Seoul campaign in 1950 [52] or attrition during the battle of the Ardennes in 1944 [53, 54]. All these results suggests that the Lanchester’s laws are useful to understand if casualties are more influenced by quantitative or qualitative factors. Some authors suggested that the models should introduce dynamic parameters such as variable fighting values or fatigue [44]. Case study: the evolution of combat However, as some of these works highlights, a pure Bayesian framework could hardly cope with the mathematical difficulties added by this new complexity. The dataset. The dataset used in this study is based on Allen’s list of battles, originally compiled in a previous work [55]. The introduction of weapons with longer ranges over 300 years should be reflected in a gradual increase in the validity of the squared model over the lin- ear model. In order to test this idea the span has been divided in four periods, based on prior opinions of decisive transitions in the evolution of warfare [56]: 1. Pike and Musket (1620–1701). The first period was characterised by deep formations of sol- diers (i.e. tercios and regiments) armed with muskets and pikes. 1. Pike and Musket (1620–1701). The first period was characterised by deep formations of sol- diers (i.e. tercios and regiments) armed with muskets and pikes. 2. Linear warfare (1702–1792). The War of the Spanish Succession (1702–1714) saw a shift in battle tactics and technological innovations. Armies were deployed in thin formations exclu- sively armed with muskets, while pikes were substituted by bayonets. 3. Napoleonic Wars (1793–1860). The French Revolution forced another major transition in warfare, which was mainly adopted during the Napoleonic wars. The new concept of citizen armies allowed the states to increase the size of their forces up to the limits imposed by pre- industrial logistics. 4. American Civil War (1861–1905). The impact of industry development became explicit on the battlefield during the American Civil War. The size of armies and the lethality of their weapons steadily increased until fully industrialised armies were deployed in the Russo-Jap- anese War. This conflict was the prelude of what would be seen during the two world wars. Exploratory Data Analysis has been used to identify structural patterns in the dataset. A time series of the number of battles can be seen in Fig 1, while size and casualty ratios are depicted in Fig 2. These visualisations shows how the dataset has relative small sample size and high variance. These are common properties seen in historical data. The figures suggest that the number of battles remained constant during the 300 years with the exception of the Napo- leonic Wars. At the same time, the gradual increase on average army size seems linked to a decrease on casualty ratios. PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 The model selection framework Standard Bayesian inference updates a set of prior beliefs considering new evidence and a given likelihood function. Prior beliefs aggregate the existing knowledge of a given topic, and the degree of credibility of this knowledge. These beliefs are translated into parameters of the model. The possible values for each parameter receive an initial probability following a specific statistical distribution. The likelihood function is used to compute the probabilities of any given result considering the value of the input parameters. The updated knowledge (i.e. the the 5 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 simplicity and lack of assumptions makes it perfect for illustrative purposes. It is defined as follows: simplicity and lack of assumptions makes it perfect for illustrative purposes. It is defined as follows: 1. Initialise parameters sampling the prior distributions 2. Run the model and compute the distance to evidence 3. If distance is within the closest runs below a tolerance level τ keep values of parameters; oth- erwise discard them. This algorithm is executed a large number of runs, and the set of kept parameter values is used as the posterior distribution. Model Selection in Historical Research Using ABC Fig 1. Number of battles by decade. The three identified transitions correlate with periods of intensive warfare. doi:10.1371/journal.pone.0146491.g001 Fig 1. Number of battles by decade. The three identified transitions correlate with periods of intensive warfare. Fig 1. Number of battles by decade. The three identified transitions correlate with periods of intensive warfare. doi:10.1371/journal.pone.0146491.g001 doi:10.1371/journal.pone.0146491.g001 posterior distribution) is then computed following Bayes’ rule: posterior distribution) is then computed following Bayes’ rule: PðyjDÞ ¼ PðDjyÞ PðyÞ PðDÞ PðyjDÞ ¼ PðDjyÞ PðyÞ PðDÞ PðDÞ being θ the considered value and D the observed data. This can be translated as (following [57]): being θ the considered value and D the observed data. This can be translated as (following [57]): posterior ¼ likelihood prior evidence posterior ¼ likelihood prior evidence A barrier to the adoption of Bayesian inference is the difficulty to derive likelihood functions when the examined model is not a standard statistical distribution. This constraint limits the use of the framework for computer simulations encapsulating complex dynamics such as the ones explored in Model-Based History. A major breakthrough to this issue is the recent devel- opment of ABC [58, 59]. ABC comprises a family of computationally-intensive algorithms able to approximate pos- terior distributions without using likelihood functions. These methods identify the regions of the prior space producing the closest results to the evidence. This capability of extending the Bayesian framework to any computer simulation has exponentially increased the popularity of ABC during the last decade, including the other historical disciplines: biology [60–63], and archaeology [31, 33, 64, 65]). The analysis performed in this work implements the simplest ABC method: the rejection algorithm [66]. It is not the most efficient ABC method (see [67, 68] for alternatives), but its 6 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC Fig 2. Size and casualty ratio by battle. The total number of soldiers involved in each battle is defined in the Y axis while the size of each point shows the casualty ratio of the battle. Fig 2. Size and casualty ratio by battle. The total number of soldiers involved in each battle is defined in the Y axis while the size of each point shows the casualty ratio of the battle. The total number of soldiers involved in each battle is defined in the Y axis while the size of each point shows the doi:10.1371/journal.pone.0146491.g002 doi:10.1371/journal.pone.0146491.g002 Definition of competing models We will evaluate the plausibility of four different variations of the Lanchester equations: the two original laws (linear and squared), the popular logarithmic variation and a new model add- ing fatigue effects. For convenience the models have been here transformed to difference equa- tions as seen in Eqs 4, 5 and 6: Linear: Btþ1 ¼ Bt rBtRt Rtþ1 ¼ Rt bRtBt ð4Þ ð4Þ 7 / 18 7 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC Squared: Squared: Btþ1 ¼ Bt rRt Rtþ1 ¼ Rt bBt ð5Þ ð5Þ Logarithmic: Btþ1 ¼ Bt rBt Rtþ1 ¼ Rt bRt ð6Þ ð6Þ The fourth model adds fatigue to the logarithmic model. This factor is modelled as a gradual decrease in the efficiency of the armies as defined in Eq 7): Fatigue: Btþ1 ¼ Bt rBt logðe þ tÞ Rtþ1 ¼ Rt bRt logðe þ tÞ ð7Þ ð7Þ Fighting value b is scaled to the maximum number of casualties that B can inflict to R in a time step. In order to avoid disparate values b is defined following Eq 8 for the linear model and Eq 9 for the other three. Linear law: Linear law: b ¼ 100 Bt¼0 Rt¼0 ð8Þ ð8Þ Other models: b ¼ 100 maxðBt¼0; Rt¼0Þ ð9Þ ð9Þ The enemy’s fighting value r is then defined as b multiplied by an odds ratio P. In this way the individual value of a Red soldier is expressed as a ratio of Blue’s value (e.g. P = 2 would mean that each Red soldier is as lethal as two Blue soldiers). Distinctive dynamics for each model are observed in Fig 3. All models are initialised as a battle where an army is being opposed by a smaller force with higher fighting value. In the lin- ear model the forces have similar casualty rates, while size has a bigger impact in the squared model. The logarithmic model increases the weight of fighting value over size as the smaller force finishes with more soldiers. The fatigue model generates similar casualties than the loga- rithmic model, but they are distributed over a longer period of time. Experiment Design Previous authors suggested that the deterministic nature of the original laws was too rigid to perform a proper comparison with long-term observations. Using a fixed P for a large number of battles would ignore any slight variation on the fighting value odds from one engagement to the next one. The issue has been solved introducing stochasticity in P, which is sampled every battle from a gamma distribution with shape κ and scale θ. For convenience the input parame- ters are expressed as mean μ and standard deviation σ, which are then used to compute k ¼ m s 2 and y ¼ s2 m . The outcome of each battle is generated using as parameters the sampled P 8 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC and initial army sizes Bt = 0, Rt = 0 set to historical values. The chosen Lanchester variant as deﬁned in Eqs 4–7 is then iterated until one of the forces has suffered as many casualties as recorded in the historical data. The entire workﬂow is depicted in Fig 4. The rejection algorithm calculates a distance between the results of a single run and obs tions A popular approach is the comparison of summar statistics aggregating the outcom Fig 3. Time series of attrition. Casualties of two forces as computed by the four competing models with P = 1.5, Bt = 0 = 15000 and Rt = 0 = 10000. doi:10.1371/journal.pone.0146491.g003 Fig 3. Time series of attrition. Casualties of two forces as computed by the four competing models with P = 1.5, Bt = 0 = 15000 and Rt = 0 = 10000. doi:10 1371/journal pone 0146491 g003 Fig 3. Time series of attrition. Casualties of two forces as computed by the four competing models with P = 1.5, Bt = 0 = 15000 and Rt = 0 = 10000. doi:10.1371/journal.pone.0146491.g003 doi:10.1371/journal.pone.0146491.g003 doi:10.1371/journal.pone.0146491.g003 and initial army sizes Bt = 0, Rt = 0 set to historical values. The chosen Lanchester variant as deﬁned in Eqs 4–7 is then iterated until one of the forces has suffered as many casualties as recorded in the historical data. The entire workﬂow is depicted in Fig 4. The rejection algorithm calculates a distance between the results of a single run and observa- tions. Experiment Design A popular approach is the comparison of summary statistics aggregating the outcome of a run against the evidence. However, this solution has theoretical issues which are currently The rejection algorithm calculates a distance between the results of a single run and observa- tions. A popular approach is the comparison of summary statistics aggregating the outcome of a run against the evidence. However, this solution has theoretical issues which are currently 9 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC Example for a experiment using 1000 runs and tolerance τ = 0.01. Left side illustrates the rejection algorithm of the Lanchester model. he ABC framework. Example for a experiment using 1000 runs and tolerance τ = 0.01. Left side illustrates t details the simulation of the Lanchester model. 146491 004 g 4. Flowchart for the ABC framework. Example for a experiment using 1000 runs and tolerance τ = 0.01. L hile the green panel details the simulation of the Lanchester model. 4. Flowchart for the ABC framework. Example for a experiment using 1000 runs and tolerance τ = 0.01. Left side illustrates the rejection algorithm le the green panel details the simulation of the Lanchester model. 10 1371/journal pone 0146491 g004 Fig 4. Flowchart for the ABC framework. Example for a experiment using 1000 runs and tolerance τ = 0.01. Left s while the green panel details the simulation of the Lanchester model. Fig 4. Flowchart for the ABC framework. Example for a experiment using 1000 runs and tolerance τ = 0.01. Left side illustrates the rejection algorithm while the green panel details the simulation of the Lanchester model. doi:10.1371/journal.pone.0146491.g004 doi:10.1371/journal.pone.0146491.g004 10 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC being discussed [69]. This experiment avoids the debate by directly comparing the set of casu- alties for each battle and side. The distance between a simulation run and evidence is the abso- lute difference between simulated and historical casualties divided by historical casualties, thus normalising the weight of all battles regardless their total size. This comparison is performed identifying both in the evidence and simulation the Red army R as the side with lower casualty ratio in each battle. Uninformed prior beliefs were used for the two parameters (μ and σ). Model selection The fatigue model is decisively selected for all periods when using the lowest τ = 0.0005 (see Fig 5 left). The two original models (linear and squared) are not present in this set comprising the best 500 runs, while the logarithmic is only present for the XVIIth century. Larger tolerance levels increase the relevance of the linear and logarithmic models, while the squared model is never selected. The estimation of distances in Fig 6 shows that the plausibility of the models is not constant over the different periods. The four models followed the same trend, as their ranks remain con- stant over the different phases. In addition, all of them performed much worse for the battles of the third period (i.e. Napoleonic wars). Experiment Design The limits of their uniform distributions were defined as Uð0; 5Þ, based on Allen’s results. Each competing model was ran 1 million times for each period. Sensitivity to tolerance levels was accounted by storing posterior distributions for different thresholds (τ = 0.05, τ = 0.005 and τ = 0.0005). The model selection method is based on Bayes Factors. They quantify the relative likelihood of different competing models against the evidence expressed as an odds ratio [70]. This ratio was quantified with the common method of introducing a third parameter m as a model index variable [59]. It was used within a hierarchical model where m identified which of the four vari- ants of the Lanchester’s laws was used during the run. Bayes Factors are then computed as the posterior distribution of m within the tolerance level τ. Model Selection in Historical Research Using ABC Fig 6. Distance from fatigue model to evidence with τ = 0.0005. Absolute distances (Y axis) of the best 500 runs ordered by rank (X axis, being 1 the best one), model (colour) and historical period (left to right). doi:10.1371/journal.pone.0146491.g006 Fig 6. Distance from fatigue model to evidence with τ = 0.0005. Absolute distances (Y axis) of the best 500 runs ordered by rank (X axis, being 1 the best one), model (colour) and historical period (left to right). doi:10.1371/journal.pone.0146491.g006 The parameter μ exhibits a dynamic of gradual decrease over the three centuries. Three main blocks can be observed: the oldest period (1620–1701) has the largest mean value (2.4), while the following 150 years (second and third period) have smaller means (around 1.9) and the latest period has the lowest peak (1.6). The σ distribution is similar for all periods except for the oldest one. The combination of the two posterior distributions as seen in Fig 9 illustrates the interaction between μ and σ. The dispersion of the posterior distribution for the first period is much larger than the rest of the examined periods. In addition all results follow a distinctive pattern: the largest values of σ are only selected if the μ value is also large. Parameter estimation The posterior distribution for parameters μ and σ is now examined for the fatigue model at τ = 0.0005. Fig 7 and Fig 8 show that both parameters follow unimodal distributions for all periods. The complete set of posterior distributions can be observed in SI 1, where similar pat- terns are observed for the other models (see S1 Fig for parameter μ and S2 Fig for parameter σ). Fig 5. Model selection for different tolerance levels. Proportion of the models used in the best runs for the four historical periods and three τ values (corresponding to the selection of left: 500, centre: 5000 and right: 50000 best runs). doi:10 1371/journal pone 0146491 g005 Fig 5. Model selection for different tolerance levels. Proportion of the models used in the best runs for the four historical periods and three τ values (corresponding to the selection of left: 500, centre: 5000 and right: 50000 best runs). doi:10.1371/journal.pone.0146491.g005 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 11 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Discussion These results confirm that the original Lanchester’s laws (i.e. linear and squared) are a poor match to historical evidence. The outcome is similar to other studies, which highlighted the better match of the logarithmic model [51]. Beyond this replication of past results, the use of the ABC framework provides new insights to the discussion. The decisive advantage of the fatigue model shows that this formulation is better supported by historical evidence than the rest of the models. The extreme psychological and physical stress conditions in the battlefield caused a gradual decrease on the efficiency of the armies. The better fit of the fourth model would suggest that this process had an impact in the final outcome. The performance of the logarithmic model is similar to the fatigue model, even though it shows slightly lower match to evidence. The explanatory power of the two classical models is much lower, as they are consistently below the best runs for any tolerance level. The credibility of the models is not constant over the entire time span. The best matches are the oldest and more recent periods, while the third period (1793-1860) is revealed as more unpredictable. The period was dominated by the French Revolutionary Wars and the Napole- onic Wars, where traditional European tactics were transformed at a scale not previously seen. PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 12 / 18 Model Selection in Historical Research Using ABC Fig 7. Posterior distribution of μ. Results for the fatigue model and τ = 0.0005. doi:10.1371/journal.pone.0146491.g007 Fig 7. Posterior distribution of μ. Results for the fatigue model and τ = 0.0005. doi:10.1371/journal.pone.0146491.g007 doi:10.1371/journal.pone.0146491.g007 This outcome would suggest that the generalist approach undertaken by the Lanchester’s laws is not suited to study transition periods with higher rates of change. Posterior distributions for parameters μ and σ suggest a gradual decrease of the relevance of individual fighting value. In particular, P values calculated for XVIIth century battles are larger and more diverse than the rest of the dataset. This result suggests that the non-professional armies of this era produced a much wider set of results under similar conditions, as the fighting value of the soldiers was much relevant than their numbers. The gradual standardisation of tac- tics and training would give more relevance to the size because individual fighting value was equalised between all armies. PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Discussion The variability of fighting value P within the same period is basi- cally constant after XVIIth century. Mean values are similar for the second and third period, while showing a significant decrease after 1861. This would suggest that the evolution of war- fare, now dominated by mass-production, would give even more relevance to sheer numbers while differences between individuals would then become a minor factor. Beyond the examined scenario, the case study illustrates how Model-Based History could benefit from a Bayesian-inspired framework. The use of a meta-model to compute Bayes Fac- tors allows the researcher to compare hypotheses while generating credible posterior PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 13 / 18 Model Selection in Historical Research Using ABC Fig 8. Posterior distribution of σ. Results for the fatigue model and τ = 0.0005. doi:10.1371/journal.pone.0146491.g008 g Fig 8. Posterior distribution of σ. Results for the fatigue model and τ = 0.0005. doi:10.1371/journal.pone.0146491.g008 Fig 8. Posterior distribution of σ. Results for the fatigue model and τ = 0.0005. doi:10.1371/journal.pone.0146491.g008 doi:10.1371/journal.pone.0146491.g008 doi:10.1371/journal.pone.0146491.g008 distributions. It also shows how the original framework can be easily extended to test new hypotheses, as seen in the fatigue model. It is worth mentioning that Bayes Factors already take into account parsimony because complex models with larger number of parameters will gener- ate wider posterior distributions. As a result, models with more parameters will be more times below the tolerance threshold, thus promoting simpler models. distributions. It also shows how the original framework can be easily extended to test new hypotheses, as seen in the fatigue model. It is worth mentioning that Bayes Factors already take into account parsimony because complex models with larger number of parameters will gener- ate wider posterior distributions. As a result, models with more parameters will be more times below the tolerance threshold, thus promoting simpler models. Fig 9. Relation between μ and σ values with τ = 0.0005. Large σ values are only selected when μ value is also large. doi:10 1371/journal pone 0146491 g009 Fig 9. Relation between μ and σ values with τ = 0.0005. Large σ values are only selected when μ value is also large. Fig 9. Relation between μ and σ values with τ = 0.0005. Large σ values are only selected when μ value is also large. Discussion doi:10.1371/journal.pone.0146491.g009 doi:10.1371/journal.pone.0146491.g009 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 14 / 18 Model Selection in Historical Research Using ABC The study of different tolerance levels also provides a cautionary tale on the use of ABC. As its name indicates it approximates the posterior distributions, and the method needs additional parameters such as the tolerance level τ. It means that τ also needs to be explored, as any other parameter. Results of the case study are a good example of the need of this exploration, as Bayes Factors for τ = 0.05 are radically different than the other two values. Any study using ABC should acknowledge this issue and integrate this discussion in the experiment design. Computational models are becoming a relevant quantitative tool for historical research. This new approach allows historians to evaluate the plausibility of competing hypotheses beyond what has been discussed in natural language. It is clear that History presents a unique set of issues and challenges to formal modelling, often related to the uncertainty of the datasets collected by the researchers. In this context, the integration of model selection methods such as ABC with new datasets and computer models can provide solutions to some of the current debates of the discipline. Supporting Information S1 Fig. Complete parameter estimation for parameter μ. Parameter μ posterior distribu- tion for the four models and historical periods. Results obtained from the four initial experi- ments with τ = 0.0005. (TIFF) S2 Fig. Complete parameter estimation for parameter σ. Parameter σ posterior distribution for the four models and historical periods. Results obtained from the four initial experiments with τ = 0.0005. S1 File. Dataset and Source code. Dataset. Dataset is distributed under the terms of a Creative Commons Attribution-ShareAlike 4.0 International License. The models and the ABC rejec- tion algorithm were implemented in Python programming language. Source code of the model is licensed under a GNU General Public License. Last versions for both data and source code can be downloaded from https://github.com/xrubio/lanchester. (ZIP) Author Contributions Conceived and designed the experiments: XRC. Performed the experiments: XRC. Analyzed the data: XRC. Contributed reagents/materials/analysis tools: XRC. Wrote the paper: XRC. Acknowledgments We would like to thank Mark Madsen and two anonymous reviewers for their comments on previous versions of the manuscript. We would also like to thank Enrico R. Crema for his con- tributions in many discussions regarding the method, Victor Pascual for his suggestions on data visualization, Maria Yubero for comments on the text and Francesc Xavier HernÃ ndez Cardona on the evolution of warfare. References 1. Epstein JM. Why model? Journal of Artificial Societies and Social Simulation. 2008; 11(4):12. Available from: http://jasss.soc.surrey.ac.uk/11/4/12.html 1. Epstein JM. Why model? Journal of Artificial Societies and Social Simulation. 2008; 11(4):12. 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https://openalex.org/W2802804821	https://europepmc.org/articles/pmc5908164?pdf=render	English	null	What does the general public understand about prevention and treatment of dementia? A systematic review of population-based surveys	PloS one	2,018	cc-by	10,856	RESEARCH ARTICLE Monica Cations1,2, Gorjana Radisic1,2, Maria Crotty1,2, Kate E. Laver1,2 Monica Cations1,2, Gorjana Radisic1,2, Maria Crotty1,2, Kate E. Laver1,2 1 Department of Rehabilitation, Aged and Extended Care, Flinders University, Adelaide, South Australia, Australia, 2 Cognitive Decline Partnership Centre, The University of Sydney, Sydney, New South Wales, Australia a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * monica.cations@flinders.edu.au Data Availability Statement: All data used for this review are presented in Table 1, S1 to S4 Tables, and S1 Fig. Data Availability Statement: All data used for this review are presented in Table 1, S1 to S4 Tables, and S1 Fig. Methods j p Editor: Perla Werner, University of Haifa, ISRAEL Received: January 31, 2018 Accepted: April 5, 2018 Published: April 19, 2018 Copyright: © 2018 Cations 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. MEDLINE, EMBASE, PsycINFO, and grey literature were searched for English language entries published between 2012 and May 2017. Survey questions were grouped using an inductive approach and responses were pooled. Editor: Perla Werner, University of Haifa, ISRAEL Received: January 31, 2018 Accepted: April 5, 2018 Published: April 19, 2018 Objectives Citation: Cations M, Radisic G, Crotty M, Laver KE (2018) What does the general public understand about prevention and treatment of dementia? A systematic review of population-based surveys. PLoS ONE 13(4): e0196085. https://doi.org/ 10.1371/journal.pone.0196085 To synthesise results of population surveys assessing knowledge and attitudes about pre- vention and treatment of dementia. Results Copyright: © 2018 Cations 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. Thirty-four eligible studies and four grey literature items were identified. Surveys were con- ducted in Europe, the US, Eastern Asia, Israel, and Australia. Nearly half of respondents agreed that dementia is a normal and non-preventable part of ageing, but belief in the poten- tial for prevention may be improving over time. The role of cardiovascular risk factors was poorly understood overall. Less than half of respondents reported belief in the availability of a cure for dementia. The value of seeking treatment was highly endorsed. * monica.cations@flinders.edu.au What does the general public understand about prevention and treatment of dementia? A systematic review of population-based surveys Monica Cations1,2*, Gorjana Radisic1,2, Maria Crotty1,2, Kate E. Laver1,2 Introduction Paraxel, and Merck. Maria Crotty receives funding from Novartis for trials of hip fracture involving treatments for sarcopenia. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Approximately 47 million people worldwide are living with dementia (otherwise known as major neurocognitive disorder) and a new diagnosis is given every three seconds [1]. While research has not yet discovered a cure, there is accumulating evidence about the potential to prevent approximately one third of cases of dementia with management of risk factors such as poor educational attainment, hypertension, and depression [2]. In addition, both pharmaco- logical and non-pharmacological treatments exist that can delay functional and cognitive decline [3,4], help to manage behaviour change [5,6], and improve wellbeing [7,8]. The recently adopted World Health Organization (WHO) Global Action Plan on Dementia urges all countries to implement campaigns to raise awareness about dementia [9]. The plan includes a global target that all member countries will have “at least one functioning public awareness campaign on dementia to foster a dementia-inclusive society by 2025” [9]. This focus reflects that population risk reduction and appropriate treatment for dementia rely on a contemporary understanding of these factors among the general public. Optimism about potential treatments can encourage early diagnosis, which allows for future planning and facil- itates access to peer support, known to protect against psychological distress [10]. Understand- ing the modifiable risk factors for dementia may encourage preventative health behaviours in early and mid-life, ultimately reducing late-life incidence (and associated costs). However, misconceptions about dementia have been present for many years, including that dementia is a normal part of ageing and that there is no value in pursuing treatment [11]. These miscon- ceptions have been noted to contribute to diagnostic delay as health professionals, people with symptoms and their families believe nothing can be done [12]. They also alleviate pressure on policy makers to devote funding to prevention and treatment services [13]. A systematic review of papers published to mid-2014 conducted by Cahill and colleagues [11] identified 40 studies of dementia literacy and reported only fair to moderate knowledge of dementia and a sparsity of evidence available in low- and middle-income countries. What does the general public understand about prevention and treatment of dementia? Conclusions Funding: Funding for this work was provided by the National Health and Medical Research Council Cognitive Decline Partnership Centre (CDPC1327) and the National Health and Medical Research Council National Institute for Dementia Research (1135667) to KEL. Results suggest that knowledge about the potential for dementia prevention and treatment remains poor but may be improving over time. Knowledge among those living in low- and middle-income countries are largely unknown, presenting challenges for the development of National action plans consistent with World Health Organization directives. Competing interests: Monica Cations is currently employed to assist with data collection for Alzheimer’s disease drug trials funded by Janssen, 1 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 Data sources and searches We searched MEDLINE, EMBASE and PsycINFO for English language studies published between 2012 and May 2017. We searched this timeframe only to report how closely contem- porary views resemble recent available evidence about dementia prevention and treatment. The search strategy is available in S2 Table. Reference lists of all included studies were hand- searched for additional records. We also searched grey literature via a general internet search, Open Grey Europe, the Grey Literature Report, Web of Science, and report publications from Alzheimer’s Disease International, national peak dementia organisations and the World Health Organisation. Methods The review protocol was registered on the PROSPERO database (CRD42017062286), and we report according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A checklist of PRISMA items is presented in S1 Table. Introduction Since that time, major milestones in research and policy have occurred including the publication of hall- mark reviews establishing the potential of dementia prevention [14], the proliferation of ‘dementia friendly community’ initiatives [15], and the establishment of dementia as a global health priority by WHO [9]. Public awareness campaigns have also become more prolific and have been delivered across a wider variety of platforms including social media [16]. However, many of these campaigns still focus on either the ‘catastrophic’ consequences of dementia or deliver overly simplistic or confusing messaging [17]. Whether such campaigns result in improved literacy about dementia prevention and treatment can inform future campaigns. This is particularly pertinent to the many low- and middle-income countries in the process of developing their first dementia action plans in response to the WHO directives. Previous reviews have not provided clear guidance about the key areas on which these campaigns should focus. The aim of this review was to build on the work conducted by Cahill and colleagues by searching for more recent studies examining the population’s knowledge and understanding of dementia, and using this data to identify key target areas for public health focus We deliber- ately included only studies published in the past five years to represent contemporary thinking and explored whether there have been improvements in literacy over time. We endeavoured to understand whether the general public understand dementia as a preventable and treatable condition consistent with currently available evidence. 2 / 18 What does the general public understand about prevention and treatment of dementia? PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 Eligibility criteria Studies were included if they: Studies were included if they: (a) Reported quantitative results of a survey (conducted via any method) of the general population; (b) Included at least one question regarding views, knowledge, beliefs, or attitudes about pre- vention and/or treatment of dementia. Outcomes included knowledge about the exis- tence of prevention or treatments strategies for dementia, as well as perceived efficacy of specific strategies, and; (c) Were published from 2012 onwards. (c) Were published from 2012 onwards. Conference abstracts were included only if they provided quantitative data that could be used in the analysis. Studies were excluded if they: (a) Surveyed a specific population, such as people with dementia, carers of people with dementia, particular health professionals, or University students; (b) Assessed attitudes to ageing in general or non-dementia conditions; (c) Assessed fear of dementia, willingness to be screened for dementia (e.g. genetic testing), or stigma about dementia, unless the question directly related to prevention or treatment of dementia (e.g. “I would be screened for dementia because there are there are treat- ments to slow progression of the disease”); (d) Reported results qualitatively only. Where a study reported mixed-methods results, only quantitative data was extracted and included; (e) Reported results of a subset of participants from another, larger included study, or; (e) Reported results of a subset of participants from another, larger included study, or; (f) Were published in a language other than English. (f) Were published in a language other than English. We requested raw data from authors where only synthesised results of a validated scale were reported (e.g. the Alzheimer’s Disease Knowledge Scale [18]). We requested raw data from authors where only synthesised results of a validated scale were reported (e.g. the Alzheimer’s Disease Knowledge Scale [18]). 3 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 What does the general public understand about prevention and treatment of dementia? Quality assessment Risk of bias was assessed at the study level. Two authors independently assessed the methodo- logical quality of included studies using the ‘Qualitative descriptive studies’ section of the Mixed Methods Appraisal Tool (MMAT) [19]. The tool asks four yes/no/unclear questions: (1) is the sampling strategy relevant to address the quantitative research question? This crite- rion was met where the sampling method was clearly stated and appropriate to recruit a repre- sentative sample; (2) Is the sample representative of the population under study? This criterion was met where the inclusion and exclusion criteria were clearly explained, and participant characteristics were described; (3) Are measurements appropriate? Measures were considered appropriate where the question asked is clearly defined and asked similarly to all participants, and; (4) Is there an acceptable response rate? This criterion was satisfied with a response rate of 60 per cent or above. Quality assessment data were not used in data synthesis (see below) but were considered during interpretation of results to identify potential bias. Study selection and data extraction Study titles, abstracts, and full-texts were independently accessed and reviewed for eligibility by two authors. A data extraction form was developed and piloted with five studies before being finalised and used with the remaining studies. Two authors extracted the data, and this was then checked by a third author. Extracted data included authors, year of survey and publi- cation, study aims, study design, sampling method, data collection method, participant details, and results. Data synthesis Two authors synthesised data by grouping similar responses into categories using an inductive approach modelled from McCullough et al [20]. Fixed responses (and the proportion of respondents endorsing them) were extracted and first grouped into six overarching categories that emerged from the data: general knowledge about dementia prevention, risk factors, pro- tective factors, general knowledge about dementia treatment, pharmacological treatment, and non-pharmacological treatment. Data within these six categories were then organised into more specific groups where similar concepts were referenced. Groupings were checked by a third author. The specific items and categories used for prevention and treatment studies are available in S3 and S4 Tables respectively. Where studies reported a percentage of the sample agreeing with a statement, these were pooled and a median, interquartile range (IQR) and range were calculated. Answers were reverse coded where necessary. We plotted trends over time (by survey year) and compared continent responses (Europe, North America, Asia, Aus- tralia) to the four most commonly reported statements: ‘Dementia is a normal part of ageing’; ‘Dementia is not preventable’, ‘There is a cure for dementia’, and ‘Effective treatments exist for dementia’. Results The search strategy identified 1364 unique records, and one additional citation was identified through hand-searching and grey literature searches. Following title and abstract screening, 1365 records were excluded due to non-relevance or not meeting inclusion criteria. One-hun- dred-and-one articles were accessed in full-text (S1 Fig). Of these, 33 met all inclusion criteria and were included in the review. Reasons for exclusion included that records did not discuss knowledge of treatment or prevention (n = 30), dementia (n = 3), or knowledge or attitudes (n = 6), were conference abstracts and provided insufficient detail (n = 7), included qualitative PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 4 / 18 What does the general public understand about prevention and treatment of dementia? data only (n = 3), included data reported by a specific group (e.g. carers of people with demen- tia, medical professionals; n = 12), used the same data as another included record (n = 5), or did not provide raw scores (n = 2). The grey literature search revealed a conference abstract by Mi-Ra et al [21], a conference presentation by Dos Santos et al [22], and a research report by Dementia Australia [23]. Thirty-one studies reported data suitable for pooling. In total, the included studies surveyed 36,519 participants. Results of quality assessment Results of the quality assessment are presented in S5 Table. Most studies were well-designed to gauge the views of the broad population of interest, and largely reported sample characteristics and results appropriately. All studies used standardised questions to gauge knowledge and atti- tudes and asked these consistently. However, only 17 reported their response rate and nine of these were below the ‘accepted’ threshold of 60 per cent. Characteristics of included articles Studies were published between 2012 and 2017 but reported on surveys conducted between 2008 and 2017 (Table 1). Study samples ranged from 50 to 3006 participants. Six of the eligible studies asked about the feasibility of treatments for dementia, seven asked about dementia prevention, and the remaining 19 included questions about both. Most surveys were conducted in Europe (n = 12), (UK, France, Germany, Poland, Spain, Portugal, Northern Ireland, Denmark, Italy). Eleven were conducted in the US, seven in Asia (China, South Korea, Singapore, Israel), and two in Australia. Africa, South and Central America, Canada, and Southern and Western Asia were not represented in any study. Six studies gathered views of specific ethnic groups within their country, including Black and Caribbean British communities [24], South Asian people living in the UK [25], Polish, Turkish, Danish, Pakistani people living in Denmark [26], or Chinese Ameri- cans [27–29]. Participants in most studies were randomly sampled via digit dialling or online access panels (n = 14) or convenience sampled via health and community services (n = 10). PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 What does the general public understand about prevention and treatment of dementia? Table 1. Included study characteristics. First author, year Treatment or prevention? Country Survey year N Sampling Recruitment source Assessment method Response rate Participant details Almeling, 2014 [30] Prevention USA 2011 2100 Random Access panel Self-administered internet survey 39% Gender 53% female; Age M = 50 (range n/r); Ethnicity 75% white, 10% African American Dementia Australia, 2017 [23] Both Australia 2017 1049 Random Access panel Self-administered internet survey n/r Gender n/r; Age n/r, Ethnicity n/r Ayalon, 2013 [31] Prevention USA 2010 1230 Random Wave of prospective cohort study Interviewer- administered face-to-face or telephone survey 100% Gender 57.7% female; Age White 24.5% >75 years, Latino 8.8% >75 years, Black 23.4% >75 years (M, range n/ r); Ethnicity 939 White, 120 Latino, 171 Black Berwald, 2016 [24] Treatment England 2014 50 Purposeful Community organisations Face-to-face interview n/r Gender 60% female; Age M = 33 years (18–65); Ethnicity Black African 56%, Black Caribbean 28%, Black British 14%, Indo-Caribbean 2% Blendon, 2012 [32] Treatment France, Germany, Poland, Spain, US 2011 2678 Random Random digit dialling Telephone interview n/r Gender n/r; Age n/r; Ethnicity n/r; Bowes, 2012 [33] Prevention UK 2009 402 Purposeful Employer organisations and online single interest discussion forums Self-administered internet survey Varies by item Gender 50.7% female; Age 50–65 years (M n/r); Ethnicity n/r Breining, 2014 [34] Both France 2008 2013 Random Random digit dialling Telephone interview 10.90% Gender 51.9% female; Age 11% 18–24 years, 15.4% 25–34 years, 29.9% 35–49 years, 22.7% 60–64 years, 21.1% 65 years or older (M n/r); Ethnicity n/r Diamond, 2014 [35] Both USA n/r 151 Convenience Seminar attendees Self-administered pen-and-paper survey 88% Gender 72% female; Age 40– 64 years (M n/r); Ethnicity Chinese Dos Santos, 2015 [22] Both Portugal 2011 1476 Random Random digit dialling Telephone interview n/r Gender 79.7% female; Age M = 38.83 (18–94); Ethnicity n/r Fowler, 2015 [36] Treatment USA n/r 400 Purposeful Health services Telephone interview n/r Gender 66.9% female (St. Vincent Health) 73.8% female (Community Health Network); Age >65 years (M n/r); Ethnicity 78.1% White, 20.9% African American (St. Knowledge about dementia prevention Twenty-six studies asked respondents about dementia prevention (Fig 1) [21–23,26,27,29– 31,33–35,37–39,41,42,45–50,52–54]. Nearly half of respondents agreed that dementia is a nor- mal part of ageing (from 13 studies; Median 48%, range 39–74%, n = 12,026) [23,27,28,35,38, 41–43,45,47,51–53] and that dementia is not preventable (from six studies; Median 48%, range 19–59%, n = 9869) [21,38,47,49,53,55]. Consistent with this, one Australian study additionally reported that only 42 per cent of participants believed they could act to reduce their own risk (42%, n = 1003) [49]. However, two studies (one in the US and one in the UK) reported high levels of agreement that genetic factors only partially account for the development of dementia (Median 83%, range 83–84%, n = 629) [37,50]. Belief that specific non-genetic factors increase the risk for dementia was highest with alcohol consumption (Median 71%, range 67–88%, n = 1736) [22,26], stroke (Median 62%, range 33–71%, n = 4137) [22,26–28,35,42,47], stress (Median 56%, range 38–83%, n = 4347) [22,31,56], and infection (Median 53%, range 14–58%, n = 1736) [22,26]. Fewer than half of respondents believed that risk for dementia was associated with high cholesterol (Median 47%, range 25–60%, n = 1014) [29,37,50], hypertension (Median 46%, range 25–60%, n = 1014) [29,37,50], drug consumption (Median 43%, n = 1476) [22], air pollution (Median 41%, range 26–56%, n = 4013) [34,53], emotional trauma (Median 31%, n = 1476) [22], or psychiatric or psychological illness (Median 26%, range 21–55%, n = 4063) [22,45]. Despite its well-estab- lished relationship with dementia, only six per cent of respondents agreed that low education PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 5 / 18 (Continued) What does the general public understand about prevention and treatment of dementia? Table 1. (Continued) First author, year Treatment or prevention? Vincent Health) 96.7% White, 2.5% African American (Community Health Network) Hailstone, 2017 [25] Treatment United Kingdom n/r 51 Purposeful Community organisations Self-administered internet or pen- and-paper survey n/r Gender 66.7% female; Age M = 50.6 years (18–85); Ethnicity South Asian Hudson, 2012 [37] Both United Kingdom n/r 312 Convenience Government institutions Self-administered pen-and-paper survey n/r Gender 63% female; Age M Women = 53.3 years, M Men = 42.3 years (17–82); Ethnicity n/r (Continued) Table 1. Included study characteristics. PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 6 / 18 Country Survey year N Sampling Recruitment source Assessment method Response rate Participant details Leon, 2015 [38] Both France 2008 and 2013b 2008: 2013; 2013: 2509 Random Random digit dialling Telephone interview n/r Gender 52.2% female (2008), 52.4% female (2013); Age 2008: 50.1% 35–64 years, 21.2% >65 years; 2013: 51.5% 35–65 years, 21.8% 65 years and over (M n/r); Ethnicity n/ r Luck, 2012 [39] Prevention Germany 2011 1002 Random Random digit dialling Telephone interview 50.9% Gender 50.3% female; Age M = 50.3 years (18–92); Ethnicity n/r Ludecke, 2016 [40] Both Germany 2012 1795 Random Access panel Self-administered pen-and-paper survey 78% Gender 52% female; Age 31.4% 20–39 years, 37.8% 40– 59 years, 30.8% 60–79 years (M n/r); Ethnicity n/r McParland, 2012 [41] Both Northern Ireland 2010 1204 Random Random digit dialling Telephone interview 58% Gender 52% female; Age 18– 75 years (M n/r); Ethnicity 98% white Mi-Ra, 2015 [21] Both South Korea 2014 926 n/r n/r Questionnaire n/r Gender n/r; Age n/r; Ethnicity n/r Nguyen, 2016 [42] Both United States n/r 102 Convenience Community health fairs Self-administered pen-and-paper survey n/r Gender 63.7% female; Age M = 50.56 years (range n/r); Ethnicity Vietnamese Nielsen, 2016 [26] Both Denmark 2013 260 Mixed convenience / random National registration system and snowball recruitment Telephone interview 73% Gender Danish 49% female, Polish 57% female, Turkish 35% female, Pakistani 39% female; Age >50 years, Danish M = 64.5, Polish M = 65.1, Turkish = 60.1, Pakistani = 58.3; Ethnicity Polish, Turkish, Danish, Pakistani Park, 2016 [43] Treatment United States n/r 626 Random Access panel Self-administered internet survey 16.7% Gender 47% female; Age M = 74.3 years (65–92); Ethnicity White, not Hispanic 95.8%, Hispanic 0.5%, Black 1.9%, Asian or Pacific Islander 0.5%, American Indian, Eskimo, and Aleut 0.3%, Others 1.0% Picco, 2016 [44] Treatment Singapore 2014– 2015 3006 Random National registration system Face-to-face interview 71% Gender 49.1% female; Age M = 40.9 years (18–65); Ethnicity Chinese 74.7% Riva, 2012 [45] Both Italy 2008– 2009 1111 Convenience Hospital waiting rooms Self-administered internet or pen- and-paper survey n/r Gender 63% female; Age 51% 18–40 years, 39% 41–64 years, 10% >65 years (M n/r); Ethnicity n/r Roberts, 2014 [46] Prevention USA 2010 1641 Random Wave of prospective cohort study Interviewer- administered face-to-face or telephone survey 89.2% Gender 53.6% female; Age M = 64.4 (range n/r); Ethnicity 8% Hispanic, 10.3% Black, 81.7% white Seo, 2015 [47] Both South Korea 2011 2189 Convenience Health services Self-administered pen-and-paper survey 78.2% Gender: 65.1% female; Age: 10–70+ years (M n/r); Ethnicity: n/r Shinan- Altman, 2017 [48] Prevention Israel n/r 236 Convenience n/r Face-to-face interview n/r Gender 49.4% female; Age M = 59 years (50–86); Ethnicity n/r (Continued) Table 1. Country Survey year N Sampling Recruitment source Assessment method Response rate Participant details Smith, 2014 [49] Prevention Australia 2012 1003 Random Random digit dialling Telephone interview 58% Gender 57.1% female; Age M = 47.6 (range n/r); Ethnicity n/r Sites, 2016 [50] Both USA 2013 317 Random Access panel Self-administered survey (method n/r) n/r Gender 49% female; Age Median 49 years (M n/r); Ethnicity 80% White-non- Latino) Sun, 2014 [29] Both USA n/r 385 Purposeful Community organisations Face-to-face interview n/r Gender 64.2% female; Age M = 72.4 years (range n/r); Ethnicity Chinese American Tan, 2012 [51] Prevention Singapore n/r 338 Convenience Hospital waiting rooms Self-administered pen-and-paper survey 91.4% Gender: 66.9% female; Age: 28.9% <65 years, 71.1% >65 years (M, range n/r); Ethnicity: 88.1% Chinese Woo, 2013 [28] Both United States n/r 288 Convenience n/r Self-administered pen-and-paper survey 88.0% Gender 67.7% female; Age 69.8% <65 years, 30.2% >65 years (M, range n/r); Ethnicity Chinese American Yang, 2015 [52] Both China 2014 140 Convenience Community organisations Self-administered pen-and-paper survey n/r Gender 59.3% female; Age M = 56.19 years (20–75); Ethnicity n/r Zeng, 2015 [53] Both China 2013 2000 Random Direct approach at public places Face-to-face interview n/r Gender 59.3% female; Age 35% 18–34 years, 53.8% 35–64 years, 11.2% >65 years (M, range n/r); Ethnicity n/r Zheng, 2016 [27] Both USA n/r 316 Convenience Seminar attendees Self-administered pen-and-paper survey 88.3 Gender 67.1% female; Age >55 years (M, range n/r); Ethnicity Chinese American n/r = Not reported; M = mean https://doi.org/10.1371/journal.pone.0196085.t001 What does the general public understand about prevention and treatment of dementia? PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 8 / 18 Table 1. (Continued) Table 1. (Continued) Table 1. (Continued) First author, year Treatment or prevention? Country Survey year N Sampling Recruitment source Assessment method Response rate Participant details Smith, 2014 [49] Prevention Australia 2012 1003 Random Random digit dialling Telephone interview 58% Gender 57.1% female; Age M = 47.6 (range n/r); Ethnicity n/r Sites, 2016 [50] Both USA 2013 317 Random Access panel Self-administered survey (method n/r) n/r Gender 49% female; Age Median 49 years (M n/r); Ethnicity 80% White-non- Latino) Sun, 2014 [29] Both USA n/r 385 Purposeful Community organisations Face-to-face interview n/r Gender 64.2% female; Age M = 72.4 years (range n/r); Ethnicity Chinese American Tan, 2012 [51] Prevention Singapore n/r 338 Convenience Hospital waiting rooms Self-administered pen-and-paper survey 91.4% Gender: 66.9% female; Age: 28.9% <65 years, 71.1% >65 years (M, range n/r); Ethnicity: 88.1% Chinese Woo, 2013 [28] Both United States n/r 288 Convenience n/r Self-administered pen-and-paper survey 88.0% Gender 67.7% female; Age 69.8% <65 years, 30.2% >65 years (M, range n/r); Ethnicity Chinese American Yang, 2015 [52] Both China 2014 140 Convenience Community organisations Self-administered pen-and-paper survey n/r Gender 59.3% female; Age M = 56.19 years (20–75); Ethnicity n/r Zeng, 2015 [53] Both China 2013 2000 Random Direct approach at public places Face-to-face interview n/r Gender 59.3% female; Age 35% 18–34 years, 53.8% 35–64 years, 11.2% >65 years (M, range n/r); Ethnicity n/r Zheng, 2016 [27] Both USA n/r 316 Convenience Seminar attendees Self-administered pen-and-paper survey 88.3 Gender 67.1% female; Age >55 years (M, range n/r); Ethnicity Chinese American n/r = Not reported; M = mean increased risk in the one study in which it was included (n = 1111) [45]. Dos Santos et al [22] reported that 25 per cent of respondents in Portugal believed that ‘use of medications’ could increase the risk for dementia, but did not specify to which medications this referred (n = 1476). Most respondents in six studies did not believe that there are medications available to pre- vent or reduce the risk of dementia (Median 37%, range 17–53%, n = 6370) [29–31,37,55,56]. On the other hand, 75 per cent of participants in two US studies [31,46] believed vitamins are available to prevent or reduce risk for dementia (n = 2571). Most respondents in eight studies agreed that risk for dementia was reduced with mental activity (Median 61%, range 34–95%, n = 9313) [31,34,37,40,49,50,55,56]. (Continued) Table 1. (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 7 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 What does the general public understand about prevention and treatment of dementia? increased risk in the one study in which it was included (n = 1111) [45]. Dos Santos et al [22] reported that 25 per cent of respondents in Portugal believed that ‘use of medications’ could increase the risk for dementia, but did not specify to which medications this referred (n = 1476). Most respondents in six studies did not believe that there are medications available to pre- vent or reduce the risk of dementia (Median 37%, range 17–53%, n = 6370) [29–31,37,55,56]. On the other hand, 75 per cent of participants in two US studies [31,46] believed vitamins are available to prevent or reduce risk for dementia (n = 2571). Most respondents in eight studies agreed that risk for dementia was reduced with mental activity (Median 61%, range 34–95%, n = 9313) [31,34,37,40,49,50,55,56]. However, when Bowes et al [33] specifically asked why respondents aged 50–65 years old participated in mental activities, very few reported ranked dementia risk reduction as their primary aim (1–8%). Belief in physical activity to reduce risk for dementia was moderate overall (Median = 41%, n = 11,966) but endorsement in individual studies ranged from 14 to 94 per cent [21,30,31,34,47,49,55,56]. Those with lower endorsement may be more representative as they tended to employ random digit dialling for recruitment [30,39] while those with higher endorsement were already involved in a prospective cohort study of ageing [31] or were recruited from health services [47]. All other non-pharmacologi- cal prevention strategies were poorly endorsed, including eating a healthy diet (Median 37%, range 9–89%, n = 10453) [26,30,31,34,49,55,56], not smoking (Median 21%, range 3–39%, n = 3016) [34,49], social activity (Median 13%, range 12–43%, n = 3481) [22,49,55], and Table 1. (Continued) First author, year Treatment or prevention? Synthesis of public knowledge and beliefs about prevention of dementia. https://doi.org/10.1371/journal.pone.0196085.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 Fig 1. Synthesis of public knowledge and beliefs about prevention of dementia. https://doi.org/10.1371/journal.pone.0196085.g001 moderate alcohol consumption (Median 5%, n = 1003) [49]. Three per cent of participants in Luck et al [39] agreed that ‘scientific research’ could reduce the risk for dementia but did not specify to what research this referred (n = 1002). Finally, Shinan-Altman et al [48] did not report raw scores and could not be pooled, but reported below average endorsement on a scale of one (strongly disagree) to five (strongly agree) that AD is attributable to risk factors (M = 2.47), psychological factors (M = 2.05), and immunity (M = 1.83). However, when Bowes et al [33] specifically asked why respondents aged 50–65 years old participated in mental activities, very few reported ranked dementia risk reduction as their primary aim (1–8%). Belief in physical activity to reduce risk for dementia was moderate overall (Median = 41%, n = 11,966) but endorsement in individual studies ranged from 14 to 94 per cent [21,30,31,34,47,49,55,56]. Those with lower endorsement may be more representative as they tended to employ random digit dialling for recruitment [30,39] while those with higher endorsement were already involved in a prospective cohort study of ageing [31] or were recruited from health services [47]. All other non-pharmacologi- cal prevention strategies were poorly endorsed, including eating a healthy diet (Median 37%, range 9–89%, n = 10453) [26,30,31,34,49,55,56], not smoking (Median 21%, range 3–39%, n = 3016) [34,49], social activity (Median 13%, range 12–43%, n = 3481) [22,49,55], and PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 8 / 18 What does the general public understand about prevention and treatment of dementia? Fig 1. Synthesis of public knowledge and beliefs about prevention of dementia. https://doi org/10 1371/journal pone 0196085 g001 Fig 1. Synthesis of public knowledge and beliefs about prevention of dementia. moderate alcohol consumption (Median 5%, n = 1003) [49]. Three per cent of particip Luck et al [39] agreed that ‘scientific research’ could reduce the risk for dementia but d specify to what research this referred (n = 1002). Finally, Shinan-Altman et al [48] did report raw scores and could not be pooled, but reported below average endorsement o of one (strongly disagree) to five (strongly agree) that AD is attributable to risk factors (M = 2.47), psychological factors (M = 2.05), and immunity (M = 1.83). Knowledge about dementia treatment Twenty-five studies included questions about the efficacy of potential treatments for d (Fig 2) [21–28,32,34–38,40–42,44,45,47,50,52,53,57,58]. The most common question a studies concerned the availability of a cure, to which 42 per cent agreed (range 6–69% 14,036) [21,26,29,32,34,37,41,44,47,52,53,57,58]. Two studies asked specifically about tions to cure dementia, but belief that these existed was very low (Median 17%, range n = 2421) [40,43]. Despite the general consensus that a cure was not available, there was a high level o ment that people should seek help for memory problems (Median 89%, range 26–95% n = 3794) [22,24,32,52]. This was accompanied by a generalised belief that ‘effective tr exist’ (Median 55%, range 28–88%, n = 7846) though participants were less convinced effective treatments exist to slow the progression of the disease (Median 42%, range 27 n = 5617) [22,32,34]. There was a strong belief that an effective treatment is available t Fig 1. Synthesis of public knowledge and beliefs about prevention of dementia. https://doi.org/10.1371/journal.pone.0196085.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 Fig 1. Synthesis of public knowledge and beliefs about prevention of dementia. moderate alcohol consump Luck et al [39] agreed that ‘ specify to what research thi report raw scores and could of one (strongly disagree) t (M = 2.47), psychological fa Knowledge about dem Twenty-five studies include (Fig 2) [21–28,32,34–38,40 studies concerned the avail 14,036) [21,26,29,32,34,37,4 tions to cure dementia, but n = 2421) [40,43]. Despite the general cons ment that people should se n = 3794) [22,24,32,52]. Th exist’ (Median 55%, range 2 effective treatments exist to n = 5617) [22,32,34]. There Fig 1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 Knowledge about dementia treatment Twenty-five studies included questions about the efficacy of potential treatments for dementia (Fig 2) [21–28,32,34–38,40–42,44,45,47,50,52,53,57,58]. The most common question across studies concerned the availability of a cure, to which 42 per cent agreed (range 6–69%, n = 14,036) [21,26,29,32,34,37,41,44,47,52,53,57,58]. Two studies asked specifically about medica- tions to cure dementia, but belief that these existed was very low (Median 17%, range 13–24%, n = 2421) [40,43]. Despite the general consensus that a cure was not available, there was a high level of agree- ment that people should seek help for memory problems (Median 89%, range 26–95%, n = 3794) [22,24,32,52]. This was accompanied by a generalised belief that ‘effective treatments exist’ (Median 55%, range 28–88%, n = 7846) though participants were less convinced that effective treatments exist to slow the progression of the disease (Median 42%, range 27–84%, n = 5617) [22,32,34]. There was a strong belief that an effective treatment is available to PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 9 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 What does the general public understand about prevention and treatment of dementia? Fig 2. Synthesis of public knowledge and beliefs about treatments for dementia. htt //d i /10 1371/j l 0196085 002 Fig 2. Synthesis of public knowledge and beliefs about treatments for dementia. htt //d i /10 1371/j l 0196085 002 improve the wellbeing of people with dementia in one study with two cohorts (Median = 82%, range 80–84%, n = 4522) [38]. Two studies reported that participants were mostly aware that pharmacological treatments are available for dementia (Median 77%, range 63–90%, n = 2587) [22,45], and five studies reported a general belief that these treatments are effective (Median = 77%, range = 58–84%, n = 7960) [21,41,44,45]. However, when Picco et al [44] asked about specific medications, belief in their efficacy ranged from 10% for antibiotics to 50% for antidepressants. Belief in the efficacy of alternate therapies was moderate overall, including 41% endorsing tonics and 48% endorsing supplements. No studies asked about the utility of acetylcholinesterase inhibitors. Seven studies included questions about the potential for non-pharmacological treatments to be helpful in the treatment of dementia [21,24,29,37,44,50,52]. Of these, social activity [44,52] and physical activity [44] were considered most beneficial overall and were both endorsed by a median of 82% of respondents (social activity range 72–91%, n = 3146; physical activity n = 3006), followed by relaxation activities (Median 76%, n = 3006) [44], in-person psychotherapy or counselling (Median 74%, range 70–90%, n = 4330) [44,50,58,59], cutting out alcohol (Median 62%, n = 3006) [44], and eating a healthy diet (Median 59%, range 34– 78%, n = 4330). Picco et al [44] additionally noted that most respondents endorsed seeking help from close family (Median 84%) and friends (Median 78%), and yoga or meditation (Median 68%). Religious or spiritual methods (Median 34%, range 22–45%, n = 3056) [24,44] and natural therapies (Median 18%, n = 50) [24] were endorsed by fewer than half of Fig 2. Synthesis of public knowledge and beliefs about treatments for dementia. https://doi.org/10.1371/journal.pone.0196085.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 10 / 18 improve the wellbeing of pe range 80–84%, n = 4522) [38 Two studies reported tha are available for dementia (M reported a general belief tha n = 7960) [21,41,44,45]. How belief in their efficacy range the efficacy of alternate ther and 48% endorsing supplem inhibitors. What does the general public understand about prevention and treatment of dementia? respondents included. The value of educating the person with dementia about their illness was recognised in two studies both conducted in Asia (Median 76%, range 65–89%, n = 5670) [21,44]. Two studies asked respondents about specific health professionals who could be helpful in the treatment of dementia [24,44]. Both reported a high level of belief that a general practi- tioner can be helpful (Median 73%, range 73–74%, n = 3056). Picco et al [44] additionally reported a moderate to high level of endorsement for psychiatrists (Median 83%), psycholo- gists (Median 74%), and social workers (Median 66%), but not for traditional Chinese medi- cine practitioners (Median 29%), among the general public in Singapore. Hailstone et al [25] reported results on a scale from 1 (strongly disagree) to 7 (strongly agree) and so could not be pooled, but participants overall agreed that they would want to see their doctor if they had memory problems, that seeking help from their doctor would be beneficial, valuable, and good, and that their doctor would be able to provide treatments to help with memory problems. https://doi.org/10.1371/journal.pone.0196085.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 Seven studies included q to be helpful in the treatmen [44,52] and physical activity endorsed by a median of 82 activity n = 3006), followed psychotherapy or counsellin out alcohol (Median 62%, n 78%, n = 4330). Picco et al [ help from close family (Med (Median 68%). Religious or and natural therapies (Medi Fig 2. Synthesis of public knowledge and beliefs about treatments for dementia. https://doi.org/10.1371/journal.pone.0196085.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 Fig 2. Synthesis of public knowledge and beliefs about treatments for dementia. improve the wellbeing of people with dementia in one study with two cohorts (Median = 82%, range 80–84%, n = 4522) [38]. Two studies reported that participants were mostly aware that pharmacological treatments are available for dementia (Median 77%, range 63–90%, n = 2587) [22,45], and five studies reported a general belief that these treatments are effective (Median = 77%, range = 58–84%, n = 7960) [21,41,44,45]. However, when Picco et al [44] asked about specific medications, belief in their efficacy ranged from 10% for antibiotics to 50% for antidepressants. Belief in the efficacy of alternate therapies was moderate overall, including 41% endorsing tonics and 48% endorsing supplements. No studies asked about the utility of acetylcholinesterase inhibitors. Seven studies included questions about the potential for non-pharmacological treatments to be helpful in the treatment of dementia [21,24,29,37,44,50,52]. Of these, social activity [44,52] and physical activity [44] were considered most beneficial overall and were both endorsed by a median of 82% of respondents (social activity range 72–91%, n = 3146; physical activity n = 3006), followed by relaxation activities (Median 76%, n = 3006) [44], in-person psychotherapy or counselling (Median 74%, range 70–90%, n = 4330) [44,50,58,59], cutting out alcohol (Median 62%, n = 3006) [44], and eating a healthy diet (Median 59%, range 34– 78%, n = 4330). Picco et al [44] additionally noted that most respondents endorsed seeking help from close family (Median 84%) and friends (Median 78%), and yoga or meditation (Median 68%). Religious or spiritual methods (Median 34%, range 22–45%, n = 3056) [24,44] and natural therapies (Median 18%, n = 50) [24] were endorsed by fewer than half of PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 10 / 18 Trends by time, location Survey administration year (where reported) was plotted against endorsement of the four most commonly included statements across studies (Fig 3). A downward trend was noted in belief that there is a cure for dementia, while belief that effective treatments exist appears to have increased over time. The understanding that dementia is a preventable disease also appears to be increasing. However, belief that dementia is a normal part of ageing has remained relatively steady over the eight-year period. Pooled responses to these same questions were stratified by continent (Table 2). No striking patterns emerged, and continents were relatively homogenous in their knowledge. European and American respondents were more likely than Asians and Australians to believe dementia Fig 3. Trends by time. https://doi.org/10.1371/journal.pone.0196085.g003 371/journal.pone.0196085 April 19, 2018 11 / 18 Fig 3. Trends by time. PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 11 / 18 Discussion The aim of this review was to compare the general public’s understanding of dementia as a preventable and treatable condition to contemporary scientific evidence. Thirty-three eligible surveys of the general population were identified, conducted predominantly in Europe and the US and occasionally in Eastern Asia and Australia. Results suggest that knowledge about the potential for dementia risk reduction and treatment of symptoms remains poor but may be improving over time. Knowledge and attitudes of those living in low- and middle-income countries are largely unknown. is a normal part of ageing. Six studies gathered views of specific ethnic groups within high- income countries, but results were unremarkable relative to other studies. is a normal part of ageing. Six studies gathered views of specific ethnic groups within high- income countries, but results were unremarkable relative to other studies. PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 What does the general public understand about prevention and treatment of dementia? Table 2. Trends by continent. Pooled median (range) Europe US Asia Australia Dementia is a normal part of ageing 53% (28–74) 53% (14–72) 43% (16–66) 39% (n/a) n = 6837 n = 1483 n = 4667 n = 1049 Dementia is not preventable 53% (45–54) n/a 28% (19–50) 59% (n/a) n = 5524 n = 5115 n = 1003 There is a cure for dementia 56% (6–69) 38% (16–64) 38% (13–62) n/a n = 5828 n = 1100 n = 8261 Effective treatments exist for dementia 40% (27–88) 59% (46–81) n/a n/a n = 10100 n = 1346 n/a = Not available https://doi org/10 1371/journal pone 0196085 t002 What does the general public understand about prevention and treatment of dementia? behaviours more generally (as has been demonstrated in the past [63]) was not explored and is an important avenue for future research There were large geographic and cultural gaps in the available literature. Low-income coun- tries were not represented, and populous countries with rapidly ageing populations were nota- bly missing (e.g. Japan, India). Given the socio-economic homogeneity of countries where data is available, it is not surprising that no major trends emerged in comparisons by conti- nent. The lack of data in low- and middle-income countries was also noted by Cahill and col- leagues in their earlier review [11]. That little progress has been made to fill this gap in the subsequent years is troubling particularly because most people with dementia live in low- and middle-income regions [1] and because structural barriers to awareness and help-seeking are more common in those regions [13]. Gathering a baseline understanding of public knowledge and attitudes in low- and middle-income regions is essential for development of targeted pub- lic awareness campaigns. Strengths and limitations of review and included studies This review benefited from a robust search strategy that was complemented by a thorough examination of the grey literature. It is the first to inductively pool similar responses and pres- ent findings visually for ease of interpretation and analysis of trends over time. Nonetheless, the results should be interpreted in the context of important limitations. First, studies pub- lished in languages other English were excluded and this may have precluded the representa- tion of many regions and cultural groups. This is particularly important given how little is known about knowledge and attitudes in non-English speaking regions, and the structural bar- riers to awareness raising that exist in these regions. Second, nearly half of the included studies did not report their response rate. Of those that did (n = 17), only eight reported a rate above 60 per cent. Non-response bias is possible and the reported views may overestimate general population knowledge. On the other hand, publication bias and selective reporting within studies is possible if authors choose to publish findings only where knowledge is ‘remarkably’ low. This is less likely given the variety in knowledge levels reported by the included studies, but some bias may still exist. Third, synthesising the data via quantitative meta-analysis was precluded by heterogeneity of the questions posed. Pooling ‘like’ themes was considered more appropriate in this case. Finally, an analysis of themes from qualitative studies in subsequent reviews will add depth to our understanding of public awareness, including the socio-political factors that allow misconceptions to persist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 Main findings Twenty-five of the studies included in this review were published since a narrative review of public knowledge and attitudes about dementia was published in 2014 [11]. Results of that review were strikingly similar to those reported here, including the common misconception that dementia is a normal part of ageing and is not preventable. As authors of the earlier review point out, these misconceptions have been documented for decades even among health profes- sionals who diagnose and treat dementia [60]. There were some positive signs in terms of improvements in awareness over time, particularly among ethnic minority groups living in high-income countries. Cognitive leisure activities in particular appear to be well understood as good candidates for dementia prevention or delay, consistent with some evidence that they can delay the onset of dementia [2]. On the other hand, the importance of formal educational attainment and management of cardiovascular were acknowledged by fewer than half of respondents who were asked about them. Despite the generalised understanding that dementia is usually a terminal condition with- out an available cure belief in the value of seeking treatment was high in almost all studies and suggests a positive shift in attitudes away from fatalistic beliefs that have acted as a barrier to help seeking in the past [61]. It was noted, however, that the perceived value in treatment lay mostly in its potential to support wellbeing rather than slow the progression of symptoms. This is contrary to evidence that both pharmacological and non-pharmacological methods can delay functional and cognitive decline [3,62]. The general public was positive overall about the value of practicing healthy behaviours after a diagnosis of dementia. Whether these strategies were believed to limit progression of disease or were simply viewed as valuable health PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 12 / 18 Implications Results of this study suggest several key areas of need in general public dementia literacy. The view that dementia is a normal part of ageing with few treatment options is a demonstrated barrier to both preventive health behaviours and to help-seeking and diagnosis in the event that symptoms emerge [12]. Stigmatisation occurs in the absence of accurate understanding, and contributes to social isolation and emotional distress for people with dementia and their carers [13]. While the proliferation of public awareness campaigns and dementia-friendly community initiatives in high-income countries appears to be having a positive impact, gaps in knowledge remain and present key target areas for future campaigns. First, a significant underestimation of the importance of non-genetic cognitive and cardiovascular risk factors is evident and is not helped by confusing messaging about what is and is not harmful [17]. Dementia is a complex syndrome for which the impact of risk factors can vary depending on the type of dementia, timing of exposure, confluence of risks, and pre-existing genetic susceptibilities [6]. The PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 13 / 18 What does the general public understand about prevention and treatment of dementia? quality of evidence regarding individual risks varies, and conflicting findings regularly emerge. In this context, researchers have a responsibility to disseminate their findings to the public in ways that do not promote misunderstandings, including working in partnership with the mainstream media. In the meantime, simple messaging about the net benefit of healthy behav- iours over the life course may be most beneficial. Second, there appears to be a misconception that available treatments are useful only for maintaining the wellbeing of people with dementia and are not able to slow progression of dis- ease. This may be related to the focus in public awareness campaigns on the serious conse- quences of dementia. While ‘fear appeal’ campaigns can promote investments in research and care [64], they also (by nature) promote fear of the illness. This messaging must be comple- mented by evidence-based campaigns emphasising the value of seeking a diagnosis and treatment. Third, the public tended to endorse poorly supported risk reduction strategies (like vita- mins) over more powerful but also more effortful strategies (like exercise). Promotion of realis- tic risk reduction messages necessitates debunking less relevant strategies to reduce noise. Implications Finally, the misconception that dementia is a normal part of ageing is persisting despite decades of public health efforts contrasting this message. New strategies are clearly required to address this. Policy-makers in low and middle-income countries, especially those developing their first dementia action plans, will benefit from a better understanding of the barriers to knowledge in their countries and cost-effective methods to overcome these. The potential reach of public awareness campaigns is ever-increasing as technology makes information more accessible. At the same time, the media landscape is crowded and public health agencies must compete for attention [65]. The WHO Global Action Plan [9] advocates for both national and local public health campaigns that are community- and culture-specific and developed in consultation with people living with dementia and their carers. Educating children and young people may have particular benefits, as this approach can foster intergenerational solidarity and prepare a future generation of informal carers [13]. The introduction of alternative terminology (neuro- cognitive disorders) in the most recent iteration of the Diagnostic and Statistical Manual for Mental Disorders [66] was intended to reduce the stigma associated with ‘dementia’ (a term meaning ‘mad’ or ‘insane’ in Latin). Increasing use of this terminology may help to correct long-held misconceptions about dementia and frame a new understanding of the condition among the general public. Most importantly, creative messaging and methods of delivery must be paired with a supportive environment that enables the public to take the action advocated in the campaign [65]. Without sustainable infrastructure to facilitate risk reduction and help seeking, the benefits of improving public awareness will be stifled. S3 Table. Prevention results. M = Mean Reverse scores used for pooling. (DOCX) Acknowledgments The authors wish to acknowledge the assistance of Ms Natalie May and Ms Kayla Ninnis with data extraction. Author Contributions Conceptualization: Monica Cations, Maria Crotty, Kate E. Laver. Data curation: Monica Cations, Gorjana Radisic, Kate E. Laver. Methodology: Monica Cations, Gorjana Radisic, Kate E. Laver. Project administration: Monica Cations. Project administration: Monica Cations. Supervision: Monica Cations, Kate E. Laver. Writing – original draft: Monica Cations, Kate E. Laver. Writing – review & editing: Monica Cations, Gorjana Radisic, Maria Crotty, Kate E. Laver. Writing – review & editing: Monica Cations, Gorjana Radisic, Maria Crotty, Kate E. Laver. Supporting information Supporting information S1 Fig. PRISMA flowchart describing the process of study selection. (DOCX) S1 Table. PRISMA checklist. (DOCX) S2 Table. OVID search strategy (Medline, EMBASE, PsycINFO). (DOCX) S3 Table. Prevention results. M = Mean Reverse scores used for pooling. (DOCX) S1 Fig. PRISMA flowchart describing the process of study selection. (DOCX) S1 Fig. PRISMA flowchart describing the process of study selection. (DOCX) S1 Table. PRISMA checklist. (DOCX) S1 Table. PRISMA checklist. (DOCX) S2 Table. OVID search strategy (Medline, EMBASE, PsycINFO). (DOCX) S3 Table. Prevention results. M = Mean Reverse scores used for pooling. (DOCX) S3 Table. Prevention results. M = Mean Reverse scores used for pooling. (DOCX) 14 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 What does the general public understand about prevention and treatment of dementia? S4 Table. Treatment results. M = Mean Reverse scores used for pooling a59% ‘disagree’ or ‘strongly disagree’, 20% ‘don’t know’ b91% disagree, 2% uncertain. (DOCX) PLOS ONE \| https://doi.org/10.1371/journal.pone.0196085 April 19, 2018 References 1. Prince MJ (2015) World Alzheimer Report 2015: the global impact of dementia: an analysis of preva- lence, incidence, cost and trends: Alzheimer’s Disease International. 2. 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https://openalex.org/W4309982799	https://zenodo.org/record/7365150/files/2Mejorasemisionesdegases%20Arias%20et%20al.pdf	es		Mejoras en las emisiones de gases y en la eficiencia de una caldera con la introducción de la tecnología magnética	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	5,060	Universidad Centroccidental “Lisandro Alvarado” Decanato de Agronomía Programa de Ingeniería Agroindustrial Revista Científica Agroindustria, Sociedad y Ambiente (A.S.A) ISSN: 2343-6115 Deposito Legal No ppl201302LA4406 Mejoras en las emisiones de gases y en la eficiencia de una caldera con la introducción de la tecnología magnética Arias Gilart Ramón1*, Fong Casas Fredy2, Rojas Lores Yuliani2, Conde García Rebeca1, Melek Campos Sofia1 1 National Center for Applied Electromagnetism, Universidad de Oriente, Santiago de Cuba, Cuba 2 Santiago de Cuba Rum Factory, Santiago de Cuba, Cuba https://orcid.org/0000-0003-2050-9712 ramonariasgliart@gmail.com https://orcid.org/0000-0002-3821-5117 fredyfong@cubaron.com https://orcid.org/0000-0002-9216-1762 yuliani@cubaronstgo.co.cu https://orcid.org/0000-0003-2810-4525 rebeca@uo.edu.cu https://orcid.org/0000-0003-2741-8821 melek@uo.edu.cu ASA/Artículo doi: http://doi.org/10.5281/zenodo.7365150 Recibido: 29-01-2022 Aceptado: 26-09-2022 RESUMEN La aplicación de tratamientos magnéticos a los combustibles es una de las alternativas más utilizadas en la actualidad con el objetivo de reducir las emisiones de gases contaminantes y aumentar el ahorro de combustible. En este trabajo se evaluó el efecto del tratamiento magnético del fuel oil utilizado en una caldera de producción de vapor. Se utilizaron dispositivos con una inducción magnética promedio de 0,36 T. Con la introducción de la tecnología magnética en una caldera para el tratamiento de fuel oil, se redujeron las emisiones de CO2 en aproximadamente un 10,18 %, lo que contribuye a reducir la carga contaminante de estos equipos. Las emisiones de CO de este equipo disminuyeron aproximadamente un 60,07 %, por lo que se obtiene una combustión más completa. El tratamiento magnético utilizado provocó una disminución del 13,84 % en la temperatura de los gases de escape, por lo que se reducen las pérdidas de calor latente en los gases de escape. En las condiciones experimentales, la tecnología magnética permite aumentar la eficiencia del proceso de combustión en un 1,6 %, lo que podría significar un mayor aprovechamiento de energía en el sistema y un potencial ahorro de combustible. Palabras Clave: Tratamiento magnético, tratamiento de fuel oil, emisiones de calderas. 23 Esta obra está bajo una licencia de creative commons reconocimiento-No comercial 4.0 internacional. Permite copiar, distribuir y comunicar públicamente la obra con fines no comerciales. A cambio, se debe reconocer y citar al autor original. (CC BY-NC-SA 4.0) Mejoras en las emisiones de gases y en la eficiencia de una caldera con la introducción de la tecnología magnética Arias Gilart Ramón, Fong Casas Fredy, Rojas Lores Yuliani, Conde García Rebeca y Melek Campos Sofia Improvements in exhaust gas emissions and boiler efficiency with the introduction of magnetic technology ABSTRAC The application of magnetic treatment to fuels is one of the most widely used alternatives today with the aim of reducing polluting gas emissions and increasing fuel savings. In this work, the effect of the magnetic treatment of fuel oil used in a steam production boiler was evaluated. Devices with an average magnetic induction of 0.36 T were used. With the introduction of magnetic technology in a boiler for the treatment of fuel oil, CO2 emissions were reduced by approximately 10.18 %, which contributes to reducing the contaminant load of these equipments. The CO emissions of this equipment decreased by approximately 60.07 %, so a more complete combustion is obtained. The magnetic treatment used caused a decrease of 13.84 % in the temperature of the exhaust gases, so the latent heat losses in the exhaust gases are reduced. Under the experimental conditions, the magnetic technology allows to increase the efficiency of the combustion process by 1.6 %, which could mean a greater use of energy in the system and potential fuel savings. Keywords: Magnetic treatment, fuel oil treatment, boiler emissions. Agroindustria, Sociedad y Ambiente/ ISSN: 2343-6115/ Vol. 19 N° 2/ julio-diciembre 2022/Barquisimeto, Venezuela/ Universidad Centroccidental “Lisandro Alvarado” pp. 23-36. 24 Mejoras en las emisiones de gases y en la eficiencia de una caldera con la introducción de la tecnología magnética Arias Gilart Ramón, Fong Casas Fredy, Rojas Lores Yuliani, Conde García Rebeca y Melek Campos Sofia or steam generators used in the production of heat and steam are some of the main causes of INTRODUCTION pollution associated with gas emissions from F ossil fuels account for 80% of the combustion. These equipments fundamentally world's use fuel oil or diesel as fuel and are used in energy supply; however, detectable oil fields are expected to most of the world's industries. decrease over the years (Mutezo & Mulopo, 2021). World oil production could begin to decline rapidly as early as 2022. In addition, approximately 65% of the world's oil reserves are in Middle Eastern countries, and this is expected to accelerate the imbalance between supply and demand. non-producing countries, since they depend heavily on oil imports (Demirbas, 2007). The consumption of alternative energy sources globally is increasing sharply due to depletion of oil resources and serious environmental pollution caused by exhaust gases in fossil energy use (Lee et al. 2021). Another of the reasons that multiplies the use of renewable energy sources is that strict rules are applied that regulate and control the emissions of this type of polluting gases; fundamentally in European countries, but with In recent decades, one of the technologies proposed to reduce the consumption of fossil fuels and the reduction of gas emissions resulting from combustion is the application of magnetic treatment to fuels. Current studies suggest that magnetic technology has a positive effect on combustion processes because it modifies some of the physical and chemical properties of fuels (Arias et al. 2018b; Arias et al. 2018c; Dinza et al. 2020; Gilart et al. 2020; Niaki et al. 2020; Sidheshware et al. 2020). Most of the studies on this subject have been carried out on internal combustion engines, in which considerable reductions in polluting gas emissions and a reduction in fuel consumption are obtained (Arias et al. 2018a; Chandrasekaran et al. 2020; Khajure et al.; 2014; Notti & Sala, 2014; Tipole et al. 2017). a great tendency to generalize worldwide. In addition to renewable energy sources, several In boilers or steam generators, this technology alternatives or innovations are currently being has been used with the aim of reducing scale in investigated that allow the reduction of the pipes that transport water (de la Cruz & polluting the García, 2016; Esmaeilnezhad et al. 2017; combustion of fossil hydrocarbons. The boilers Vidaurre, 2015). Salih and Ahmed (2016) gas emissions caused by Agroindustria, Sociedad y Ambiente/ ISSN: 2343-6115/ Vol. 19 N° 2/ julio-diciembre 2022/Barquisimeto, Venezuela/ Universidad Centroccidental “Lisandro Alvarado” pp. 23-36. 25 Improvements in exhaust gas emissions and boiler efficiency with the introduction of magnetic technology explored the effect of magnetized fuel on the Arias Gilart Ramón, Fong Casas Fredy, Rojas Lores Yuliani, Conde García Rebeca y Melek Campos Sofia Magnetic devices combustion performance of a diesel fuel fired boiler. The analysis was made from fuel consumption and exhaust emissions. The magnetic field was placed in the diesel supply line to the burner. In this study, gas emissions and fuel consumption decreased after the application of the magnetic field (Salih & Ahmed, 2016). However, no research reports have been found that evaluate the influence or effect of magnetically treated fuel oil on gas emissions from this type of equipment. That is why the objective of this work is to evaluate the effect of applying magnetic technology to fueloil fuel on gas emissions from a boiler or steam To produce the static magnetic field, magnetic conditioners made from permanent Neodymium-Iron-Boron magnets were used (Figure 1). The average magnetic induction of these conditioners is 0.36 T, with a deviation of 0.005 T. A dipole configuration was used to ensure even distribution of field induction lines throughout the fuel flow area. The magnetic devices were in the pipe that leads the fuel oil to the burner at the pump outlet and as close as possible to the fuel atomization. The selection of the material of the devices and of the magnetic induction was carried out considering the most relevant results described in the generator. consulted literature (Kartik et al. 2019; Kushal & Basavaraj, 2015; Salih & Ahmed, 2016). MATERIALS AND METHODS This research includes two measurement stages, Higher inductions are very difficult and expensive to obtain using permanent magnets. the determination of gas emissions before and after the installation of magnetic conditioners for fuel treatment in the boiler. The subsequent analysis of the data allows elucidating the effect that this type of technology produces on the combustion processes and on the levels of pollution that they generate. The equipment and Figure 1. Novamag magnetic conditioner procedures used are described below. Agroindustria, Sociedad y Ambiente/ ISSN: 2343-6115/ Vol. 19 N° 2/ julio-diciembre 2022/Barquisimeto, Venezuela/ Universidad Centroccidental “Lisandro Alvarado” pp. 23-36. 26 Improvements in exhaust gas emissions and boiler efficiency with the introduction of magnetic technology Steam generator Arias Gilart Ramón, Fong Casas Fredy, Rojas Lores Yuliani, Conde García Rebeca y Melek Campos Sofia Table 1. Characteristics of the Boiler used in the test The boiler or steam generator used in the study carried out (Figure 2) is located in the Base Parameter Boiler Brand LEON Business Unit (UEB) Distillery, belonging to the Santiago de Cuba Rum Factory of the CubaRon.SA Corporation. Its characteristics Type Firetube Model CFDC350 Country of origin Mexico are shown in Table 1. Nominal Production (t) 5 Nominal Pressure (Kg/cm2) 8 Fuel type Fuel oil Gas measurements A Testo 350 Gas Analyzer was used to measure the concentration of the gases produced by combustion CO, CO2, NOX and the temperature of the gases. This equipment is a professional instrument for gas analysis. It has been Figure 2. Image of the boiler used designed for the following applications: • Service and adjustment of industrial incinerators • Control of emissions and verification of compliance with regulatory emission values. • Service and commissioning of burners and industrial boilers. Agroindustria, Sociedad y Ambiente/ ISSN: 2343-6115/ Vol. 19 N° 2/ julio-diciembre 2022/Barquisimeto, Venezuela/ Universidad Centroccidental “Lisandro Alvarado” pp. 23-36. 27 Improvements in exhaust gas emissions and boiler efficiency with the introduction of magnetic technology • Arias Gilart Ramón, Fong Casas Fredy, Rojas Lores Yuliani, Conde García Rebeca y Melek Campos Sofia Measurements in gas turbines, boilers 𝑞𝐴 = 𝐾 𝐹𝑇−𝐴𝑇 𝐶𝑂2 ……………………(2) and industrial stationary engines. 𝐶𝑂 𝑞𝐼 = 60 𝐶𝑂+𝐶𝑂 ……………..….. (3) The measurement ranges and the resolution of 2 the equipment are shown in Table 2. Equation 2 is used to calculate the sensible heat In addition to these variables, the Combustion losses in the exhaust gases, expressed in % Performance (𝑅𝐸𝑁) was also determined, using relative to the Lower Caloric Value of the fuel, the parameters provided by the Testo 350 Gas 𝐹𝑇 and 𝐴𝑇 being the exhaust gas temperature Analyzer and the atmospheric temperature, respectively. and the following equations 𝐾 is the specific factor of the fuel used. (González-González et al. 2014): Equation 3 calculates the unburned losses (𝑞𝐼) 𝑅𝐸𝑁 = 100 − 𝑞𝐴 − 𝑞𝐼 ……….. (1) expressed in % with respect to the lower Where REN is the efficiency of the combustion calorific value. CO and CO2 are the contents of process, 𝑞𝐴 and 𝑞𝐼 are the sensible heat losses these elements in the exhaust gases, expressed in the chimney and unburned, respectively. in ppm and volume percentage, respectively. These losses are calculated using the following mathematical expressions: Table 2. Characteristics of the gas analyzer used Parameter Measuring range Resolution O2 0-25 Vol. % 0,01 Vol. % CO 0-10000 ppm 1 ppm NO2 0-500 ppm 0,1 ppm CO2 0-25 Vol. % 0,01 Vol. % Gas Temperature -40 - 1200 0C 1% Agroindustria, Sociedad y Ambiente/ ISSN: 2343-6115/ Vol. 19 N° 2/ julio-diciembre 2022/Barquisimeto, Venezuela/ Universidad Centroccidental “Lisandro Alvarado” pp. 23-36. 28 Improvements in exhaust gas emissions and boiler efficiency with the introduction of magnetic technology Procedure Arias Gilart Ramón, Fong Casas Fredy, Rojas Lores Yuliani, Conde García Rebeca y Melek Campos Sofia emissions obtained in the two conditions in The analyzes were carried out in two which the boiler operated. moments, under normal operating conditions The CO2 gas is one of the most damaging of the boiler, working in the same work greenhouse gases (GHG), but it is also one of regime. Determinations made before the the most important combustion products. installation of magnetic technology are 13 for The 12 measurements made after the installation of 11 No Magnetic Treatment. the magnetic technology are represented by CO2 ( %) represented by the letters (STM) which stands 11.79 10.59 10 the letters (TM) which means with Magnetic 9 Treatment and were made three months after 8 7 the installation of the magnetic devices in the 6 pipes. STM TM In total, 20 concentration values of the analyzed gases, temperature and combustion Figure 3. Behavior of CO2 emissions from the boiler in the conditions analyzed performance were obtained for both moments (STM and TM). With these data, the statistical comparison of the average values was made using a probability value of p ≤ 0.5. In Figure 3 it can be seen that CO2 emissions decrease after applying the magnetic treatment. The boiler was operating under normal operating conditions in both conditions (STM and TM) the same amount of steam was produced. This means that the RESULTS AND DISCUSSION The gas emissions, temperature and combustion performance obtained with the fuel without magnetic treatment (STM) were used as control in all the analyzes carried out. CO2, CO and NOX emissions were considered due to their importance from the environmental and combustion efficiency point of view. Figure 3 shows the CO2 decrease in CO2 emissions obtained with the TM is due to a reduction in the fuel consumption of the boiler. Several authors associate these results with an improvement in the atomization process and a reduction in the size of the fuel droplets after receiving the magnetic treatment (Arias et al., 2018c; Gilart et al., 2020). These changes are also Agroindustria, Sociedad y Ambiente/ ISSN: 2343-6115/ Vol. 19 N° 2/ julio-diciembre 2022/Barquisimeto, Venezuela/ Universidad Centroccidental “Lisandro Alvarado” pp. 23-36. 29 Improvements in exhaust gas emissions and boiler efficiency with the introduction of magnetic technology Arias Gilart Ramón, Fong Casas Fredy, Rojas Lores Yuliani, Conde García Rebeca y Melek Campos Sofia explained due to the differences in the results are due to the dissimilar magnetic physical-chemical the treatment systems used and the structural hydrocarbons that have been magnetically differences between the fuels used in the treated. Some of these properties that are experimentation. In our research, the effects affected by magnetic fields and that influence of the application of magnetic technology in the combustion process are: surface tension, boilers or steam generators fed with fuel oil viscosity and rheological behavior (Arias et are reported for the first time. Under these al. 2018b; Arias et al. 2018c; Jiles et al. conditions, the combustion process was more 2015). efficient, so a greater use of the fuel's properties of From the environmental point of view, a chemical energy was achieved. decrease in the emissions of this gas indicates In the combustion process of the different that the pollutant load of this boiler is types of fuel, the chemical reaction is not considerably reduced, as it stops emitting complete, so gases such as CO are obtained. 10.18 % of this GHG. Similar results have The behavior of the CO emissions from the been obtained in several investigations but boiler with and without the application of using internal combustion engines in the magnetic technology is shown in Figure 4. experiments, such is the case of (Chen et al. 2017; Kushal & Basavaraj, 2015). These authors attribute the reduction in emissions to greater combustion efficiency, which is caused by the action of the magnetic field by weakening intermolecular interactions in hydrocarbons, improving the atomization and mixing process, as well as increasing their reactivity with oxygen from the air (Chen et With the magnetic treatment of fuel oil, the CO emissions generated by the boiler are reduced by 60.07 %, so a more complete combustion is obtained, and the quality of this process is increased. In addition, energy losses due to incomplete combustion are considerably eliminated, which can lead to considerable fuel savings in this process. al. 2017). Other authors have obtained contradictory results, that is, increases in CO2 emissions with magnetic technology, but using other magnetic configurations and different types of fuel (Al-Khaledy, 2008; Faris et al., 2012). The contradictions in the Agroindustria, Sociedad y Ambiente/ ISSN: 2343-6115/ Vol. 19 N° 2/ julio-diciembre 2022/Barquisimeto, Venezuela/ Universidad Centroccidental “Lisandro Alvarado” pp. 23-36. 30 Improvements in exhaust gas emissions and boiler efficiency with the introduction of magnetic technology Samadi & Heidarbeigi, 2020; Wibowo et al. 18 16 Arias Gilart Ramón, Fong Casas Fredy, Rojas Lores Yuliani, Conde García Rebeca y Melek Campos Sofia 14.65 2020). CO (ppm) 14 12 Other 10 processes are NOX (NO and NO2). The 8 5.85 6 produced in combustion chemical mechanism for the formation of these gases during combustion processes is 4 due to more than 100 elementary chemical 2 0 GHGs STM TM reactions. These reactions fundamentally depend on changes in temperature, the Figure 4. Behavior of CO emissions from the boiler in the conditions analyzed stoichiometric ratio and the nitrogen species With the application of magnetic fields in the 2017). The behavior of the emissions of this pipes through which the fuel flows, the gas in the boiler with and without the molecular associations in the form of a cage application of the magnetic treatment is present in the hydrocarbons are broken down shown in Figure 5. into smaller particles; which causes, among other things, the decrease in viscosity and surface tension (Patel et al. 2014). That is why smaller droplets are produced in the atomization process and the fuel-air mixture becomes more homogeneous. This process allows a greater penetration and reaction of oxygen, thus producing a more complete combustion inside the boiler. Similar results have been described by dissimilar researchers using internal combustion engines, generally present in the combustion chamber (Yamin, These gases are produced by excess air, by the high temperature of the gases in the postcombustion zone and by the chemical compounds present in the mixture after combustion (Patel et al. 2014). NOx emissions generally do not depend on the physical chemical characteristics of the fuel, which is why, as Figure 5 shows, the emissions of this gas with and without the application of magnetic technology are similar. diesel engines, and various types of magnetic treatments for experimentation (Dinza et al. 2020; Gilart et al. 2020; Niaki et al. 2020; Oommen & GN, 2020; Perdana et al. 2020; Agroindustria, Sociedad y Ambiente/ ISSN: 2343-6115/ Vol. 19 N° 2/ julio-diciembre 2022/Barquisimeto, Venezuela/ Universidad Centroccidental “Lisandro Alvarado” pp. 23-36. 31 Improvements in exhaust gas emissions and boiler efficiency with the introduction of magnetic technology In general, the TGE decreases around 13.84 275 270 267.25 267.75 265 NOX (ppm) Arias Gilart Ramón, Fong Casas Fredy, Rojas Lores Yuliani, Conde García Rebeca y Melek Campos Sofia % when the magnetically treated fuel is used. In boilers, the heat losses in the exhaust gases 260 represent approximately 50 to 80 % of the 255 250 total sensible heat loss of the boiler (Jin et al. 245 2019). 240 A reduction in the outlet temperature of the 235 230 STM TM gases is due to a considerable increase in the efficiency of the boiler and a reduction in fuel Figure 5. Behavior of NOx emissions from the boiler in the conditions analyzed consumption (Fialko et al. 2019). As exhaust Another important parameter in the evaluation reduced and thermal efficiency of steam of boilers is the temperature of the exhaust generation is improved (Habib et al. 2008). gases (TGE). Figure 6 shows the variation of This result indicates that the magnetic this parameter in the boiler with and without treatment caused variations in the chemical- the use of magnetic fuel treatment. physical properties of the fuel that enabled an gas temperature decreases, dry gas loss is increase in the efficiency of the combustion 280 273.79 process and a better use of the chemical TGE (0C) energy of the fuel. These changes are evident 260 in the reduction of CO emissions and in the 235.90 240 decrease in the outlet temperature of the gases from the boiler, obtained with the magnetically treated fuel. 220 Another of the parameters evaluated in this 200 STM TM investigation was Combustion performance, the behavior of this parameter is shown in Figure 6. Behavior of the outlet temperature of the boiler gases in the conditions analyzed. Figure 7. Agroindustria, Sociedad y Ambiente/ ISSN: 2343-6115/ Vol. 19 N° 2/ julio-diciembre 2022/Barquisimeto, Venezuela/ Universidad Centroccidental “Lisandro Alvarado” pp. 23-36. 32 Improvements in exhaust gas emissions and boiler efficiency with the introduction of magnetic technology these types of equipment contribute to the 92.5 91.95 92 REN (%) Arias Gilart Ramón, Fong Casas Fredy, Rojas Lores Yuliani, Conde García Rebeca y Melek Campos Sofia environment. 91.5 91 CONCLUSIONS 90.495 90.5 In the present study, the effect of 90 magnetically treated fuel oil on gas emissions 89.5 from a boiler or steam generator was STM TM Figure 7. Combustion efficiency of the boiler in the conditions analyzed evaluated for the first time. The analyzes were carried out using magnetic conditioners with an average magnetic induction of 0.36 T. The determinations made in the boiler allow us to As can be seen in the figure, with the conclude that: application of the magnetic treatment of the fuel, an increase in the efficiency of the • The magnetic treatment of the fuel oil used combustion process of 1.6 % was obtained. in boilers allows CO2 emissions to be reduced This result is related to the decreases obtained by approximately 10.18 %, which contributes in CO emissions and in the TGE. According to reducing the contaminant load that these to equations 1, 2 and 3, a decrease in CO and types TGE (which translates into a lower amount of environment. latent heat loss, both due to unburned fuel and exhaust gas temperature) would produce an increase in process performance. An increase in the efficiency of the combustion process by 1.6 % could mean greater use of energy in the of equipment contribute to the • The magnetic treatment of the fuel oil used in boilers allows CO emissions of these type of equipment to be reduced by approximately 60.07 %, for which a more complete combustion is obtained. system and potential fuel savings. All of the above indicates that the magnetic treatment of • The magnetic treatment used caused a the fuel can be considered as an alternative to decrease of 13.84 % in the temperature of the improve the combustion process and to exhaust gases, thus reducing the loss of latent increase its efficiency. In addition, it also heat in the exhaust gases. allows reducing the contaminant load that • The magnetic treatment, under the experimental conditions, makes it possible to Agroindustria, Sociedad y Ambiente/ ISSN: 2343-6115/ Vol. 19 N° 2/ julio-diciembre 2022/Barquisimeto, Venezuela/ Universidad Centroccidental “Lisandro Alvarado” pp. 23-36. 33 Improvements in exhaust gas emissions and boiler efficiency with the introduction of magnetic technology increase the efficiency of the combustion process by 1.6 %, which could mean greater use of energy in the system and potential fuel savings. ACKNOWLEDGEMENTS The authors would like to thank the management of Santiago de Cuba Rum Factory for the support received for this work and acknowledge thankfully the permission and help provided by the management of UEB Destileria where the experimental research was carried out. REFERENCES Al-Khaledy, A. A. J. (2008). 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https://openalex.org/W2143808608	https://virologyj.biomedcentral.com/counter/pdf/10.1186/1743-422X-7-254	English	null	Identification of NCAM that interacts with the PHE-CoV spike protein	Virology journal	2,010	cc-by	8,833	© 2010 Gao 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. Identification of NCAM that interacts with the PHE-CoV spike protein Wei Gao1,3†, Wenqi He1†, Kui Zhao1, Huijun Lu2, Wenzhi Ren3, Chongtao Du1, Keyan Chen1, Yungang Lan1, Deguang Song1, Feng Gao1 Abstract Background: The spike proteins of coronaviruses associate with cellular molecules to mediate infection of their target cells. The characterization of cellular proteins required for virus infection is essential for understanding viral life cycles and may provide cellular targets for antiviral therapies. Results: We identified Neural Cell Adhesion Molecule (NCAM) as a novel interacting partner of the PHE-CoV S protein. A T7 phage display cDNA library from N2a cells was constructed, and the library was screened with the soluble PHE-CoV S glycoproteins. We used a coimmunoprecipitation assay to show that only the NCAM was a binding partner of spike protein. We found that a soluble form of anti-NCAM antibody blocked association of the PHE-CoV with N2a cells. Furthermore, double-stranded siRNA targeted against NCAM inhibited PHE-CoV infection. Conclusions: A novel interaction was identified between NCAM and spike protein and this association is critical during PHE-CoV infection. * Correspondence: Songdg6301@126.com; gaofeng2010852010@yahoo.cn † Contributed equally 1College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, PR China Full list of author information is available at the end of the article Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 Animals Specific pathogen-free lines of piglets were purchased from the Centre for Medicine Animal Research (Jilin, China). Animal procurement and transportation into the HEPA-ventilated caging systems and performance of the experimental-challenge tests were performed in accor- dance with the guidelines for animal experimentation of Jilin University. Viruses and cell culture The 67N strain of PHE-CoV [13] was propagated and assayed by the plaque method in N2a cell culture, as described previously [14], and the titres were expressed as plaque-forming units (PFU). The cell lines were obtained from the American Type Culture Collection (ATCC), N2a (ATCC CCL-131) and 293T (ATCC CRL- 11268). These cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% cosmic calf serum (HyClone, Logan, UT) and 2 mM L-glutamine. All of the cell cultures were maintained at 37°C in 5% CO2. Panning In order to screen the clones that display the adhesion protein, the cDNA library from N2a cells was panned with the S protein. The 96-well plates were coated with 200 μl of the purified S protein (2 mg/ml) in coating buffer (50 mM NaHCO3 pH 9.6) overnight at 4°C. Non- specific sites were blocked with 5% bovine serum albu- min for 1 h at 37°C, and a 100 μl aliquot of the T7 phage display library (containing 6.4 × 1010 PFU/ml) was added to the wells and incubated for 2 h at 37°C. Following this, the wells were washed five times with PBST (phosphate-buffered saline containing 0.1% [v/v] Tween-20) to discard any unbound phages. The bound phages were eluted with 200 μl of T7 elution buffer (TBS in 1% sodium dodecyl sulfate [SDS]) and amplified by infecting Escherichia coli BLT5403 [15]. The ampli- fied phages were then subjected to another four rounds of panning as described above, to enrich the clones that were highly specific for the S protein of PHE-CoV. Sequence analysis After five rounds of panning, the final enriched specific clones were plated and single pure plaques were iso- lated. The cDNA inserts in these plaques were amplified by PCR using primers (T7 Select Up primer: 5’- GGAGCTGTCGTATTCCAGTC-3’; T7 Select Down primer: 5’-AACCCCTCAAGACCCGTTTA-3’) flanking the inserts. Each PCR consisted of 30 cycles of dena- turation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min. The reaction also included an initial denaturation step at 94°C for 5 min and a final extension step at 72°C for 7 min. After PCR Background superfamily are the receptors for the murine coronavirus mouse hepatitis virus [4]. The aminopeptidase N (APN) glycoproteins are the receptors for human coronavirus 229E (HCoV-229E), the transmissible gastroenteritis virus of swine, and the feline coronavirus of genetic group 1 [7-10]. Porcine hemagglutinating encephalomyelitis coronavirus (PHE-CoV) is a member of the Coronaviridae family, which causes porcine encephalomyelitis [1]. The mechanisms by which PHE-CoV infects cells and causes disease are not well characterized, nor are the factors known which determine the host and tissue specificity. The cellular receptor which is a crucial determinant of the tropism of several viruses, is not known in the case of PHE-CoV. PHE-CoV has a strong tropism for the central nervous system (CNS) [11]. The virus spreads via peripheral nerves to the CNS. PHE-CoV propagates mainly in the CNS, and nerve cells are a main target for virus replica- tion [12]. The molecular mechanisms and specific pro- teins involved in adhesion of PHE-CoV to host cells have not yet been elucidated. The spike glycoprotein of coronavirus is a major determinant of neurovirulence [2-5]. The coronavirus spike glycoprotein is responsible for viral attachment to the cellular receptor and fusion of the viral and cellular membranes, resulting in virus entry [4]. Several types of receptors for coronavirus have been previously identified [6]. The murine carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) and related murine glycopro- teins in the carcinoembryonic antigen family of the Ig In this work, we discovered that the PHE-CoV S pro- tein interacted with NCAM by screening a T7 phage cDNA library from Neuro-2a (N2a) cells. It is necessary to investigate these interactions with host-cell proteins, as discovering these interactions may be helpful in the identification of host proteins participating in important stages of the virus life cycle, such as virus entry, virion morphogenesis, and virion release. In addition, estab- lished protein contacts could serve as targets for anti- viral chemotherapy. * Correspondence: Songdg6301@126.com; gaofeng2010852010@yahoo.c † Contributed equally 1College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, PR China Full list of author information is available at the end of the article Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 Page 2 of 11 Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 μg mRNA, following the manufacturer’s instructions for the OrientExpress Random Primer cDNA Synthesis kit (Novagen, Madison, WI), with some modifications. Protein production The recombinant S protein of PHE-CoV was obtained using a Pichia pastoris yeast expression system. The S gene was subcloned by PCR. The forward primer for the S gene (5’-CGGAATTCGTGCCATCTATTAGCTCT- GAAGT-3’) and the reverse primer for the S gene (5’- TTGCGGCCGCAAGTATGCCCTGGCCTGTAATG-3’) introduced EcoRI and NotI sites, respectively. Following gel purification, using the QIAquick gel extraction kit (Qiagen, Valencia, CA), the purified PCR products were ligated into the EcoRI and NotI sites of the pPICZaA vector (Invitrogen, San Diego, CA), yielding pPICZaAS. GS115 yeast cells, transformed with pPICZaAS (Invitro- gen, San Diego, CA), were grown at 30°C in 100 ml liquid Buffered Methanol Complex Medium (BMMY) (Invitrogen, San Diego, CA) with 0.1 mg/ml Zeocine (Invitrogen, San Diego, CA). Production of the His6- tagged fusion S protein was induced with 1% methanol. After 5 d, the protein was collected from the superna- tant. The His6-tagged recombinant S protein was puri- fied by nickel affinity chromatography with the HisTrap HP column (Amersham Biosciences AB, Uppsala, Sweden). Background The first and second strand cDNA syntheses are simple reac- tions that are carried out sequentially in the presence of 5-methyl dCTP, which protects any internal EcoR I and Hind III restriction sites from digestion. The cDNA was treated with T4 DNA polymerase to blunt the ends, and EcoR I/Hind III Directional Linker was added at the end. Following, the cDNA fragments were digested with EcoRI and HindIII. The Mini Column Fractionation Kit (Novagen, Madison, WI) is used for rapid and effective size fractionation of DNA and removal of small mole- cules (< 300 bp) from DNA solutions by gel filtration. The cDNA fragments were ligated to the arms of T7 Select 10-3b and packaged in vitro using a T7 packaging extract (Novagen, Madison, WI), according to the man- ufacturer’s directions. The packaged phage were ampli- fied in liquid media with the host Escherichia coli BLT5403. Transfections and co-immunoprecipitation The PHE-CoV 67N strain did not infect the 293T cell line. To investigate the interactions between the PHE- CoV 67N strain and the chimeric protein, 293T cells were transfected with the pcDNA3.1 (+) (Invitrogen, Carlsbad, CA) expression plasmid containing the chi- meric gene, using Lipofectamine 2000 (Invitrogen, Carls- bad, CA). The transfections were performed following the manufacturers’ protocols [16,17]. After 24 h, the cells were replated in selective media containing 50-100 μg/ml ampicillin [18], and single ampicillin-resistant clones were selected. For co-immunoprecipitation, cells were lysed in 500 μl of radioimmune precipitation buffer (150 mm NaCl, 5 mg/ml sodium deoxycholate, 50 mm Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.1% SDS) supplemented with freshly added protease inhibitors. After rotating for 1 h at 4°C, cell lysates were cleared by centrifugation at 8000 × g for 10 min at 4°C. The 100-ml aliquot of lysate was incubated with 3 ml of glutathione-Sepharose beads conjugated with His6-tagged fusion S (6 mg). Cell lysates were electrophoresed through 12% sodium dode- cyl sulfate-polyacrylamide gels and transferred to polyvi- nylidene difluoride membranes. The blots were blocked at room temperature for 3 h with 3% BSA in PBS con- taining Tween 20 (0.05%) and then incubated overnight with a 1:2,000 dilution of the rabbit anti-S protein anti- body. The blot were washed again and exposed to films [19]. After a 24 h transfection, the PHE-CoV 67N strain was added to N2a KD cells. As control, the virus was added to mock-transfected siRNA N2a cells. At the indi- cated timings, culture supernatants were collected for plaque assay. Preparation of the T7 phage display library from N2a cells Total RNA from the N2a cells was extracted using stan- dard methodology, while mRNA was purified using the poly (A) Quick mRNA Isolation Kit (Promega, South- ampton, UK). A cDNA library was constructed with 10 Page 3 of 11 Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 with 20 μl of rabbit anti-PHE-CoV antiserum at 1:1,000 per well for 1 h at 37°C. The cells were washed three times in PBS (pH7.4). Fluorescein (FITC)-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratory, West Grove, PA) was added to the N2a cell mixtures for 30 min. After 48 hours, the samples were analyzed on a BD FACSAria flow cytometer [6]. amplification, the products were purified by Qiaquick columns (Qiagen, Hilden, Germany) and were then sequenced. The nucleotide sequence of the protein that was most predominantly recognized by the S protein of PHE-CoV was a hypothetical gene of N2a cells http://www.ncbi. nlm.nih.gov/blast. Transfection of siRNAs and PHE-CoV infection Double-stranded siRNA were designed based on the NCAM gene sequence to various regions of the genome using the Ambion siRNA Design tool http://www. ambion.com. Sequences were designed using (NN) N19 nt (where N is any nucleotide) and a GC content of less than 50%. The siRNAs targeted against the NCAM gene were synthesized at Sangon Biotech Co, Ltd. RNAs were deprotected and annealed using the Silencer siRNA Construction Kit (Ambions,Austin,USA). Double- stranded siRNA transfect into N2a cells using RNAimax (Invitrogen, Carlsbad,CA) as the transfection reagent. Before transfection, the cells were washed and resus- pended in 900 μl of RPMI 1640 medium. Cationic lipid complexes, prepared by incubating 2 μM siRNA duplexes with 3 μl of oligofectamine in 100 μl of RPMI 1640 medium, were added to the wells. The effect of gene silencing was examined by indirect immunofluores- cence. The resulting N2a cells were named N2a KD cells. Results Display of the cDNA library from N2a cells on T7 phage T7 phage was enumerated using the plaque assay method on LB semi-solid medium. Based on the PFU after in vitro packaging, the T7 phage display library from the N2a cells was calculated to contain 1.5 × 107 independent clones. The amplified library with a titer of 6.4 × 1010 pfu/mL was used for the subsequent screen- ing. Amplification of the inserts in randomly selected clones revealed that the library contained >90% recom- binants, with an average insert size of >300 bp. Because the size of the phage display library exceeded the esti- mated number, most of the expressed genes were repre- sented in this library (Fig. 1). Flow Cytometry We investigated whether a soluble form of the rabbit anti-NCAM antibody (Santa Cruz, California, USA, CATALOG: SC-10735) could inhibit PHE-CoV binding to N2a cells. The anti-NCAM antibody was diluted and added to N2a cells. The cells were incubated with 100 μl of soluble anti-NCAM antibody (10-25 μg/ml) for 1 h at 37°C. The controls included cells with goat IgG (1:1000) (Maixin, Fuzhou, China). Following this, the wells were washed five times with PBS (phosphate-buf- fered saline). After 2 hours, the PHE-CoV 67N strain (diluted to yield 20 to 40 plaques/well in 20 μl) was added to N2a cells that had been grown at a plating density of 105 cells per well in 24-well plates. After a 48-h infection, PHE-CoV binding was detected with the Rabbit PHE-CoV antiserum. The N2a cells were coated Affinity selection and sequence analysis of specific genes recognized by the S protein Affinity selection and sequence analysis of specific genes recognized by the S protein The entire screening process was repeated for five rounds. After each round of panning, there was an increase in the number of clones, suggesting that the Page 4 of 11 Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 M 1 28S RNA 18S RNA 5S RNA M 1 mRNA M 1 2 3 4 5 6 7 8 9 10 A B C Figure 1 The results of display of the cDNA library from N2a cells on T7 phage. (A) Lane1:The result of extracted total RNA of N2a cells. The electrophoresis results show 28 S and 18 S bands were clear, indicating the total RNA extraction without degradation. M: DL2000 Marker. (B) Lane1:The result of purified mRNA of N2a cells. OD260/OD280 = 1.950. The data show that the purified mRNA could be used for cDNA synthesis. M: DL2000 Marker. (C) Lane1 to 10: The PCR identified result of randomly picked phage clones of the library. Amplification of inserts in randomly selected clones revealed that the library contained >90% recombinants with average insert size of >300 bp. M: DL2000 Marker. M 1 28S RNA 18S RNA 5S RNA A M 1 2 3 4 5 6 7 8 9 10 Figure 1 The results of display of the cDNA library from N2a cells on T7 phage. (A) Lane1:The result of extracted total RNA of N2a cells. The electrophoresis results show 28 S and 18 S bands were clear, indicating the total RNA extraction without degradation. M: DL2000 Marker. (B) Lane1:The result of purified mRNA of N2a cells. OD260/OD280 = 1.950. The data show that the purified mRNA could be used for cDNA synthesis. M: DL2000 Marker. (C) Lane1 to 10: The PCR identified result of randomly picked phage clones of the library. Amplification of inserts in randomly selected clones revealed that the library contained >90% recombinants with average insert size of >300 bp. M: DL2000 Marker. procedure enriched for specific clones (Table 1). By the end of the fifth round of panning, there was a 320-fold increase in specific clones compared to the number of clones that were obtained after the first round. However, there was no further enrichment after additional rounds of panning. Expression of NCAMSf3b2, Hdac2 and RPS13 The full lengths cDNA of these genes (NCAM: GenBank no. Affinity selection and sequence analysis of specific genes recognized by the S protein NM_001081445Sf3b2: NM_030109, Hdac2: NM_008229 and RPS13: NM_026533) was used to con- struct the transfect plasmid. These four protein expres- sion levels were detected by a BioPhotometer Plus (Eppendorf, Hamburg, Germany). The correct expres- sion of NCAM, Sf3b2, Hdac2 and RPS13 in 293T cells was studied by immunoblotting. The four proteins anti- bodies were purchased from Santa Cruz biotechnology, inc (NCAM antibody CATALOG: SC-10735; Sf3b2 anti- body: SC-101133; Hdac2 antibody: SC-7899; RPS13 anti- body: SC-162098). The polypeptides migrated to a molecular weight corresponding to NCAM (140 kDa), Sf3b2 (100 kDa), Hdac2 (55 kDa) and RPS13 (17 kDa), respectively (Fig. 3). Approximately 100 clones were randomly picked from individual plaques, and the DNA sequences of clones were amplified by PCR and analyzed on an agarose gel to determine the insert size. Approximately 38% of the phage clones had an insert size of 830 bp, 25% had an insert size of 750 bp, 22% had an insert size of 400 bp and 15% had an insert size of 250 bp (Fig. 2). The clones were then further sequenced. DNA sequences of the inserts from the fifth round of panning were determined and compared using BLAST analysis. Panning yielded four clones (Table 2), as fol- lows: neural cell adhesion molecule (NCAM), splicing factor 3b, subunit 2 (Sf3b2), histone deacetylase 2 (Hdac2), and ribosomal protein S13 (RPS13). Identification of NCAM as a binding partner of the S protein To identify the binding partner of S protein, co-immu- noprecipitation was performed. The 293T cell lysates were immunoprecipitated with anti-S protein antibody. Supernatants of 293T cells transfected with plasmid encoding the screened gene were immunoprecipitated with S protein and anti-S protein antibody. The 293T cells transfected with vector alone were controls. When the soluble form of NCAM was incubated with S pro- tein, a 160 kDa band was observed (Fig. 4). However, Sf3b2, Hdac2 and RPS13 were not immunoprecipitated with S protein. Moreover, the PHE-CoV spreads via per- ipheral nerves to the central nervous system. Sf3b2, Table 1 Phage enrichment results after different rounds of panning Table 1 Phage enrichment results after different rounds of panning Round of panning Phage applied (PFU/ml) Phage eluted (PFU/ml) Enrichment (fold) 1 1.6 × 1010 5.5 × 105 3.4 × 10−5 2 2.8 × 1010 5.3 × 106 1.9 × 10−4 3 2.5 × 1010 8.3 × 106 3.3 × 10−4 4 3.4 × 1010 5.1 × 107 1.5 × 10−3 5 3.8 × 1010 6.5 × 107 1.7 × 10−3 Page 5 of 11 Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 M 1 2 3 4 5 830bp M 1 2 3 4 5 750bp M 1 2 3 4 5 M 1 2 3 4 5 400bp 250bp A B C D Figure 2 Detection of inserted fragments of phage clones in the fifth round of selection library by PCR. M: DL2000 Marker; Lane 1 to 5: The PCR result of randomly picked phage clones of the fifth round of selection library. (A) Approximately 38% of the phage clones had an insert size of 830 bp. (B) 25% of the phage clones had an insert size of 750 bp. (C) 22% of the phage clones had an insert size of 400 bp. (D) 15% of the phage clones had an insert size of 250 bp. 0bp M 1 2 3 4 5 750bp M 1 2 3 4 5 bp 250bp B D M 1 2 3 4 5 M 1 2 3 4 5 A 5 250bp D 400bp C D Figure 2 Detection of inserted fragments of phage clones in the fifth round of selection library by PCR. M: DL2000 Marker; Lane 1 to 5: The PCR result of randomly picked phage clones of the fifth round of selection library. Identification of NCAM as a binding partner of the S protein (A) Approximately 38% of the phage clones had an insert size of 830 bp. (B) 25% of the phage clones had an insert size of 750 bp. (C) 22% of the phage clones had an insert size of 400 bp. (D) 15% of the phage clones had an insert size of 250 bp. Hdac2 and RPS13 are all expressed in various tissues and cells. Therefore, we did not analyze them further. The data demonstrate a specific, high-affinity association between the S protein of PHE-CoV and NCAM. noticed that, the inhibition rate reached about 95% (Fig. 5). However, the proliferation of virus could not be suppressed completely. The result was that a soluble form of the anti-NCAM antibody blocked the associa- tion of PHE-CoV with the N2a cells. The NCAM siRNAs inhibit PHE-CoV infection for prolonged periods of time We investigated whether anti-NCAM antibody could block the association of PHE-CoV with N2a cells. Virus binding was detected using PHE-CoV antiserum and FITC-conjugated goat anti-rabbit IgG (H+L). FACS ana- lysis showed that the binding rate of PHE-CoV to N2a cells with control goat IgG was 99%. However, the 10 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a cells by 75%. With the increased anti-NCAM antibody concentration in the cell blocking, it was All oligonucleotide sequences used to produce NCAM siRNA are shown in Table 3. Three siRNAs were designed based on the NCAM sequence (Accession no. NC_000075). The effect of NCAM gene silencing in N2a cells was confirmed by flow cytometry. The NCAM protein expression was completely suppressed within 72 h (Fig. 6). To determine the antiviral effects of siRNAs, N2a cells were transfected with NCAM siRNAs and challenged with the PHE-CoV 67N strain 24 hours later. The effects of the siRNAs in N2a cells stained after transfection and infection with PHE-CoV was analysed by indirect immunofluorescence (Fig. 7). After further culture for 5 days, the reduction of cell-free viral particle production was assessed by plaque assay. Plaque assay analysis of the cultures after infection revealed a corre- sponding reduction in siRNA-transfected N2a cells. The NCAM siRNAs inhibited PHE-CoV infection compared Table 2 BLAST analysis identification of fifth round-insert sequences Clone name GenBank no. Identity NC-1 NM_001081445 neural cell adhesion molecule (NCAM) NC-2 NM_030109 splicing factor 3b, subunit 2 (Sf3b2) NC-3 NM_008229 histone deacetylase 2 (Hdac2) NC-4 NM_026533 ribosomal protein S13 (RPS13) Table 2 BLAST analysis identification of fifth round-insert sequences Clone name GenBank no. Identity NC-1 NM_001081445 neural cell adhesion molecule (NCAM) NC-2 NM_030109 splicing factor 3b, subunit 2 (Sf3b2) NC-3 NM_008229 histone deacetylase 2 (Hdac2) NC-4 NM_026533 ribosomal protein S13 (RPS13) Table 2 BLAST analysis identification of fifth round-insert sequences Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 Page 6 of 11 NCAM Hdac2 Rps13 Sf3b2 Mock (Kda) 250 150 100 75 50 37 Figure 3 Western blot analysis of proteins expression in total extracts of 293T cells transfected with the pcDNA3.1 (+) expression plasmid. Lane 1: Western blot analysis of NCAM protein expression. The full lengths cDNA of NCAM gene was used to construct the transfect plasmid. Cell lysates from 293T cells were run on a 10% SDS-PAGE gel and blotted onto polyvinylidene difluoride membranes. The NCAM siRNAs inhibit PHE-CoV infection for prolonged periods of time Lane 1: Western blot analysis of NCAM protein expression. The full lengths cDNA of NCAM gene was used to construct the transfect plasmid. Cell lysates from 293T cells were run on a 10% SDS-PAGE gel and blotted onto polyvinylidene difluoride membranes. The blots were probed with a 1:10 dilution of the rabbit anti-NCAM polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-rabbit IgG antibodies and chemiluminescence. Lane 2: Immunoblots for Hdac2 protein. The blot was probed Figure 3 Western blot analysis of proteins expression in total extracts of 293T cells transfected with the pcDNA3.1 (+) expression Figure 3 Western blot analysis of proteins expression in total extracts of 293T cells transfected with the pcDNA3.1 (+) expression plasmid. Lane 1: Western blot analysis of NCAM protein expression. The full lengths cDNA of NCAM gene was used to construct the transfect plasmid. Cell lysates from 293T cells were run on a 10% SDS-PAGE gel and blotted onto polyvinylidene difluoride membranes. The blots were probed with a 1:10 dilution of the rabbit anti-NCAM polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-rabbit IgG antibodies and chemiluminescence. Lane 2: Immunoblots for Hdac2 protein. The blot was probed with a 1:10 dilution of the rabbit anti-Hdac2 polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-rabbit IgG antibodies. Lane 3: Immunoblots for RPS13 protein. The blot was probed with a 1:10 dilution of the goat anti-RPS13 polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated mouse anti-goat IgG antibodies. Lane 4: Immunoblots for Sf3b2 protein. The blot was probed with a 1:5 dilution of the mouse monoclonal anti-Sf3b2 IgG2a (100 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-mouse IgG antibodies. Lane 5: The 293T cells transfected with vector alone. protein to identify the interaction between PHE-CoV and N2a cells. Co-immunoprecipitation analysis showed that the NCAM was a binding partner of spike protein. In addition, FACS analysis demonstrated that a soluble form of the anti-NCAM antibody blocked association of PHE-CoV with N2a cells. Moreover, double-stranded siRNA targeted against NCAM inhibit PHE-CoV infec- tion. The results suggest that NCAM might participate in virus infection. to controls throughout the 84-hour period of observa- tion (Fig. 8). These results demonstrated that expression levels of NCAM correlate with PHE-CoV infection. NCAM might participate in the attachment and invasion of N2a cells. The NCAM siRNAs inhibit PHE-CoV infection for prolonged periods of time The blots were probed with a 1:10 dilution of the rabbit anti-NCAM polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-rabbit IgG antibodies and chemiluminescence. Lane 2: Immunoblots for Hdac2 protein. The blot was probed with a 1:10 dilution of the rabbit anti-Hdac2 polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-rabbit IgG antibodies. Lane 3: Immunoblots for RPS13 protein. The blot was probed with a 1:10 dilution of the goat anti-RPS13 polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated mouse anti-goat IgG antibodies. Lane 4: Immunoblots for Sf3b2 protein. The blot was probed with a 1:5 dilution of the mouse monoclonal anti-Sf3b2 IgG2a (100 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-mouse IgG antibodies. Lane 5: The 293T cells transfected with vector alone. Western blot analysis of proteins expression in total extracts of 293T cells transfected with the pcDNA3.1 (+) Figure 3 Western blot analysis of proteins expression in total extracts of 293T cells transfected with the pcDNA3.1 (+) expression plasmid. Lane 1: Western blot analysis of NCAM protein expression. The full lengths cDNA of NCAM gene was used to construct the transfect Figure 3 Western blot analysis of proteins expression in total extracts of 293T cells transfected with the pcDNA3.1 (+) expression plasmid. Lane 1: Western blot analysis of NCAM protein expression. The full lengths cDNA of NCAM gene was used to construct the transfect plasmid. Cell lysates from 293T cells were run on a 10% SDS-PAGE gel and blotted onto polyvinylidene difluoride membranes. The blots were probed with a 1:10 dilution of the rabbit anti-NCAM polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-rabbit IgG antibodies and chemiluminescence. Lane 2: Immunoblots for Hdac2 protein. The blot was probed with a 1:10 dilution of the rabbit anti-Hdac2 polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-rabbit IgG antibodies. Lane 3: Immunoblots for RPS13 protein. The blot was probed with a 1:10 dilution of the goat anti-RPS13 polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated mouse anti-goat IgG antibodies. Lane 4: Immunoblots for Sf3b2 protein. The blot was probed with a 1:5 dilution of the mouse monoclonal anti-Sf3b2 IgG2a (100 μg/ml). The antibodies Figure 3 Western blot analysis of proteins expression in total extracts of 293T cells transfected with the pcDNA3.1 (+) expression plasmid. Anti-Spike protein Ab Figure 4 The NCAM binding to PHE-CoV S protein. Lane 1-3, NCAM involves in recognition by PHE-CoV S protein. Supernatants of 293T cells transfected with plasmid encoding soluble NCAM. The 293T cells were added fusion S protein (6 mg) and incubate for 2 h at 4°C. The cells were lysed in 500 μl of radioimmune precipitation buffer. The 10-ml aliquot of lysate was incubated with 300 μl of glutathione-Sepharose beads conjugated with fusion anti-S protein antibody and gently rocking on a orbital shaker overnight at 4°C. The sepharose beads are boiled for 5 min to dissociate the immunocomplexes from the beads. The supernatant was electrophoresed through 12% sodium dodecyl sulfate- polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The blots were blocked at room temperature for 3 h with 3% BSA in PBS containing Tween 20 (0.05%) and then incubated overnight with the anti-NCAM protein antibody. The proteins was analyzed by western blotting. Lane 4-6, Sf3b2, Hdac2 and RPS13 were not immunoprecipitated with S protein. Lane 7, 293T cells transfected with vector alone were negative controls. neurons, glia, skeletal muscle and natural killer cells [20]. NCAM is a member of the immunoglobulin super- gene family of Cell adhesion molecules (CAMs) [21]. CAMs play important roles in cell-cell and cell-extracel- lular matrix interactions in both mature and developing nervous system [22]. During development, they are involved in cell migration, axon guidance, target recog- nition, and synapse formation; while in the mature ner- vous system, they maintain synaptic connections, cell- cell contacts, and neuron-glial interactions [22]. Injuries to the nervous systems break the stable state of the tis- sues and the repair of damaged tissues and regeneration of axons require the participation of CAMs both as adhesion molecules and as signal transduction molecules [22]. NCAM has been implicated as having a role in cell-cell adhesion, neurite outgrowth, synaptic plasticity, and learning and memory [23,24]. There is evidence that PHE-CoV is disseminated throughout the central nervous system by direct transfer of virus from neuron to neuron [25]. Thus, by binding to NCAM, the PHE- Figure 5 Anti-NCAM antibody inhibition of PHE-CoV binding to N2a cells. PHE-CoV binding assay using various concentrations of anti- NCAM antibody. The 10 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a cells by 75%. With the increased anti-NCAM antibody concentration in the blocking, the inhibition rate increased accordingly. NCAM NCAM NCAM Sf3b2 Hdac2 RPS13 NCAM NCAM NCAM NCAM NCAM NCAM Sf3b2 Hdac2 RPS13 160kDa Discussion In this report, we describe the discovery of a novel interaction between NCAM and spike protein of PHE- CoV. To our knowledge, this is the first study that used a phage display-based cDNA expression library for screening and affinity panning with the PHE-CoV spike Neural Cell Adhesion Molecule (NCAM, also the clus- ter of differentiation CD56) is a homophilic and hetero- philic binding glycoprotein expressed on the surface of Page 7 of 11 Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 NCAM NCAM NCAM Sf3b2 Hdac2 RPS13 Mock 160kDa Anti-Spike protein Ab The 25 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a cells by 95.7%. However, the proliferation of virus could not be suppressed completely. Figure 5 Anti-NCAM antibody inhibition of PHE-CoV binding to N2a cells. PHE-CoV binding assay using various concentrations of anti- NCAM antibody. The 10 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a cells by 75%. With the increased anti-NCAM antibody concentration in the blocking the inhibition rate increased accordingly The 25 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a Figure 5 Anti-NCAM antibody inhibition of PHE-CoV binding to N2a cells. PHE-CoV binding assay using various concentrations of anti- NCAM antibody. The 10 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a cells by 75%. With the increased anti-NCAM antibody concentration in the blocking, the inhibition rate increased accordingly. The 25 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a cells by 95.7%. However, the proliferation of virus could not be suppressed completely. Figure 5 Anti-NCAM antibody inhibition of PHE-CoV binding to N2a cells. PHE-CoV binding assay using various concentrations of anti- NCAM antibody. The 10 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a cells by 75%. With the increased anti-NCAM antibody concentration in the blocking, the inhibition rate increased accordingly. The 25 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a cells by 95.7%. However, the proliferation of virus could not be suppressed completely. Page 8 of 11 Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 Table 3 Oligonucleotides for siRNA construction siRNA strand Sequence SiNCAM79 Antisense 5’-AAGGTCTTTGCAAAGCCCAAACCTGTCTC-3’ Sense 5’-AATTTGGGCTTTGCAAAGACCCCTGTCTC-3’ SiNCAM81 Antisense 5’-AAGTCTATGTGGTAGCTGAAACCTGTCTC-3’ Sense 5’-AATTTCAGCTACCACATAGACCCTGTCTC-3’ SiNCAM90 Antisense 5’-AACTCTGTCGAACCTCACAAACCTGTCTC-3’ Sense 5’-AATTTGTGAGGTTCGACAGAGCCTGTCTC-3’ siCtrl Antisense 5’-AATTTGGGCTTTGCAAAGACCTTCCTGTCTC-3’ Sense 5’-AATTCCAGAAACGTTTCGGGTTTCCTGTCTC-3’ Table 3 Oligonucleotides for siRNA construction siRNA strand Sequence SiNCAM79 Antisense 5’-AAGGTCTTTGCAAAGCCCAAACCTGTCTC-3’ Sense 5’-AATTTGGGCTTTGCAAAGACCCCTGTCTC-3’ SiNCAM81 Antisense 5’-AAGTCTATGTGGTAGCTGAAACCTGTCTC-3’ Sense 5’-AATTTCAGCTACCACATAGACCCTGTCTC-3’ SiNCAM90 Antisense 5’-AACTCTGTCGAACCTCACAAACCTGTCTC-3’ Sense 5’-AATTTGTGAGGTTCGACAGAGCCTGTCTC-3’ siCtrl Antisense 5’-AATTTGGGCTTTGCAAAGACCTTCCTGTCTC-3’ Sense 5’-AATTCCAGAAACGTTTCGGGTTTCCTGTCTC-3’ Table 3 Oligonucleotides for siRNA construction Table 3 Oligonucleotides for siRNA construction siRNA strand Sequence surfaces, that in turn induces growth promoting mechanisms in the nerve process [26]. The close genetic and antigenic relatedness among the group 2 coronaviruses human coronavirus OC43 (HcoV-OC43), bovine coronavirus (BCV), and porcine hemagglutinating encephalomyelitis virus (PHE-CoV) suggests that these three viruses with different host spe- cificities diverged fairly recently [1]. HcoV-OC43, BCV and PHE-CoV recognize sialic acid-containing receptors similar to those of influenza C viruses [28-32]. Polysialic acid (PSA) is a developmentally regulated carbohydrate composed of a linear homopolymer of a-2,8-linked sialic acid residues [33]. Anti-Spike protein Ab NCAM undergoes post-translational modification during development, leading to the abun- dant addition of PSA chains on its extracellular domain [34]. PSA on NCAM is developmentally regulated thus playing a prominent role in different forms of neural plasticity spanning from embryonic to adult nervous sys- tem, including axonal growth, outgrowth and fascicula- tion, cell migration, synaptic plasticity, activity-induced plasticity, neuronal-glial plasticity, embryonic and adult neurogenesis [35]. CoV might increase the probability of gaining access form peripheral nervous system to the central nervous system. Affected piglets show the clinical symptoms such as generalized muscle trembling, abnormal walking, lack of co-ordination, ears held back, convulsions and lying on the side and paddling legs. If PHE-CoV bind to NCAM, certain aspects of the clinical symptoms may be readily explained. NCAM is expressed in the surface of devel- oping muscle with a spatiotemporal pattern that is con- sistent with a role in neuromuscular junction (NMJ) formation [26]. Only NCAM of the CAMs appears on the surface of muscle cells in parallel with the ability of the muscle cell surface to accept synapses [27]. Levels of NCAM in muscle are regulated in parallel with the sus- ceptibility of muscle to innervation. NCAM-induced sprouting is thought to be induced via homophilic bind- ing between NCAMs in the neural and the muscle The entry of coronaviruses is a multi-step process that involve: docking on the plasma membrane, binding to a receptor or co-receptors and delivery of the viral gen- ome into the host cell. Docking of viruses on the plasma membrane of a susceptible cell is the first step during virus entry. Docking involves non-specific interactions between the viral envelope protein and carbohydrate moieties like heparan sulfate or sialic acid on the surface of cells. These initial docking interactions may lead to 0 20 40 60 80 100 24 48 72 96 hours post-transfection The positive rate of cells(%) Mock siNCAM79 siNCAM81 siNCAM90 Figure 6 Double-stranded siRNA could effectively inhibit NCAM expression in N2a cells. N2a cells were transfected with siRNA targeted against NCAM. The cells were harvested after siRNA transfection and analyzed by FACS with rabbit anti-NCAM antibody and FITC-conjugated goat anti-rabbit IgG (H+L). Mock-transfected siRNA N2a cells served as a control. There appeared to be a slight decrease of the positive rate of N2a KD cells compared to that of controls within 72 hours. Anti-Spike protein Ab hours post-transfection Mock siNCAM79 siNCAM81 siNCAM90 Mock siNCAM79 siNCAM81 siNCAM90 Figure 6 Double-stranded siRNA could effectively inhibit NCAM expression in N2a cells. N2a cells were transfected with siRNA targeted against NCAM. The cells were harvested after siRNA transfection and analyzed by FACS with rabbit anti-NCAM antibody and FITC-conjugated goat anti-rabbit IgG (H+L). Mock-transfected siRNA N2a cells served as a control. There appeared to be a slight decrease of the positive rate of N2a KD cells compared to that of controls within 72 hours. Figure 6 Double-stranded siRNA could effectively inhibit NCAM expression in N2a cells. N2a cells were transfected with siRNA targeted against NCAM. The cells were harvested after siRNA transfection and analyzed by FACS with rabbit anti-NCAM antibody and FITC-conjugated goat anti-rabbit IgG (H+L). Mock-transfected siRNA N2a cells served as a control. There appeared to be a slight decrease of the positive rate of N2a KD cells compared to that of controls within 72 hours. Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 Page 9 of 11 A C D E F B Figure 7 The NCAM siRNAs inhibit PHE-CoV infection by indirect immunofluorescence. After a 48 h viral infection, the N2a cells were fixed with 80% acetone for 10 min at -20°C, rehydrated in PBS, labeled with rabbit PHE-CoV antiserum, and washed three times with PBS. FITC- conjugated goat anti-rabbit IgG (H+L) (1:50 dilution) was added to the N2a cell mixtures for 30 min at room temperature, and the cells were washed and observed with an Olympus FV1000 laser scanning confocal microscope. Microscopic magnification, 400×. (A) Mock transfection (stained with PHE-CoV-positive serum); (B) Mock transfection (stained with PHE-CoV-negative serum); (C) siCtrl transfection; (D) siNCAM79 transfection; (E) siNCAM81 transfection; (F) siNCAM90 transfection. B E Figure 7 The NCAM siRNAs inhibit PHE-CoV infection by indirect immunofluorescence. After a 48 h viral infection, the N2a cells were fixed with 80% acetone for 10 min at -20°C, rehydrated in PBS, labeled with rabbit PHE-CoV antiserum, and washed three times with PBS. FITC- conjugated goat anti-rabbit IgG (H+L) (1:50 dilution) was added to the N2a cell mixtures for 30 min at room temperature, and the cells were washed and observed with an Olympus FV1000 laser scanning confocal microscope. Microscopic magnification, 400×. (A) Mock transfection (stained with PHE-CoV-positive serum); (B) Mock transfection (stained with PHE-CoV-negative serum); (C) siCtrl transfection; (D) siNCAM79 transfection; (E) siNCAM81 transfection; (F) siNCAM90 transfection. References 1. Vijgen L, Keyaerts E, Lemey P, Maes P, Van Reeth K, Nauwynck H, Pensaert M, Van Ranst M: Evolutionary history of the closely related group 2 coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus OC43. J Virol 2006, 80:7270-7274. 2. Iacono KT, Kazi L, Weiss SR: Both spike and background genes contribute to murine coronavirus neurovirulence. Journal of Virology 2006, 80:6834-6843. 3. Li WH, Moore MJ, Vasilieva N, Sui JH, Wong SK, Berne MA, Somasundaran M, Sullivan JL, Luzuriaga K, Greenough TC, et al: Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003, 426:450-454. 3. Li WH, Moore MJ, Vasilieva N, Sui JH, Wong SK, Berne MA, Somasundaran M, Sullivan JL, Luzuriaga K, Greenough TC, et al: Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003, 426:450-454. 4. Miura TA, Travanty EA, Oko L, Bielefeldt-Ohmann H, Weiss SR, Beauchemin N, Holmes KV: The spike glycoprotein of murine coronavirus MHV-JHM mediates receptor-independent infection and spread in the central nervous systems of Ceacam1a(-/-) mice. Journal of Virology 2008, 82:755-763. Finally, identification of the NCAM that interacts with PHE-CoV spike protein will facilitate the description of the binding domain of the spike protein, which will pre- sumably be the most effective target epitope for a spike protein-based subunit vaccine. In addition, it is likely that a cell line approved for vaccine production, and one that is made permissive for viral replication through expression of NCAM, will be the most efficient large- scale producer of whole-killed or attenuated virus for use as a vaccine. There are a number of chronic neuro- logic diseases, such as myasthenia gravis, subacute scler- osing panencephalitis, and Alzheimer’s disease, for which some evidence of viral etiology exists [39]. One explanation for these diseases is that after a virus binds to a cellular constituent acting as a receptor, the recep- tor might be altered. Identification of the specific neuro- nal constituents to which neurotropic viruses bind will allow for an analysis of the potential effects of these interactions on functional or antigenic alterations of receptors [40]. 5. Phillips JJ, Chu MM, Lavi E, Weiss SR: Pathogenesis of chimeric MHV4/ MHV-A59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence. Journal of Virology 1999, 73:7752-7760. 6. g We thank American Journal Experts for excellent grammar revisions of this paper. This work was supported by the National Natural Science Foundation of China (No. 30671551 and No. 31072134). Authors’ contributions WG and WH carried out most of the experiments and wrote the manuscript. HL participated in the protein production. KZ carried out the co- immunoprecipitation assay. WR and CD participated in the sequence alignment. YL participated in the design of the NCAM siRNAs. KC participated in the design of the study. FG and DS conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript. Competing interests h h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 28 June 2010 Accepted: 24 September 2010 Received: 28 June 2010 Accepted: 24 September 2010 Published: 24 September 2010 Received: 28 June 2010 Accepted: 24 September 2010 Published: 24 September 2010 Additionally, porcine hemagglutinating encephalomye- litis is an infectious disease affecting mainly pigs under 3 weeks old [37]. During the embryonic development of the brain, NCAM undergoes posttranslational modifica- tions leading to the addition of a-2,8-polysialic acid (PSA) chains on its extracellular domain [38]. This embryonic highly PSA-NCAM is expressed abundantly throughout the brain until early postnatal period and is involved in neurite extension and synaptogenesis [38]. In the adult brain, however, PSA-NCAM expression is considerably reduced, although it has been shown to be expressed in certain areas (e.g. the olfactory bulb and hippocampus) [34]. Published: 24 September 2010 Author details 1 1College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, PR China. 2Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun 130062, PR China. 3Laboratory Animal Center, Jilin University, Changchun 130062, PR China. Anti-Spike protein Ab Reovirus strains that have sialic acid-binding activity attach to cells with 5-fold more avidity than strains that do not bind sialic acid, and their infectivity is enhanced 50-100 fold [36]. After docking at the sur- face of a susceptible cell, the virus binds a receptor molecule(s) that in turn triggers conformational changes that result in virus entry. We speculate that the entry of PHE-CoV is a multi-step process. The Hemagglutinin- esterase (HE) protein of PHE-CoV binds to polysialic acid (PSA) moieties, while the spike (S) protein of PHE- CoV binds to NCAM at the plasma. Anti-Spike protein Ab Figure 8 The NCAM siRNAs could inhibit PHE-CoV infection in a period of time. Culture supernantants were collected 120 hours after the PHE-CoV challenge. The supernatants harvested at indicated timings were subjected to plaque assay. There was significant difference (p < 0.05) in virus titres. Knock-down of NCAM caused a marked reduction of PHE-CoV infection within 84 hours. Figure 8 The NCAM siRNAs could inhibit PHE-CoV infection in a period of time. Culture supernantants were collected 120 hours after the PHE-CoV challenge. The supernatants harvested at indicated timings were subjected to plaque assay. There was significant difference (p < 0.05) in virus titres. Knock-down of NCAM caused a marked reduction of PHE-CoV infection within 84 hours. Figure 8 The NCAM siRNAs could inhibit PHE-CoV infection in a period of time. Culture supernantants were collected 120 hours after the PHE-CoV challenge. The supernatants harvested at indicated timings were subjected to plaque assay. There was significant difference (p < 0.05) in virus titres. Knock-down of NCAM caused a marked reduction of PHE-CoV infection within 84 hours. Page 10 of 11 Page 10 of 11 Page 10 of 11 Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 Gao et al. Virology Journal 2010, 7:254 http://www.virologyj.com/content/7/1/254 concentration of virus at the plasma membrane of a sus- ceptible cell that in turn may enhance the infectivity of the virus by facilitating the interactions of the envelope protein with a cellular receptor that promotes virus entry. Reovirus strains that have sialic acid-binding activity attach to cells with 5-fold more avidity than strains that do not bind sialic acid, and their infectivity is enhanced 50-100 fold [36]. After docking at the sur- face of a susceptible cell, the virus binds a receptor molecule(s) that in turn triggers conformational changes that result in virus entry. We speculate that the entry of PHE-CoV is a multi-step process. The Hemagglutinin- esterase (HE) protein of PHE-CoV binds to polysialic acid (PSA) moieties, while the spike (S) protein of PHE- CoV binds to NCAM at the plasma. concentration of virus at the plasma membrane of a sus- ceptible cell that in turn may enhance the infectivity of the virus by facilitating the interactions of the envelope protein with a cellular receptor that promotes virus entry. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 32. 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https://openalex.org/W4290998792	https://ejournal.stismu.ac.id/ojs/index.php/qolamuna/article/download/663/359	Indonesian	null	MANAJEMEN MUTU PENINGKATAN DAYA SAING LULUSAN PERGURUAN TINGGI BERBASIS PESANTREN	Qolamuna	2,022	cc-by	3,550	Abstract Abstract This article reveals the contribution of the education quality system in increasing the competitiveness of Miftahul Ulum Lumajang Islamic Boarding School graduates. This type of research is qualitative with a case study approach, data obtained from interviews, observations, and documentation, then analyzed by condensing, displaying data, and drawing conclusions. The research findings show that quality of the Miftahul Ulum Lumajang Islamic Boarding School education system begins with the integration of visions between institutions under the auspices of the Miftahul Ulum Lumajang Foundation. They were followed by setting goals and strategies for improving the academic quality of students through three models of quality development, namely coaching in the recitation, extracurricular activities, and activities in the pesantren environment. p Keywords: quality managemeny, the competitiveness of graduates, College University, pesantren. MANAJEMEN MUTU PENINGKATAN DAYA SAING LULUSAN PERGURUAN TINGGI BERBASIS PESANTREN Ahmad Zarkasyi STIS Miftahul Ulum Lumajang zarkazee@gmail.com Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Qolamuna : Jurnal Studi Islam Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Abstrak Artikel ini mengungkap kontribusi sistem mutu pendidikan dalam meningkatkan daya saing lulusan Pondok Pesantren Miftahul Ulum Lumajang. Penelitian berjenis kualitatif dengan pendekatan studi kasus, data diperoleh dari hasil wawancara, observasi dan dokumentasi, kemudian dianalisis dengan tahap kondensasi, diplay data dan penarikan kesimpulan. Temuan penelitian menunjukkan bahwa manajemen mutu pendidikan STIS Miftahul Ulum Lumajang diawali dengan integrasi visi antar lembaga yang berada dibawah naungan Yayasan Miftahul Ulum Lumajang. Dilanjutkan dengan menetapkan tujuan, strategi peningkatan mutu akademik santri melalui tiga model pembinaan kualitas, yaitu pembinaan pada pengajian, ekstrakulikuler, kegiatan di lingkungan pesantren. g g g Kata Kunci : manajemen mutu, daya saing lulusan, perguruan tinggi, pesantren 116 Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Pendahuluan Manajemen peningkatan daya lulusan dalam konteks pendidikan diukur dari : mutu input mahasiswa, mutu proses pendidikan, mutu output dan mutu outcome lulusan(Ganseuer and Randhahn 2017). Mutu input pendidikan adalah segala sesuatu karakteristik yang tersedia dari pondok pesantren karena dibutuhkan untuk berlangsungnya proses input sumber daya meliputi: sumber daya manusia (kiai, guru, karyawan, dan siswa) dan sumber daya selebihnya (peralatan, perlengkapan, dana, bahan dan sebagainya) (Sonia 2021). Mutu input perangkat lunak meliputi struktur pesantren atau sekolah, peraturan tata tertib, deskripsi tugas, rencana, program, dan sebagainya. Mutu proses pendidikan berupa berupa visi, misi, tujuan dan sasaran-sasaran yang ingin dicapai oleh perguruan tinggi dalam menjalankan proses pendidikan (Komsiyah 2021). Sedangkan mutu outcome pesantren diukur dari kiprah para alumni dan sumbangsihnya di masyarakat baik lokal, nasional dan internasional merupakan bukti kesuksesan output pesantren dalam mendidik dan memberikan kompetensi serta membangun karakter (caracter building) mahasiswa (Sahid et al. 2021) . Sementara dari perspektif Pendidikan pesantren, Zamakhsari, menyebutkan pondok pesantren dengan berbagai harapan dan predikat yang diletakkan padanya, sesungguhnya berujung pada tiga fungsi yang senantiasa diemban, yaitu: sebagai pusat pengkaderan pemikir-pemikir agama (Hardianto 2019). Sebagai lembaga yang mencetak sumber daya manusia, sebagai lembaga yang memiliki kekuatan melakukan pemberdayaan kepada masyarakat Penelitian ini dilakukan di Sekolah Tinggi Ilmu Syariah Miftahul Ulum Lumajang, dengan jumlah mahasiswa 1500, sehingga menjadi pesantren dengan peminat terbesar di Kabupaten Lumajang. Di samping tetap mempertahankan nilai-nilai salaf juga sangat responsif terhadap perkembangan zaman. Hal ini dibuktikan dengan dibukanya lembaga pendidikan formal di lingkungan pesantren, mulai dari Madrasah Ibtidaiyah hingga perguruan tinggi (Observasi Lapangan, 2022). gg p g gg ( p g ) Berdasar arsip laporan Instansi Urusan Tugas Mengajar Tugas Belajar (TMTB) dan Dai Miftahul Ulum, dapat dirinci bahwa pada tahun 2019 mengirim sebanyak 63 Guru/Ustadz Tugas dan Dai, selanjutnya pada tahun 2020 mengirim sebanyak 67 Guru/Ustadz Tugas dan Dai, terahir pada tahun ajaran 2021 resmi melepas 74 Dai ke Enam wilayah di Indonesia. Enam wilayah tersebut yakni; Jawa Timur, Jawa Tengah, Kalimantan Barat, Nusa Tenggara Timur (NTT), Bali, dan Papua 117 Faktor tersebut menjadi keunikan dan daya tarik terhadap pondok pesantren Miftahul Ulum Lumajang bagi masyarakat, sehingga ini merupakan sarana promosi yang efektif bagi masyarakat untuk menyantrikan anak- anaknya ke pondok pesantren Miftahul Ulum Lumajang. Selain faktor jumlah santri yang dididik, pesantren ini juga memiliki lulusan dengan kontribusi pembinaan penguatan aspek keagamaan masyarakat secara nyata melalui penguatan sistem mutu pendidikan. Beberapa penelitian dan artikel terdahulu seperti yang dilakukan oleh (Sahid et al. Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Pendahuluan 2021), (Mukhtar, Hidayat, and Ulfah 2020), (Nasser et al. 2022), (Komsiyah 2021), (Nurzannah, Ginting, and Setiawan 2020), (Muchith 2019), (Darojat 2019), umumnya meneliti dampak dan peran pondok pesantren dalam pendidikan karakter, moral, kurikulum dan metode pembelajaran hingga sufistik. Padahal, senada dengan yang disinggung oleh (Ijah, Florentinus, and Sudana 2021), bahwa pesantren telah mampu mengadaptasi manajemen modern sebagai standar mutu pengelolaan sistem pendidikan. Pesantren dengan ciri khas salafnya terus melakukan inovasi yang terus menerus dalam manajemen input, proses, output hingga outcome daya saing lulusan (Ilyasin 2020). Perbedaan artikel ini dibanding beberapa penelitian sebelumnya adalah pada masuknya analisis daya saing alumni pesantren dalam pembinaan civiel society masyarakat yang ditinjau dengan teori sistem mutu pendidikan. Lebih jauh, artikel ini mencoba mengeksplorasi dan menganalisis lebih jauh tentang konstribusi alumni dalam penguatan lulusan berdaya saing dalam bidang sosial ekonomi kemasyarakatan. Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Metode Penelitian Metode yang digunakan dalam penelitian ini adalah metode kualitatif dengan pendekatan analisis deskriptif (R.Sherman 2006). Adapun sumber data primer dalam penelitian ini yaitu person dan place dengan prosedur dan teknik pengumpulan data melalui wawancara dan observasi, adapun sumber data sekunder dalam penelitian ini yaitu paper melalui kegiatan studi dokumen (Rahi 2017). Sumber utama penelitian adalah pimpinan Yayasan Pondok Pesantren Miftahul Ulum Lumajang, kepala pesantren, asatidz, dosen, santri, dan masyarakat sebagai sumber data tambahan untuk memperkuat jawaban dan menguji kebenaran. g j Untuk memperoleh data yang komprehensif peneliti melakukan observasi (Morgan and Harmon 2001) untuk melihat situasi secara umum di Pondok Pesantren khususnya terkait proses dan hasil manajemen mutu dalam meningkatkan kualitas lulusan. Studi dokumen dilakukan untuk memperoleh dokumen profil Pondok Pesantren Miftahul Ulum Lumajang, beserta visi dan misi, data santri, data tenaga pendidik, data kependidikan, struktur organisasi, data sarana prasarana pesantren, rencana Pondok Pesantren yang terkait 118 dengan peningkatan kualitas santri, program kerja, nilai santri, dan jadwal kegiatan kegiatan rutin pesantren. Dokumen-Dokumen tersebut digunakan untuk melengkapi data penelitian sehingga dapat ditampilkan gambaran tentang objek penelitian secara komprehensif (R.Sherman 2006). Peningkatan kualitas manusia pada dasarnya menjadi visi utama didirikannya pondok pesantren ini, dari dasar itulah maka peneliti akan menjabarkan sejauh mana kontribusi manajemen mutu pendidikan dalam meningkatkan daya saing lulusan STIS Miftahul Ulum Lumajang. Hasil Penelitian Berdasar hasil penelitian dalam menjamin keberhasilan proses pendidikan dan pengajaran, Pondok Pesantren Miftahul Ulum memiliki beberapa lembaga yang menyelenggarakan proses ini, baik secara langsung maupun tidak. Yayasan pesantren, sebuah badan legislatif yang bertanggung jawab penuh terhadap pelaksanaan dan perkembangan setiap jenjang pendidikan yang berada dalam naungan dan pengawasan yayasan, pengasuh mengamanatkan kepada ketua yayasan Pesantren Miftahul Ulum sebagai mandatarisnya untuk menjamin keberlangsungan proses pendidikan dan pengajarannya, sebagaimana pemaparan berikut : 1. Manajemen Mutu Keterpaduan Kelembagaan Secara akademis, ada dua jenjang pendidikan yang diselenggarakan oleh Pesantren Miftahul Ulum, yaitu jenjang non formal madrasah diniah dan jenjang formal yaitu Madrasah Ibtidaiah, Mts, MA hingga perguruan tinggi dengan nama STIS Miftahul Ulum Lumajang (Profil STIS Miftahul Ulum, 2021). Di lingkup non formal, ada dua lembaga yang secara langsung menanganin pendidikan dan pengajaran, yaitu Madrasah diniah (Madin) Miftahul Ulum dan Pengasuhan Santri. Kegiatan intrakurikuler dilaksanakan oleh Madin, dan dipimpin oleh kepala pesantren sedangkan kegiatan ekstrakurikuler dan sebagian kokurikuler, dilaksanakan oleh lembaga Kepengasuhan Santri, dan dipimpin langsung oleh pengasuh santri yang juga Pimpinan Pondok. Namun, dalam pelaksanaan hariannya, diurus oleh asatidz yang bernama, “Pengasuhan Santri” (Observasi, 2021). y g g ( ) Di tingkat tinggi, sebagaimana perguruan tinggi Islam swasta yang lain, STIS Miftahul Ulum dipimpin oleh seorang ketua beserta jajarannya, yang juga bertanggung jawab kepada ketua yayasan Pesantren Miftahul Ulum. Berbeda dengan Madin, kurikulum Mts, MA dan STIS Miftahul Ulum menggunakan kurikulum KKNI (Kerangka Kualifikasi Nasional Indonesia). Pengembangan sistem mutu pendidikan di kedua jenjang pendidikan berlangsung independen dan terbebas dari intervensi pihak mana pun, karena yayasan Pesantren Miftahul Ulum mandiri dalam menyelenggarakan pendidikan dan pengajarannya (Observasi, 2021). 119 Sebagai contoh, kurikulum disusun secara mandiri disesuaikan dengan program Pondok secara keseluruhan. Sistem Madin Miftahul Ulum merupakan lembaga yang mengurus aktivtas akademis para santri, di mana sistem perjenjangannya sudah diterapkan sejak tahun 1946. Untuk memastikan berjalannya dan meningkatnya kualitas akademik, Madin memiliki bagian- bagian tertentu. Seperti Bagian Proses Belajar-Mengajar (PBM), Bagian Penelitian dan Pengembangan (Litbang) Kurikulum, Bagian Karir asatidz, Perpustakaan, Tata Usaha dan Peralatan (inventaris) (Kepala Pesantren, Hasil Wawancara, 2021). Terkait dengan manajemen peningkatan mutu, “Continous Improvement”, adalah motto yang selalu dipegang, agar Madin Miftahul Ulum selalu dinamis dan mampu meningkatan program akademiknya. Karena itu, koordinasi antar bagian dalam Madin Miftahul Ulum menjadi satu keharusan yang tak terelakkan. Karena pentingnya koordinasi tersebut, Madin Miftahul Ulum telah menetapkan jadwal pertemuan/rapat rutin antarbagian Madin Miftahul Ulum sebulan sekali (Kepala Pesantren, Hasil Wawancara, 2021).. Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Hasil Penelitian Pertemuan tersebut sekaligus sebagai sarana evaluasi program yang sudah dilakukan dan perencanaan program yang akan dilakukan. Tak cukup itu, masing-masing bagian juga mengalokasikan waktu khusus untuk mengadakan pertemuan internal pada tiap pekan (Observasi, 2021). g p p p p ( ) Pondok Pesantren Miftahul Ulum di dalam pendidikan pesantrennya memfokuskan pada dua konsentrasi kompetensi santri, yaitu pada kajian- kajian kitab kuning (Pesantren Salafi), penguatan bahasa dan pada hafalan Al- Qur’an (Pesantren Tahfidz), dari ketiga konsentrasi tadi wali santri dan santri boleh memilih salah satu pada saat pendaftaran, sehingga nantinya para santri dibimbing dan dibina sesuai dengan konsentrasi kompetensi yang dipilihnya. 2. Manajemen Mutu Keterpaduan Akademik Untuk mengetahui sejauh mana santri menguasai materi yang telah diberikan, Pondok Pesantren Miftahul Ulum mengadakan tes evaluasi pengajian di akhir semester, selanjutnya diketahuilah hasilnya melalui rapot santri itu sendiri. Untuk pesantren tahfidz, ujian yang dilaksanakan terus berkesinambungan baik dari ujian mingguan yaitu santri melanjutkan ayat yang dibacakan Ustadz, ujian bulanan yaitu santri melakukan Sima’an dengan membaca hafalan didepan santri yang lain, dan terakhir ujian tahunan yang dilaksanakan di akhir semester adalah dengan menggunakan Tes Tamrinatul’am, minimal hafalan untuk santri setiap tahunnya adalah dua juz (Observasi, 2021). Sistem bandongan adalah strategi khas yang biasa dijumpai di Pesantren salafi, didalam strategi ini seluruh santri berkumpul di Majlis untuk menyimak ustadz membacakan kitab kuning beserta artinya lalu santri melogat (menulis) kembali artinya setelah itu ustadz menjelaskan isi dari kitab yang sudah 120 dibacakan (Kepala Pesantren, Hasil Wawancara, 2021). Sistem klasikal adalah metode yang diambil dari pendidikan kontemporer atau modern, dimana santri dibagi menjadi beberapa tingkat kemudian pada masing-masing tingkatan dikaji ilmu-ilmu agama yang bersumber dari kitab kuning. g j g y g g Tingkatan dan materi pelajaran yang ada di Pondok Evaluasi menjadi tahapan yang penting karena dengan diadakannya evaluasi pengelola dapat mengetahui strategi yang dilakukan terlaksana dengan tepat dan mencapai tujuan. Evaluasi strategi pada intinya adalah untuk mengetahui sejauh mana keberhasilan strategi yang dilakukan dalam meningkatkan kualitas santri itu sendiri. 3. Manajemen Mutu Kontrol Moral dan Etika Individu Sebagaimana ciri khas Pondok Pesantren Miftahul ulum tergambar dari visi STIS Miftahul Ulum yaitu mencetak sarjana profisional dan berkarakter ulama berlandasakan nilai ahlussunnah wal jama’ah (Profil STIS Miftahul Ulum, 2021). Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Hasil Penelitian Keseriusan Pesantren STIS Miftahul Ulum dalam pembentukan akhlaq ditunjukan dengan adanya budaya akhlaqul karimah yang dijalankan, apabila ada santri yang kurang baik akhlaqnya ke Kiai, Guru bahkan ke tamu maka santri yang lain akan mengingatkan santri yang bersangkutan, inilah yang disebut pembentukan budaya pesantren agar santri menjadi terbiasa berbuat baik (Observasi, 2021).. ( ) Untuk mengukur kualitas secara individual ini, peneliti mengamati dari tingkat kedisiplinan santri mengikuti kegiatan-kegiatan pesantren seperti shalat subuh berjamaah, pengajian empat kali dalam sehari dan shalat dhuha. Sebagaimana dalam pengalaman pribadi penulis, salah satu peningkatan kualitas ibadah santri itu ditandai dengan kegiatan ibadahnya pada dini hari, sebelum subuh dan menjelang subuh. Penulis mengamati bahwa pada kegiatan ini pesantren tidak ada mekanisme yang ketat agar seluruh santri shalat berjamaah Subuh, namun penulis menyaksikan hampir semua santri laki-laki ikut melaksanakan Shalat Subuh berjamaah bahkan sebelum adzanpun santri sudah melakukan ibadah-ibadah yang lain (Observasi, 2021). Salah satu langkah sistem mutu pendidikan individu mahasiswa adalah dengan memfokuskan santri-santri belajar kitab-kitab klasik/kitab kuning. Pesantren salafi yang dimaksudkan adalah pendidikan kepesantrenan dengan menggunakan kurikulum sederhana bermuatan mata pelajaran-mata pelajaran yang disusun berdasarkan kebutuhan dan kemampuan santri, metode yang khas, dan sumber belajar kitab-kitab kuning. Hal ini masuk pada katagori ciri khas pendidikan agama Islam. 4. Manajemen Mutu Pendidikan Sosial dan Budaya Pondok Pesantren Miftahul Ulum sendiri dalam bidang sosial, mengikut sertakan santri-santrinya dalam pembangunan Sumber daya manusia dan pengembangan taraf hidup masyarakat sekitar, Miftahul Ulum berupaya 121 melatih santri agar hidup berdampingan dengan masyarakat sekitar melalui berbagai kegiatan seperti kerja bakti di lingkungan pesantren, mengadakan bakti sosial berupa santunan kepada yatim piatu, khitanan masal, pengobatan gratis dan lain sebagainya. Ini semua merupakan kebijakan strategis yang dilakukan oleh kepesantrenan dalam melatih santri-santri berperan aktif serta hidup berdampingan dengan masyarakat (Kepala Pesantren, Hasil Wawancara, 2021). Pondok Miftahul Ulum sebagai benteng dakwah membekali santri- santrinya selain dengan pemahaman agama namun juga memiliki kemampuan dalam praktek muamalah dengan masyarakat. Pesantren Miftahul Ulum menyebutnya ilmu-ilmu kemasyarakatan seperti dakwah dan berorganisasi, yang nantinya santri siap dan tidak kaget menjadi solusi menghadapi fenomena di masyarakat. Pembahasan Hasil penelitian menunjukkan mutu sistem pendidikan Pondok Pesantren Miftahul Ulum Lumajang diawali dengan integrasi visi antar lembaga yang berada dibawah naungan yayasan Miftahul Ulum Lumajang. Hal ini ditandai dengan telah berdirinya lembaga pendidikan formal (madrasah) dari jenjang Madrasah Ibtidaiyyah, Madrasah Tsanawiyah, Madrasah Aliyah dan juga Perguruan Tinggi (STIS Miftahul Ulum Lumajang). Sebagaimana pendapat (Umulkulsum and Suaji 2020) bahwa perubahan tersebut dimaksudkan untuk mencetak lulusan yang mampu berdaya saing dalam konteks perkembangan zaman di satu sisi dan santri yang tetap mempertahankan nila-nilai ahlussunah wal jama’ah. Tahap kedua melalui mutu sistem pendidikan peningkatan mutu akademik santri di Pesantren Miftahul Ulum, santri dibina pada tiga pembinaan kualitas, yaitu pembinaan pada pengajian, kedua pembinaan pada ekstrakulikuler, ketiga pembinaan pada kegiatan di lingkungan pesantren. Dalam implementasinya, mata pelajaran tersebut disisipkan pada sistem pengajiannya, ada dua sistem pengajian yang diterapkan di Pesantren Madin Miftahul Ulum yaitu sistem bandongan atau pengajian umum dan sistem klasikal atau tingkatan-tingkatan (Nugraheni 2021). Hasil dari penelitian ini adalah manajemen mutu dilakukan dengan cara pembinaan kedisiplinan, keteladanan, kesederhanaan, pembiasaan dan memperbanyak kegiatan kegiatan yang bisa memaksimalkan potensi dari setiap santri. Untuk bidang kepesantrenan PPM Madin Miftahul Ulum memiliki program-program wajib seperti Wajib Berjamaah, Qiyamul-Lail, One Day One Verse, Literasi, Sholat Dhuha, Musyawarah, Ziarah, pengabsenan, belajar kosakata Bahasa Arab dan Inggris, Baca al-Qur’an, Halaqoh bersama Ketua Kepengasuhan, pelatihan public speaking. 122 Temuan di atas, sesuai dengan pendapat (Sahid et al. 2021), bahwa Pendidikan Agama Islam sebagai pendidikan yang didasarkan pada nilai-nilai ajaran Islam sebagaimana yang tercantum dalam Al-Qur’an dan Al-Hadits. Berbagai komponen dalam pendidikan dimulai dari tujuan, kurikulum, guru, metode, pola hubungan guru murid, sarana prasarana, lingkungan dan evaluasi pendidikan harus didasarkan pada nilai-nilai ajaran Islam. p p j Upaya yang dilakukan pesantren dalam mutu sistem pendidikan guna meningkatkan kepekaan sosial santri menumbuhkan rasa simpati dan empati kepada santri karena rasa simpati dan empati sangat penting di tanamkan kepada santri agar santri peka dalam situasi dan keadaan yang ada di sekitar serta saling membantu orang yang ada di sekitar, melalui berbagai kegiatan yng bersentuhan dengan berbagai lapisan masyarakat (Mukhtar, Hidayat, and Ulfah 2020),. Temuan tersebut sesuai dengan pendapat (Nasser et al. 2022), bahwa hakekat pendidikan manusia yang baik atau berkarakter Islami (akhlak karimah). Akhlak yang baik hanya dapat tumbuh dari pengulangan dan pembiasaan setiap waktu. Selain itu, penanaman akhlak harus diawasi dan dibimbing oleh guru yang berakhlak pula. Akhlak dapat terlihat dari perkataan, sikap dan tingkah laku keseharian. Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Pembahasan Upaya yang dilakukan pesantren dalam manajemen mutu pendidikan guna meningkatkan kepekaan sosial mahasantri menumbuhkan rasa simpati dan empati kepada santri karena rasa simpati dan empati sangat penting di tanamkan kepada santri agar santri peka dalam situasi dan keadaan yang ada di sekitar serta saling membantu orang yang ada di sekitar, melalui berbagai kegiatan yng bersentuhan dengan berbagai lapisan masyarakat (Komsiyah 2021). Temuan penelitian sejalan dengan penjelasan (Nurzannah, Ginting, and Setiawan 2020) dan (Muchith 2019), yang menyatakan bahwa Keterampilan Sosial (social skill) merupakan kemampuan untuk menciptakan hubungan sosial yang serasi dan memuaskan, penyesuaian terhadap lingkungan sosial dan memecahkan masalah sosial yang dihadapi serta mampu mengembangkan aspirasi dan menampilkan diri, dengan ciri saling menghargai, mandiri, mengetahui tujuan hidup, disiplin dan mampu membuat keputusan. Keterampilan sosial dapat berupa keterampilan komunikasi, solusi konflik, dengan teman yang memilik karakter berbeda di pesantren Kesimpulan Hasil penelitian menunjukkan keberhasil manajemen mutu untuj meningkatkan daya saing lulusan Perguruan Tinggi di Pondok Pesantren Miftahul Ulum Lumajang diawali dengan integrasi visi antar lembaga yang berada dibawah naungan yayasan Miftahul Ulum Lumajang. Hal ini ditandai dengan telah berdirinya lembaga pendidikan formal dari Madrasah Ibtidaiyah 123 (MI) Miftahul Ulum hingga Perguruan Tinggi (STIS Miftahul Ulum Lumajang). Tahap kedua melalui manajemen mutu akademik santri di Pesantren Miftahul Ulum, santri dibina pada tiga pembinaan kualitas, yaitu pembinaan pada pengajian, kedua pembinaan pada ekstrakulikuler, ketiga pembinaan pada akhlak para santri, dalam pembentukan akhlak ditunjukan dengan adanya budaya akhlaqul karimah yang dijalankan, apabila ada santri yang kurang baik akhlaqnya ke Kiai, Guru bahkan ke tamu maka santri yang lain akan mengingatkan santri yang bersangkutan, inilah yang disebut pembentukan budaya pesantren agar santri menjadi terbiasa berbuat baik kegiatan di lingkungan pesantren. Langkah terakhir adalah manajemen mutu pembekalan santri- dengan kemampuan dalam praktek muamalah dengan masyarakat. Implementasinya dengan cara mengikut sertakan santri-santrinya dalam pembangunan Sumber daya manusia dan pengembangan taraf hidup masyarakat sekitar, Miftahul Ulum berupaya melatih santri agar hidup berdampingan dengan masyarakat sekitar melalui berbagai kegiatan seperti kerja bakti di lingkungan pesantren, mengadakan bakti sosial berupa santunan kepada yatim piatu, khitanan masal, pengobatan gratis dan lain sebagainya. Keberhasilan strategi dalam meningkatkan kualitas santri secara akademik tidak hanya dibuktikan dengan penguasaan materi saja, prestasi santri pula dapat diukur dari keberhasilan meraih juara pada setiap perlombaan-perlombaan. 124 Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Daftar Pustaka Darojat, Zakiyatu. 2019. “Implementation Of Islamic Boarding School Rules In Establishing Student Discipline In Jagad ‘Alimusirry Islamic Student Boarding Schools.” Journal Intellectual Sufism Research (JISR) 1 (2): 19– 22.. Ganseuer, Christian, and Solveig Randhahn. 2017. Quality Management and Its Linkages to Higher Education Management. Module 5. Training on Internal Quality Assurance Series. https://doi.org/10.17185/duepublico/43226. Hardianto, Hardianto. 2019. “Reposition of Historical Pesantren, Madrasah and Integrated Islamic School.” Edumaspul: Jurnal Pendidikan 3 (2): 75–86. Ijah, Titin, Totok Sumaryanto Florentinus, and I Made Sudana. 2021. “The Quality Assurance of Islamic Boarding School Based on Total Quality Management ( TQM ).” Educational Management 10 (1): 42–49. Ilyasin, Mukhamad. 2020. “Transformation of Learning Management : Integrative Study of Islamic Boarding School Curriculum.” DINAMIKA ILMU 20 (1): 13–22. Komsiyah, Indah. 2021. “Implementation of Internal Quality Assurance to Improve the Quality of Islamic Education.” AL-ISHLAH: Jurnal Pendidikan 13 (3): 41–48. Morgan, G. A., and R. J. Harmon. 2001. “Data Collection Techniques.” Journal of the American Academy of Child and Adolescent Psychiatry 40 (8): 973–76.. Muchith, Mukhamad Saekan. 2019. “Total Quality Management Pendidikan Islam (Studi TPQ Qiro’ati Di Kabupaten Kudus Jawa Tengah).” Al-Izzah: Jurnal Hasil-Hasil Penelitian 14 (2): 17.. Mukhtar, Mukhtar, Hidayat Hidayat, and Siti Mariah Ulfah. 2020. “Implementation of Total Quality Management in Developing Santri Characters.” International Journal of Southeast Asia 1 (2): 1–12. Nasser, Asep Azis, Sutaryat Trisnamansyah, Achmad Mudrikah, and Yosal Iriantara. 2022. “Strengthening Character Education Of Madrasah Students Based On Boarding School (Case Study At MAN Insan CendekiaSerpong, South Tangerang City).” International Journal of Educational Research & Social Sciences, no. 87: 653–67. Nugraheni, Yumidiana Tya. 2021. “Model Pengembangan Pendidikan Karakter Di Pesantren Khalaf ( Studi Kasus Di Pondok Pesantren Modern Muhammadiyah Boarding School Yogyakarta ).” JOURNAL OF EMPIRICAL RESEARCH IN ISLAMIC EDUCATION 9 (1): 39–56. Nurzannah, Nurzannah, Nurman Ginting, and Hasrian Rudi Setiawan. 2020. “Implementation Of Integrated Quality Management In The Islamic 125 Education System.” Proceeding International Seminar of Islamic Studies 1 (1): 1–9.. R.Sherman, Robert. 2006. Qualitative Research in Education: Focus and Methods. Library of Congress. IX. New York, NY 10001: Falmer Press RoutledgeFalmer. Rahi, Samar. 2017. “Research Design and Methods: A Systematic Review of Research Paradigms, Sampling Issues and Instruments Development.” International Journal of Economics & Management Sciences 06 (02).. Sahid, Ujang, Iim Wasliman, Hendi Suhendraya Muchtar, and Husen Saeful Insan. 2021. Qolamuna : Jurnal Studi Islam Vol. 08 No. 1 (2022) 116-126 Available online at https://ejournal.stismu.ac.id/ojs/index.php/qolamuna Daftar Pustaka “Management of Student Characteristics Through Extracurricular Activities in The School Environment Based on Islamic Boarding Schools.” Munaddhomah: Jurnal Manajemen Pendidikan Islam 2 (2): 116–25. Sonia, Nur Rahmi. 2021. “Total Quality Management Dalam Lembaga Perguruan Tinggi.” Southeast Asian Journal of Islamic Education Management 2 (1): 125–39. Umulkulsum, Dara, and R. Achmad Drajat Aji Suaji. 2020. “A New Decade for Social Changes.” Technium Social Sciences Journal 7: 312–20. 126
https://openalex.org/W4393863659	https://jurnal.unimus.ac.id/index.php/MKI/article/download/9227/pdf	Indonesian	null	Parents income and nutritional status of toddlers during the covid-19 pandemic	Media Keperawatan Indonesia	2,023	cc-by	3,909	Jl. Kedungmundu Raya No. 18 Semarang Gedung NRC Universitas Muhammadiyah Semarang Phone: 02476740287, Fax: 02476740287 Email: mki@unimus.ac.id Research article Jl. Kedungmundu Raya No. 18 Semarang Gedung NRC Universitas Muhammadiyah Semarang Phone: 02476740287, Fax: 02476740287 Email: mki@unimus.ac.id Research article Jl. Kedungmundu Raya No. 18 Semarang Gedung NRC Universitas Muhammadiyah Semarang Phone: 02476740287, Fax: 02476740287 Email: mki@unimus.ac.id parents income and nutritional status of toddlers during the covid-19 pandemic Nur Umi Amanah1, Lantin Sulistyorini1, Peni Perdani Juliningrum1, Iis Rahmawati1, Nuning Dwi Merina1 1 Program Studi Sarjana Keperawatan, Fakultas Keperawatan, Universitas Jember, Indonesia The nutritional status of a toddler can be influenced by a parent's income because meeting the nutritional needs of a toddler, is very dependent on parent's income. This study aims to analyze the Relationship between Parent's Income and Nutritional Status of Toddlers During the Covid-19 Pandemic at the Integrated Health Post in Alassumur Kulon Village, Kraksaan District, Probolinggo Regency District, Probolinggo Regency. The research design used was an analytic observational design, with a cross- sectional research design. The number of samples was 80 respondents with the purposive sampling method. This study uses univariate analysis to determine the frequency distribution and bivariate analysis using Spearman's Rho test. The results show that most of the parents' income is in the medium category, with as many as 31 respondents (38.8%). Most of the nutritional status of toddlers is in the good nutrition category, with as many as 46 respondents (57.5%). The results showed that there was a Relationship between Parent's Income and the Nutritional Status of Toddlers During the Covid-19 Pandemic at the Integrated Health Post in Alassumur Kulon Village, Kraksaan District, Probolinggo Regency with a p- value of 0.000 (<0.05), so Ha was accepted. The conclusion is that the income of parents affects the availability of food and the fulfillment of nutritional intake which has an impact on the nutritional status of toddlers. The role of parents in efforts to fulfil children's nutritional intake is very important, managing their finances while still paying attention to the nutritional needs of children is the key to success in maintaining the good nutritional status of toddlers. Keywords: Parents income; Nutritional status; Toddlers; COVID-19 pandemic terjadinya infeksi pada sistem pernapasan yang biasa disebut dengan COVID-19. Virus Corona ini begitu cepat menyebar hingga pada berbagai belahan dunia, setidaknya diketahui terdapat 188 negara yang mengkorfirmasi telah terpapar oleh virus Corona ini [1]. Berdasarkan data resmi COVID-19 pada tanggal 19 Agustus 2020, PENDAHULUAN Di China pada akhir bulan Desember 2019 muncul suatu wabah penyakit baru, tepatnya pada tanggal 30 Desember 2019. World Health Organization (WHO) mengungkapkan bahwa Coronaviruses (Cov) merupakan virus yang menyebabkan Corresponding author: Nur Umi Amanah nurumiamanah3@gmail.com Media Keperawatan Indonesia, Vol 6 No 4, Dec 2023 e-ISSN: 2615-1669 ISSN: 2722-2802 DOI: 10.26714/mki.6.4.2023.285-292 Corresponding author: Nur Umi Amanah nurumiamanah3@gmail.com Media Keperawatan Indonesia, Vol 6 No 4, Dec 2023 e-ISSN: 2615-1669 ISSN: 2722-2802 DOI: 10.26714/mki.6.4.2023.285-292 Media Keperawatan Indonesia, Vol 6 No 4, Dec 2023/ page 277-292 286 total 74 sampel balita, sedangkan balita yang mengalami gizi buruk yang berasal dari keluarga dengan pendapatan terpenuhi sebanyak 6 balita. Selain itu, terdapat penelitian lain yang dilakukan oleh Roficha (2018) [9] tentang Pengetahuan Gizi Ibu dan Sosial Ekonomi Keluarga Terhadap Status Gizi Balita Umur 6-24 Bulan, dimana menunjukkan persentase balita yang mengalami gizi kurang dari orang tua berpendapatan kurang sebanyak 8,7% dan balita yang mengalami status gizi sangat kurang dari keluarga berpendapatan kurang sebanyak 7,2%. tercatat sebanyak 143.043 kasus yang telah terkonfirmasi COVID-19 di Indonesia. Virus ini dapat mengenai siapapun tidak memandang kelompok usia, bahkan pada kelompok usia balita sekalipun [2]. Untuk dapat mencegah tertularnya penyakit tersebut diperlukan kepatuhan dalam menerapkan pola hidup sehat serta membatasi kontak fisik dengan menjaga jarak aman, kemudian hal penting lainnya yaitu meningkatkan daya tahan tubuh dengan tercukupinya asupan zat gizi, sehingga status gizi dalam keadaan baik [3]. Status gizi merupakan keadaan tubuh berdasarkan keseimbangan antara intake asupan gizi dalam tubuh dengan zat gizi yang di butuhkan [4]. Terdapat berbagai fungsi dari terpenuhinya zat gizi/ nutrisi bagi balita yaitu sebagai sumber energi, menyokong pertumbuhan badan, memelihara jaringan tubuh, mengatur metabolisme tubuh, dan sebagai pertahanan tubuh terhadap berbagai penyakit [5]. Apabila status gizi balita buruk, maka dapat berdampak terhadap proses tumbuh kembang balita tersebut, sehingga menimbulkan berbagai masalah kesehatan, termasuk dapat menyebabkan penurunan kekebalan tubuh sampai meningkatkan risiko terjadinya penyakit infeksi [6]. Sektor perekonomian saat ini banyak yang terganggu akibat dari adanya kebijakan lockdown. Beberapa sektor perekonomian atau usaha yang terganggu, misalnya buruh migran, pedagang usaha kecil, pekerja upahan harian, dan pedagang kaki lima [10]. Sejalan dengan hal tersebut, pada dasarnya daya tahan ekonomi informal relatif lemah, terutama pada pekerja dengan penghasilan harian, grosir dan pedagang eceran yang biasanya dibayar rendah dan tak terlindungi [11]. Sedangkan di bidang sektor pariwisata juga terancam, karena di masa pandemi mengakibatkan penurunan pengunjung ke Indonesia. Nur Umi Amanah / parents income and nutritional status of toddlers during the covid-19 pandemic PENDAHULUAN Sektor yang mendukung pariwisata, seperti rumah makan, penginapan, sampai dengan pengusaha retail ikut terkena dampaknya. Selain itu, sektor usaha mikro, kecil, sampai menengah juga ikut terpengaruh, hal tersebut disebabkan karena sepinya wisatawan/ pengunjung [12]. Tujuan umum penelitian ini untuk mengetahui ada tidaknya hubungan antara pendapatan orang tua dengan status gizi balita selama masa pandemi COVID-19 di Posyandu Desa Alassumur Kulon, Kecamatan Kraksaan, Kabupaten Probolinggo. Hasil penelitian ini diharapkan dapat menjadi sumber informasi terkait hubungan pendapatan orang tua dengan status gizi balita agar dapat lebih memperhatikan pemenuhan asupan gizi yang dibutuhkan balita. Status gizi pada balita dapat dipengaruhi oleh beberapa faktor, salah satu contohnya adalah pola serta kebiasaan orang tua dalam memilih menu makanan yang kemudian berdampak bagi tumbuh kembang anak, apabila konsumsi yang diberikan tidak sesuai dengan kebutuhan maka akan menggambarkan gizi buruk bahkan mengarah pada pertumbuhan yang tidak ideal, untuk itu dalam upaya pemenuhan konsumsi kebutuhan gizi anak sangat bergantung dari pendapatan orang tua [7]. Menurut hasil penelitian yang dilakukakan oleh Kasumayanti (2020)[8] menjelaskan bahwa balita yang mengalami gizi buruk lebih banyak berasal dari keluarga dengan pendapatan yang tidak terpenuhi yaitu sebanyak 35 balita dari ur Umi Amanah / parents income and nutritional status of toddlers during the covid-19 pandemic Media Keperawatan Indonesia, Vol 6 No 4, Dec 2023/ page 277-292 287 dilakukan pengecekan kembali terkait kelengkapan kuesioner oleh tim peneliti. METODE Desain penelitian yang digunakan pada penelitian ini adalah desain penelitian observasional analitik Cross Sectional. Variabel yang diteliti yaitu pendapatan orang tua dan status gizi balita. Populasi pada penelitian ini yaitu sebanyak 387 balita yang tergabung dalam posyandu desa Alassumur Kulon, kecamatan Kraksaan, kabupaten Probolinggo. Sampel pada penelitian ini adalah balita yang memenuhi kriteria inklusi sejumlah 80 responden. Pengambilan sampel dengan teknik purposive sampling. Analisis data dengan analisis univariat dan analisis bivariat. Analisis bivariat menggunakan uji korelasi Spearman’s Rho untuk melihat apakah adanya hubungan antara kedua variabel yang berskala data ordinal dengan ketentuan Ha diterima jika p<0,05. Penelitian ini dilakukan sesuai dengan etika penelitian yang meliputi lembar persetujuan (informed consent), kerahasiaan (confidentiality), keadilan (justice) dan kemanfaatan (beneficiency). Tahapan awal atau persiapan melakukan izin etik penelitian di Fakultas Keperawatan Universitas Jember dan mengajukan surat perizinan penelitian kepada instansi Fakultas Keperawatan UNEJ, LP2M, Dinas Kesehatan Kabupaten Probolinggo, BAKESBANGPOL Kabupaten Probolinggo. Peneliti menentukan populasi, sampel, dan responden yang akan digunakan untuk penelitian sesuai dengan kriteria inklusi dan kriteria eksklusi yang telah ditentukan oleh peneliti, kemudian responden didampingi oleh pihak tim peneliti dalam menandatangani lembar persetujuan untuk menjadi responden, sebelum itu tim peneliti membacakan lembar informasi terkait penelitian yang akan dilakukan secara jelas kepada responden. HASIL Hasil penelitian menunjukkan bahwa mayoritas pendidikan orang tua balita adalah SLTA/SMA dengan jumlah 58 responden (72,5%). Pekerjaan mayoritas orang tua balita adalah wiraswasta dengan jumlah responden sebanyak 40 (50%). Lama masa kerja orang tua balita terbanyak terdapat pada kisaran waktu 1 s/d 5 tahun sebanyak 30 responden (37,5%) dan 6 s/d 10 tahun sebanyak 30 responden (37,5%). Jumlah anggota keluarga mayoritas berjumlah 4 orang yaitu sebanyak 34 responden (42,5%). Asupan gizi balita mayoritas berada pada kategori cukup yaitu sebanyak 50 responden (62,5%). Sanitasi lingkungan berada pada kategori sanitasi baik dengan jumlah 80 responden (100%). Hasil penelitian menunjukkan bahwa mayoritas pendidikan orang tua balita adalah SLTA/SMA dengan jumlah 58 responden (72,5%). Pekerjaan mayoritas orang tua balita adalah wiraswasta dengan jumlah responden sebanyak 40 (50%). Lama masa kerja orang tua balita terbanyak terdapat pada kisaran waktu 1 s/d 5 tahun sebanyak 30 responden (37,5%) dan 6 s/d 10 tahun sebanyak 30 responden (37,5%). Jumlah anggota keluarga mayoritas berjumlah 4 orang yaitu sebanyak 34 responden (42,5%). Asupan gizi balita mayoritas berada pada kategori cukup yaitu sebanyak 50 responden (62,5%). Sanitasi lingkungan berada pada kategori sanitasi baik dengan jumlah 80 responden (100%). Tahapan selanjutnya yaitu pengumpulan data, pelaksanaan peneliti dan pengumpulan data dilakukan di posyandu desa pada pertemuan rutin yang dilakukan setiap bulannya. Tim peneliti membagikan kuesioner penelitian dan ballpoint kepada setiap orang tua balita. Mengukur variabel independen yaitu pendapatan orang tua dengan memberikan kuesioner terkait data demografi dan variabel dependen status gizi balita dengan melihat data KMS beserta data kunjungan balita yang disediakan oleh kader. Pengisisian kuesioner oleh responden didampingi oleh tim peneliti. Tim peneliti mengumpulkan kembali kuesioner setelah diisi oleh responden dan Hasil penelitian menunjukkan bahwa mayoritas pendapatan orang tua balita berada pada kategori sedang sebanyak 31 responden (38,8%), yakni pada rentang pendapatan 900.000 – 1.800.000 per bulan. Hasil penelitian menunjukkan bahwa mayoritas status gizi balita di Posyandu Desa Alassumur Kulon, Kecamatan Kraksaan, Kabupaten Probolinggo berada pada kategori gizi baik sebanyak 46 responden (57,5%). ur Umi Amanah / parents income and nutritional status of toddlers during the covid-19 pandemic Media Keperawatan Indonesia, Vol 6 No 4, Dec 2023/ page 277-292 288 berdasarkan hasil tersebut dapat diartikan bahwa terdapat hubungan yang signifikan antara pendapatan orang tua dengan status gizi balita di Posyandu Desa Alassumur Kulon, Kecamatan Kraksaan, Kabupaten Probolinggo. Hasil nilai korelasi (r) yaitu 0,385 terdapat pada rentang 0,20-0,39 maka tergolong pada kategori lemah. HASIL Berdasarkan hasil dari uji korelasi Spearman’s Rho diperoleh nilai p value yaitu 0,000 (<0,05) maka Ha diterima, Tabel 1 Karakteristik responden (n=80) Indikator f % Pendidikan Orang Tua Tidak sekolah SD SLTP/SMP SLTA/SMA Sarjana - 1 13 58 8 - 1,3 16,3 72,5 10,0 Pekerjaan Orang Tua PNS Wirausaha Wiraswasta Petani/buruh tani Pegawai pemerintah/swasta Ibu rumah tangga 4 2 40 26 8 - 5,0 2,5 50,0 32,5 10,0 - Lama masa kerja 1 s/d 5 tahun 6 s/d 10 tahun 11 s/d 15 tahun 16 s/d 20 tahun >20 tahun 30 30 13 6 1 37,5 37,5 16,3 7,5 1,3 Jumlah anggota keluarga 2 orang 3 orang 4 orang 5 orang 1 30 34 15 1,3 37,5 42,5 18,8 Asupan gizi Kurang Cukup Baik 3 50 27 3,8 62,5 33,8 Sanitasi lingkungan Sanitasi baik Sanitasi buruk 80 - 100 - Pendapatan orang tua Rendah (<900.000) Sedang (900.000-1.800.000) Tinggi (>1.800.000) 30 31 19 37,5 38,8 23,8 Status gizi balita Gizi buruk Gizi kurang Gizi baik Berisiko gizi lebih Gizi lebih Obesitas 7 20 46 4 2 1 8,8 25,0 57,5 5,0 2,5 1,3 ur Umi Amanah / parents income and nutritional status of toddlers during the covid-19 pandemic Nur Umi Amanah / parents income and nutritional status of toddlers during the covid-19 pandemic Media Keperawatan Indonesia, Vol 6 No 4, Dec 2023/ page 277-292 289 Tabel 2 Hubungan pendapatan orang tua dengan status gizi balita (n=80) Status Gizi Balita Pendapatan Orang Tua Jumlah Nilai korelasi (r) p- value Rendah (< 900.000) Sedang (900.000 s/d 1.800.000) Tinggi (> 1.800.000) total % Gizi buruk (severely wasted) 5 0 2 7 8,8 0,385 0,000 Gizi kurang (wasted) 15 3 2 20 25 Gizi baik (normal) 9 23 14 46 57,5 Berisiko gizi lebih (possible risk of overweight) 0 4 0 4 5 Gizi lebih (overweight) 1 1 0 2 2,5 Obesitas (obese) 0 0 1 1 1,3 Total 30 31 19 80 100 Tabel 2 pekerjaan dapat menentukan tingkat pendapatan seseoraang [15]. HASIL Analisa hubungan pendapatan orang tua dengan status gizi balita selama masa pandemi COVID-19 di Posyandu Desa Alassumur Kulon, Kecamatan Kraksaan, Kabupaten Probolinggo tahun 2022 dilakukan dengan menggunakan uji Spearman-rho. Berdasarkan hasil dari uji korelasi Spearman’s Rho diperoleh nilai p value yaitu 0,000 (<0,05) maka Ha diterima, Analisa hubungan pendapatan orang tua dengan status gizi balita selama masa pandemi COVID-19 di Posyandu Desa Alassumur Kulon, Kecamatan Kraksaan, Kabupaten Probolinggo tahun 2022 dilakukan dengan menggunakan uji Spearman-rho. Karakteristik responden Mayoritas lama masa kerja orang tua balita dalam penelitian ini berada pada kisaran waktu 1 s/d 5 tahun dan 6 s/d 10 tahun, karena pada 2 kategori rentang waktu tersebut memiliki kesamaan jumlah yaitu 30 responden pada masing-masing kategori. Lama masa kerja juga dapat mempengaruhi pendapatan, ini dikarenakan apabila semakin lama masa kerja, maka dapat meningkatkan kemampuan dan pengalaman seseorang dalam bekerja yang kemudian juga dapat meningkatkan pendapatan [16]. Hasil penelitian ini menunjukkan mayoritas pendidikan orang tua adalah SLTA/SMA, pendidikan orang tua yang paling rendah adalah lulusan SD dan pendidikan tertinggi adalah sarjana. Pendidikan menjadi salah satu faktor yang dapat mempengaruhi pendapatan, apabila tingkat pendidikan semakin tinggi maka semakin tinggi pula pendapatan yang diperoleh, begitupun sebaliknya [13]. Tingkat pendidikan orang tua juga berkaitan dengan pemenuhan asupan gizi balita yang dapat berpengaruh terhadap status gizi balita tersebut, karena dengan pendidikan orang tua yang baik maka pemahaman dalam merawat dan mengasuh anak juga akan baik, selain itu pendidikan orang tua dapat mempengaruhi sikap serta perilaku mereka dalam memanfaatkan fasilitas pelayanan kesehatan dan menjaga kebersihan lingkungan dengan baik [14]. Mayoritas jumlah anggota keluarga responden pada penelitian ini terdiri dari 4 orang, itu berarti yang menjadi tanggungan keluarga adalah sebanyak 4 orang. Jumlah anggota keluarga dapat memberi pengaruh dalam pendapatan keluarga, apabila setiap anggota memiliki penghasilan sendiri maka akan dapat meningkatkan pendapatan keluarga, namun sebaliknya apabila jumlah anggota keluarga banyak dan yang memiliki penghasilan sedikit, maka akan menambah beban keluarga atau tanggungan keluarga [13]. Menurut Lestari, (2016) dalam Hanum, (2018)[17] menjelaskan bahwa jumlah tanggungan anggota keluarga dalam suatu rumah tangga berpengaruh terhadap tingkat konsumsi yang harus dikeluarkan karena kebutuhan keluarga yang semakin banyak [17]. Mayoritas pekerjaan orang tua balita pada penelitian ini yaitu wiraswasta, dimana wiraswasta sendiri dapat terbagi menjadi berbagai bidang pekerjaan, seperti pedagang dan peternak. Pekerjaan juga dapat menjadi salah satu faktor yang mempengaruhi pendapatan. Menurut Putri dan Nyoman, (2013) dalam Kapisa, (2021)[15] menjelaskan bahwa pekerjaan seseorang dapat mempengaruhi pendapatan yang didapatkan, karena jenis ur Umi Amanah / parents income and nutritional status of toddlers during the covid-19 pandemic Media Keperawatan Indonesia, Vol 6 No 4, Dec 2023/ page 277-292 290 pertumbuhannya terganggu [18]. Berdasarkan hasil dari kuesioner sebanyak 80 responden menjawab dengan baik, dimana dikatakan sanitasi baik apabila responden menjawab dengan benar sejumlah 11-20 soal, untuk masing–masing soal benar mendapatkan nilai 1, sedangkan dikatakan sanitasi buruk apabila responden hanya bisa menjawab 1-10 soal jawaban benar. Pendapatan orang tua selama masa pandemi COVID-19 Pendapatan orang tua balita mayoritas berada pada rentang pendapatan 900.000 – 1.800.000 per bulan, sehingga termasuk pada kategori pendapatan sedang, jumlah pendapatan tersebut dapat dipengaruhi oleh beberapa hal, seperti pendidikan, jenis pekerjaan, lama masa kerja, dan jumlah anggota keluarga. Kategori pendapatan di Desa Alassumur Kulon, Kecamata Kraksaan, Kabupaten Probolinggo termasuk rendah jika dibandingkan dengan UMK untuk kabupaten Probolinggo, dimana nominalnya mencapai Rp. 2.553.269 berdasarkan Surat Keputusan (SK) Gubernur Jawa Timur Nomor: 188/538/KPTS/013/2020 [21]. Perbedaan pendapatan ini disebabkan oleh mayoritas pekerjaan yang ada di Desa Alassumur Kulon adalah sebagai wiraswasta dan petani, dimana upahnya tidak bergantung pada UMK Probolinggo. Sanitasi lingkungan responden berada pada kategori sanitasi baik, Sanitasi lingkungan merupakan faktor secara tidak langsung yang dapat berpengaruh terhadap status gizi balita, dimana apabila kondisi sanitasi lingkungan tidak baik, maka akan memungkinkan untuk terjadinya berbagai macam penyakit, seperti diare, cacingan, serta infeksi saluran pencernaan, dan apabila anak mengalami infeksi saluran pencernaan maka akan dapat menghambat proses penyerapan zat-zat gizi yang kemudian dapat mengakibatkan Karakteristik responden Terdapat 43 responden menjawab 20 soal benar, 27 responden menjawab 19 soal benar, 9 responden menjawab 18 soal benar, dan 1 responden menjawab 17 soal benar. [18]. Asupan gizi / nutrisi pada balita yang terlibat dalam penelitian ini mayoritas berada dalam kategori cukup. Asupan gizi dapat menjadi faktor langsung yang dapat mempengaruhi status gizi balita, baik buruknya kuantitas dan kualitas asupan makanan yang dikonsumsi dapat menggambarkan baik buruknya status gizi balita [18]. terganggu Pemenuhan asupan gizi yang seimbang dapat dimulai dari 1000 hari pertama kehidupan, dimana dalam masa ini terbagi lagi menjadi 2 masa yaitu 270 hari (9 bulan) masa kehamilan dan masa 730 hari (2 tahun) setelah kelahiran bayi. Pemenuhan gizi seimbang tidak berhenti sampai usia anak 2 tahun saja, karena pada usia anak pra sekolah (3-6 tahun) juga perlu diperhatikan, dimana pada masa ini penting bagi perkembangan kognitif anak sekaligus membantu dalam mempertahankan daya tahan tubuhnya [14,19]. Dalam upaya pemenuhan asupan gizi perlu memperhatikan angka kecukupan gizi, bagi anak usia 1-3 tahun dianjurkan untuk mengkonsumsi makanan yang mengandung karbohidrat sebesar 215 gr/hari, protein sebesar 20 gr/hari, serat sebesar 19 gr/hari, lemak sebesar 45 gr/hari, dan air sejumlah 1150 ml/hari, sedangkan bagi anak yang berusia 4-6 tahun dianjurkan untuk mengkonsumsi makanan yang mengandung karbohidrat sebesar 220 gr/hari, protein sebesar 25 gr/hari, serat sebesar 20 gr/hari, lemak sebesar 50 gr/hari, dan air sejumlah 1450 ml/hari [20]. UCAPAN TERIMAKASIH Peneliti mengucapkan terimakasih kepada seluruh responden yang telah bersedia untuk berpartisipasi dalam penelitian ini dan kepada semua pihak yang telah membantu serta mendukung peneliti selama proses penelitian. Status gizi balita selama masa pandemi COVID-19 Status gizi balita di Posyandu Desa Alassumur Kulon, Kecamatan Kraksaan, Kabupaten Probolinggo mayoritas berada pada gizi baik, dimana hasil perhitungan status gizi berdasarkan IMT/U menunjukkan pada nilai -2 SD sd +1 SD, namun hasil juga menunjukkan terdapat 20 balita berada pada kategori status gizi ur Umi Amanah / parents income and nutritional status of toddlers during the covid-19 pandemic Media Keperawatan Indonesia, Vol 6 No 4, Dec 2023/ page 277-292 291 SIMPULAN kurang, dengan nilai perhitungan - 3 SD sd <- 2 SD dan 7 balita berada pada kategori status gizi buruk, dengan nilai perhitungan < -3 SD. Status gizi balita penting agar orang tua dapat memantau apakah gizi yang diberikan sudah tercukupi atau belum, apabila gizi tidak terpenuhi maka akan berdampak dalam proses tumbuh kembang, sehingga menimbulkan berbagai masalah kesehatan, termasuk dapat menyebabkan penurunan kekebalan tubuh sampai meningkatkan risiko terjadinya penyakit infeksi [6]. Berdasarkan hasil penelitian dan pembahasan diatas dapat disimpulkan bahwa terdapat hubungan antara pendapatan orang tua dengan status gizi balita selama masa pandemi COVID-19 di Posyandu Desa Alassumur Kulon, Kecamatan Kraksaan, Kabupaten Probolinggo. Hubungan aktivitas fisik dengan kualitas tidur lansia di masa pandemi COVID-19 Hasil penelitian yang dilakukan peneliti menunjukkan bahwa terdapat hubungan antara pendapatan orang tua dengan status gizi balita selama masa pandemi COVID-19 di Posyandu Desa Alassumur Kulon, Kecamatan Kraksaan, Kabupaten Probolinggo dengan nilai p value = 0,000, r = 0,385. Hasil dari penelitian ini memang terdapat hubungan antara pendapatan orang tua dengan status gizi balita namun korelasinya lemah, ini berarti tidak menutup kemungkinan bahwa asupan gizi juga menjadi salah satu faktor langsung yang dapat mempengaruhi status gizi balita, berdasarkan penelitian yang dilakukan oleh Septikasari (2016) dijelaskan bahwa apabila pendapatan orang tua dialokasikan untuk kebutuhan pangan maka ketersediaan asupan gizi keluarga terjamin, sehingga asupan gizi balita dapat terpenuhi dengan baik, namun sebaliknya jika alokasi pangan rendah maka akan meningkatkan risiko kejadian gizi kurang [14]. Berdasarkan hal tersebut maka peran orang tua dalam upaya pemenuhan asupan nutrisi anak sangatlah penting, mengatur keuangan yang dimiliki dengan tetap memperhatikan kebutuhan gizi anak menjadi kunci keberhasilan dalam mempertahankan status gizi balita yang baik. Hasil penelitian yang dilakukan peneliti menunjukkan bahwa terdapat hubungan antara pendapatan orang tua dengan status gizi balita selama masa pandemi COVID-19 di Posyandu Desa Alassumur Kulon, Kecamatan Kraksaan, Kabupaten Probolinggo dengan nilai p value = 0,000, r = 0,385. Hasil dari penelitian ini memang terdapat hubungan antara pendapatan orang tua dengan status gizi balita namun korelasinya lemah, ini berarti tidak menutup kemungkinan bahwa asupan gizi juga menjadi salah satu faktor langsung yang dapat mempengaruhi status gizi balita, berdasarkan penelitian yang dilakukan oleh Septikasari (2016) dijelaskan bahwa apabila pendapatan orang tua dialokasikan untuk kebutuhan pangan maka ketersediaan asupan gizi keluarga terjamin, sehingga asupan gizi balita dapat terpenuhi dengan baik, namun sebaliknya jika alokasi pangan rendah maka akan meningkatkan risiko kejadian gizi kurang [14]. Berdasarkan hal tersebut maka peran orang tua dalam upaya pemenuhan asupan nutrisi anak sangatlah penting, mengatur keuangan yang dimiliki dengan tetap memperhatikan kebutuhan gizi anak menjadi kunci keberhasilan dalam mempertahankan status gizi balita yang baik. REFERENSI [1] Hanoatubun S. Dampak Covid – 19 Terhadap Perekonomian Indonesia. Journal of Education, Psychology, and Counseling 2020;2. [1] Hanoatubun S. Dampak Covid – 19 Terhadap Perekonomian Indonesia. Journal of Education, Psychology, and Counseling 2020;2. [2] Kementerian Kesehatan RI. Panduan Pelayanan Kesehatan Balita Pada Masa Pandemi Covid- 19 Bagi Tenaga Kesehatan 2020. [3] Sumarmi S. Kerja Harmoni Zat Gizi dalam Meningkatkan Imunitas Tubuh Terhadap Covid-19: Mini Review. Amerta Nutrition 2020;4:250. [4] Marmi SS, Rahardjo K. Asuhan Neonatus, Bayi, Balita, dan Anak Prasekolah. Yokyakarta: Pustaka Pelajar; 2015. [5] Rahmi P. Peran Nutrisi Bagi Tubuh dan Kembang Anak Usia Dini. Bunayya: Jurnal Pendidikan Anak 2019;5. 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REFERENSI Hubungan Pendapatan Orang Tua Siswa Dengan Hasil Belajar Ips di Sdn Gugus 4 Sukowati Kabupaten Sragen 2016. [20] Kemenkes. Peraturan Menteri Kesehatan Republik Indonesia Nomor 28 Tahun 2019 Tentang Angka Kecukupan Gizi yang Dianjurkan untuk Masyarakat Indonesia 2019. [14] Septikasari M. Status Gizi Anak dan Faktor yang Mempengaruhi. Yogyakarta: UNY Press; 2018. [15] Kapisa MB, Bauw SA, Yap RA. Analisis Tingkat Pendidikan dan Jenis Pekerjaan terhadap Pendapatan Kepala Keluarga (KK) di Kampung Manbesak Distrik Biak Utara Provinsi Papua. Jurnal Lensa Ekonomi n.d.;15. [21] Akbar S. UMK Tahun 2021 Ditetapkan Sebesar Rp 2.553.265. ProbolinggokabGoId 2020. https://probolinggokab.go.id/umk-tahun- 2021-ditetapkan-sebesar-rp-2-553-265/. [16] Kosasih A. Pengaruh Pengalaman Kerja, dan Jam Kerja terhadap Pendapatan Ibu Rumah Tangga Tani Kemiri di Desa Pemunduran Kecamatan Kumpe Ulu. Skripsi 2020. Nur Umi Amanah / parents income and nutritional status of toddlers during the covid-19 pandemic Nur Umi Amanah / parents income and nutritional status of toddlers during the covid-19 pandem Nur Umi Amanah / parents income and nutritional status of toddlers during the covid-19 pandemic
https://openalex.org/W4251635506	https://www.researchsquare.com/article/rs-520083/latest.pdf	English	null	Streptococcus Vaginalis Sp. Nov., a Novel Bacterial Species Isolated From Vaginal Swabs of a Pregnant Woman With Diabetes	Research Square (Research Square)	2,021	cc-by	5,461	Research Article Keywords: Streptococcus vaginalis, diabetes, pregnancy, vaginal environment DOI: https://doi.org/10.21203/rs.3.rs-520083/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Archives of Microbiology on August 20th, 2021. See the published version at https://doi.org/10.1007/s00203-021-02532-y. Page 1/10 Page 1/10 Abstract Sequences targeted at the V3 and V4 16S rRNA hypervariable regions of a streptococcal strain (P1L01T) isolated from vaginal swabs of a pregnant woman with diabetes were 100% similar to those of Streptococcus anginosus subsp. whileyi. However, phylogenetic analysis based on 16S rRNA full-gene sequencing (1562 bp) revealed highest sequence similarity to Streptococcus periodonticum (98.65%), followed by Streptococcus anginosus subsp. whileyi (98.65 %), and Streptococcus anginosus subsp. anginosus (98.44%). Phylogenies of housekeeping genes rpoB and groEL were compared to improve classification, and the results showed a clear separation between strain P1L01T and closely related Streptococcus type strains. The complete genome of strain P1L01T consisted of 2,108,769 bp with a G+C content of 38.5 mol%. Average nucleotide identity values, based on genome sequencing, between strain P1L01T and Streptococcus periodonticum KCOM 2412T, Streptococcus anginosus subsp. whileyi CCUG 39159T, and Streptococcus anginosus subsp. anginosus NCTC 10713T were 95.48%, 94.33%, and 95.28%, respectively. The highest in silico DNA-DNA hybridization value with respect to the closest species was 66.2%, i.e., below the species cut-off of 70% hybridization. The main cellular fatty acids of strain P1L01T were 16:0, 18:1ω7c, and 14:0. On the basis of phylogenetic, genotypic and phenotypic data, we propose to classify this isolate as representative of a novel species of the genus Streptococcus, Streptococcus vaginalis sp. nov., in reference to its isolation from vaginal swabs, with strain P1L01T (=NBRC 114754T = BCRC 81289T) as the type strain. Introduction The genus Streptococcus, a heterogeneous group of Gram-positive bacteria, includes some of the most significant human pathogens – including S. pyogenes, S. pneumoniae (Megged 2020), Streptococcus suis (Yi et al. 2020), S. iniae, S. parauberis (Park et al. 2016), and S. agalactiae (Group B Streptococcus, GBS) (Opinion 2020). Of the 178 different species of species of the genus Streptococcus (https://lpsn.dsmz.de/), several are known to cause gynecological and obstetrics infections. Notably, GBS is the predominant infectious organism that causes bacteremia or sepsis in newborns and young infants (Opinion 2020). During labor and delivery, the vertical transmission of GBS from an infected mother to her fetus can also lead to pneumonia or meningitis (Scholl et al. 2016; Yu et al. 2011). For this reason, antibiotics are routinely given for preventing peripartum infections in women who test positive for GBS colonization in the anogenital region (Francois Watkins et al. 2019; Opinion 2020). However, only a limited number of studies have investigated the prevalence of other streptococcal species within the vaginal environment during pregnancy (Al Majid et al. 2020; Kolter and Henneke 2017; Rabe et al. 1988). The Streptococcus anginosus group is a subgroup of viridans streptococci that comprises three closely related species: S. intermedius, S. constellatus, and S. anginosus, of which the last named is divided into two subspecies (S. anginosus subsp. anginosus and S. anginosus subsp. whileyi). In the current study, a streptococcal strain (termed P1L01T) was isolated from vaginal swabs of a pregnant woman with type 2 diabetes at 35 weeks of gestation. 16S rRNA gene-based phylogenetic analyses were untaken, and species that showed > 98.5% similarity were selected for further characterization and comparison (Lim et al. 2019). Phylogenies of housekeeping genes rpoB (Glazunova et al. 2006; Saito et al. 2016) and groEL (Glazunova et al. 2006; Niu et al. 2017; Zbinden et al. 2012)were also analyzed to improve classification. While sequences targeted at the V3 and V4 16S rRNA hypervariable regions were 100% similar to those of Streptococcus anginosus subsp. whileyi, phylogenies of housekeeping genes rpoB and groEL revealed a clear separation between P1L01T and related type strains of Streptococcus. Herein, we investigated the taxonomic status of strain P1L01T and propose to classify this isolate as representative of a novel species of the genus Streptococcus, Streptococcus vaginalis sp. nov. Sample isolation As recommended by the National Health Bureau Care Program (Taiwan), vaginal swabs of a Taiwanese pregnant woman with type 2 diabetes were obtained at 35 weeks of gestation as part of screening for GBS colonization. Swab specimens were submitted for bacterial species identification. Under institutional review board approval (IRB number: 201701371A3), the patient gave written informed consent. Strain P1L01T was isolated from cultures grown at 37°C in brain-heart infusion (BHI) medium under anaerobic conditions. As the patient was at high risk for preterm labor, steroids and antibiotics were administered at 37 weeks of gestation to accelerate lung maturation and prevent peripartum infections. Genomic features and gene phylogeny Genomic features and gene phylogeny Page 2/10 Genomic DNA was extracted from strain P1L01T using the QIAGEN DNeasy Blood & Tissue kit (QIAGEN, Valencia, CA, USA). Phylogenies of housekeeping genes rpoB and groEL were compared to improve classification within the genus Streptococcus (Glazunova et al. 2006; Niu et al. 2017; Saito et al. 2016; Zbinden et al. 2012). Genomic sequences of the 16S rRNA, rpoB, and groEL genes were obtained from the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/) using ARSA (accession number: NZ_CP034543, NZ_CP012805, and NZ_LR134283). Neighbour-joining phylogenetic analyses, based on 16S rRNA gene sequence comparison, were performed using the EzBioCloud database (http://www.ezbiocloud.net/identify). Sequence homology analyses for the rpoB and groEL genes were undertaken using the Genetyx-Win software (version 5.1; Genetyx Corporation, Tokyo, Japan). The draft genome sequence of strain P1L01T was obtained with an Illumina MiniSeq system (Illumina, San Diego, CA, USA) using the 600-cycle MiSeq Reagent Kit v3. De novo assembly of reads was performed using the SPAdes (version 3.10.1) (Bankevich et al. 2012). Gene annotation of the draft sequence was carried out using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html) (Klimke et al. 2009). Average nucleotide identity (ANI) values was calculated with Orthologous Average Nucleotide Identity tool (Lee et al. 2016). Digital DNA-DNA hybridization (dDDH) values were determined using Formula 2 of the Genome-to-Genome Distance (GGD) Calculator (Meier-Kolthoff et al. 2013). Sequence alignment was carried out using the CLUSTAL W software (Thompson et al. 1997) and the MEGA X package (Kumar et al. 2018) was used for all analyses. Phylogenetic trees were constructed with different methods – including maximum-likelihood (ML) with the Jukes-Cantor model (Chor et al. 2006), minimum-evolution (ME) (Rzhetsky and Nei 1992) with the Tamura-Nei model (Tamura and Nei 1993) and neighbour-joining (NJ) with the Kimura’s two-parameter model (Kimura 1980; Saitou and Nei 1987) – using 1000 bootstrap replications. Phenotyping Sheep blood (final concentration, 5% in BHI medium) was used for hemolytic activity assays. Fermentation capacity was examined using a commercial kit (API 50 CHL; bioMérieux, Lyon, France) according to the manufacturer’s protocol. Halotolerance and ranges of permissive growth temperatures and pH were determined as described by Kozaki et al. (1992). The biochemical profiles of P1L01T and closely related strains (S. periodonticum, S. anginosus subsp. whileyi, and S. anginosus subsp. Anginosus) were assessed using the API ZYM and API Rapid ID 32 Strep systems (bioMérieux). Whole-cell fatty acid methyl ester (FAME) profiling was performed using the Sherlock Microbial Identification system (MIDI). After incubation of bacterial cells on solid BHI medium for 48 h at 30°C under anaerobic conditions, FAME extraction and preparation were carried out as described by Sasser (1990). Gene phylogeny The 16S rRNA gene sequences (1562 bp) of strain P1L01T were compared with those stored in the EzBioCloud database. Three species − Streptococcus periodonticum JCM 33300T (Japan Collection of Microorganisms, Ibaraki, Japan), Streptococcus anginosus subsp. whileyi DSM 25818T (DSMZ GmbH, Braunschweig, Germany), and Streptococcus anginosus subsp. anginosus BCRC 14730T (BCRC, Hsinchu, Taiwan) − showed > 98.0% similarity with strain P1L01T on sequences targeted at the 16S rRNA region (Table 1). The highest sequence similarity was observed with Streptococcus periodonticum (98.65%), followed by Streptococcus anginosus subsp. whileyi (98.65 %), and Streptococcus anginosus subsp. anginosus (98.44%; Fig. 1). Similar findings were obtained using the NJ and ME algorithms (Supplementary Figures S1 and S2). The concatenated sequences (5190 bp) of two housekeeping genes (rpoB, 3567 bp; groEL, 1,623 bp) in strain P1L01T had 93.64 − 95.07% identity with those of S. periodonticum, S. anginosus subsp. whileyi and S. anginosus subsp. anginosus (Table 1). Phylogenetic trees based on concatenated nucleotide alignments of housekeeping genes were constructed using the ML, NJ, and ME algorithms. The results showed a clear separation between P1L01T and Streptococcus reference strains (Fig. 2 and Supplementary Figures S3 and S4). Page 3/10 Table 1 Table 1 Table 1 Genotypic characteristics of Streptococcus vaginalis sp. nov. P1L01T and the closely related species and subspecies Similarity of each gene with strain P1L01T P1L01T Species Strain No. 16S rDNA rpoB groEL GenBank accession number of genome GenBank assembly accession No. G + C content (%) ANI value (%) GGD value (%) S. periodonticum KCOM 2412T 98.65% 95.63% 93.83% NZ_CP034543 GCA_003963555.1 38.95% 95.48% 66.20% [63.3– 69.1%] S. anginosus subsp. whileyi CCUG 39159T 98.65% 93.28% 94.45% NZ_CP012805 GCA_000257765.1 38.50% 94.33% 56.50% [53.8– 59.3%] S. anginosus subsp. whileyi MAS624 98.72% 93.27% 94.52% NZ_AP013072 GCA_000478925.1 38.34% 94.25% 56.70% [53.9– 59.4%] S. anginosus subsp. anginosus NCTC 10713T 98.44% 95.10% 94.82% NZ_LR134283 GCA_900636475.1 38.64% 95.28% 61.60% [58.7– 64.4%] S. anginosus subsp. anginosus ATCC 33397T 98.53% 95.04% 94.82% NA GCA_002088025.1 38.63% 95.1% 61.50% [58.6– 64.3%] S. anginosus NCTC 11062 98.72% 94.09% 95.07% NA GCA_901542445.1 38.84% 94.05% 54.90% [52.1– 57.6%] S. anginosus NCTC 11064 98.66% 96.02% 97.91% NZ_LR594037 GCA_901542485.1 38.66% 95.82% 64.10% [61.2– 66.9%] S. intermedius SK54T 96.56% 95.04% 94.82% NA GCA_000258445.1 37.58% 86.96% 32.40% [30– 34.9%] S. intermedius ATCC 27335T 96.54% 91.67% 91.19% NA GCA_000413475.1 37.66% 87.04% 32.60% [30.2– 35.1%] S. constellatus subsp. constellatus NCTC 11325T 96.48% 92.09% 92.79% NA GCA_900459125.1 38.08% 88.78% 36.40% [34– 39%] S. constellatus subsp. Gene phylogeny pharyngis CCUG 46377T 96.39% 92.43% 92.67% NA GCA_000474135.1 37.94% 89.13% 36.80% [34.4– 39.3%] NA: Not available. Genome features The complete genome of the P1L01T strain – which consisted of 2,108,769 bp with a G + C content of 38.5 mol% – contained 1,808 protein-coding genes and 53 predicted RNA genes. ANI values, based on genome sequencing, between strain P1L01T and Streptococcus periodonticum, Streptococcus anginosus subsp. whileyi, and Streptococcus anginosus subsp. anginosus were 95.48%, Genome features The complete genome of the P1L01T strain – which consisted of 2,108,769 bp with a G + C content of 38.5 mol% – contained 1,808 protein-coding genes and 53 predicted RNA genes. ANI values, based on genome sequencing, between strain P1L01T and Streptococcus periodonticum, Streptococcus anginosus subsp. whileyi, and Streptococcus anginosus subsp. anginosus were 95.48%, 94.33%, and 95.28%, respectively. The dDDH value with respect to the closest species was 66.2% (Table 1). Biochemical features Page 4/10 The biochemical characteristics of strain P1L01T were determined in parallel to those of Streptococcus periodonticum, Streptococcus anginosus subsp. whileyi, and Streptococcus anginosus subsp. anginosus strains. Significant differences for strain P1L01T were observed compared with other strains with respect to fermentation capacity and enzyme activities (Table 2). The major cellular fatty acids of strain P1L01T were C16:0 (40.7%), C18:1 ω7c (18.0%), and C14:0 (14.7%) – with significant differences with the other three strains (Supplementary Table 1). acids of strain P1L01T were C16:0 (40.7%), C18:1 ω7c (18.0%), and C14:0 (14.7%) – with significant differences with the other three strains (Supplementary Table 1). acids of strain P1L01T were C16:0 (40.7%), C18:1 ω7c (18.0%), and C14:0 (14.7%) – with significant differences with the other three strains (Supplementary Table 1). Table 2 Characteristics helpful in differentiating S. vaginalis sp. nov. P1L01T from closely related species of the genus Streptococcus. All data are from this study. Strains Characteristic 1 2 3 4 Hemolysis γ α α α Acid production from (API 50 CHL) N-Acetyl glucosamine + – + + Amygdaline + – + + Melibiose + – – – D-Raffinose + – – + β-Gentiobiose + – + + Enzyme activity (API ZYM) Alkaline phosphatase 1 3 2 4 Esterase (C4) 0 1 0 1 Cystine aminopeptidase 1 0 1 2 β-galactosidase 0 3 0 1 α-glucosidase 0 3 1 4 API Rapid ID 32 Strep α-galactosidase + – – – APPA – + + + Strains: 1, strain P1L01T; 2, S. periodonticum JCM 33300T; 3, S. anginosus subsp. whileyi DSM 25818T; 4, S. anginosus subsp. anginosus BCRC 14730T. On the basis of phylogenetic, genotypic, and phenotypic data (Tables 1 and 2), we propose to classify strain P1L01T as representative of a novel species of the genus Streptococcus. In reference to its isolation from vaginal swabs, the name Streptococcus vaginalis sp. nov. (vaginalis. L. gen. n. vaginalis, of the vagina) is suggested. Cells are Gram-stain-positive, catalase-negative, facultatively anaerobic, non-spore-forming cocci (diameter: 0.5 − 1.0 µm) that can be arranged in pairs, short chains, or small groups. The addition of 5% sheep blood to BHI agar showed γ-hemolytic activity (i.e., no hemolysis) after incubation at 37°C for 48. Growth was observed on BHI agar at 30 − 45°C under anaerobic conditions, but temperatures of 10°C and 50°C were not tolerated. Additionally, the new species tolerated 5.5% − but not 6.5% − NaCl. Biochemical features Growth was observed in a wide range of pH variations (4.5–8.5). Acid production was found from galactose, D-glucose, D-fructose, D-mannose, N-acetyl glucosamine, amygdalin, esculine, arbutine, salicine, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, D-raffinose, and β-gentiobiose. However, no production of acid was observed from glycerol, ribose, mannitol, sorbitol, erythritol, L-arabinose, D-arabinose, D-xylose, L-xylose, adonitol, β-methyl-xyloside, L-sorbose, rhamnose, dulcitol, α- methyl-D-mannoside, α-Methyl-D-glucoside, inuline, melezitose, glycogene, xylitol, D-turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L- arabitol, D-tagatose, 2-keto-gluconate, gluconate, and 5-keto-gluconate. The major cellular fatty acids were 16:0, 18:1 ω7c, and 14:0. Strain P1L01T (= NBRC 114754T = BCRC 81289T) was the type strain. Discussion Page 5/10 Page 5/10 Next-generation sequencing (NGS) is increasingly being applied for the analysis of complex microbial communities (Boers et al. 2019; Graspeuntner et al. 2018). Most published studies in the field have relied on sequences targeted at the V3 and V4 16S rRNA hypervariable regions (319F–806 R). During the analysis of vaginal swabs obtained from a Taiwanese pregnant woman with type 2 diabetes, we isolated a strain (P1L01T) that could not be identified conclusively to the species level based on 16S V3–V4 rRNA gene sequence analysis. Highest sequence homology at these regions was obtained with Streptococcus anginosus subsp. whileyi and Streptococcus anginosus subsp. anginosus (100% and 99.78%, respectively). On analyzing sequences targeted at the V6 region (926F– 1100R), strain P1L01T was distinctly different from S. anginosus subsp. whileyi (similarity: 90.23%), S. anginosus subsp. anginosus (similarity: 90.23%), and S. periodonticum (similarity: 94.25%). The highest ANI and dDDH values among strain P1L01T and the S. periodonticum type strain were 95.48 % and 66.2%, respectively. While dDDH values were lower than the commonly accepted cut-off (70%), the ANI value between strain P1L01T and S. periodonticum KCOM 2412T was of uncertain significance (Goris et al. 2007; Rossello-Mora and Amann 2015). Recent studies have suggested that bacterial isolates with ANI values < 95–96% or dDDH values < 70% with respect to type strains of their closest related species and genera should be classified in novel species Chun et al. 2018; Lim et al. 2019). Thus, a hypothesis was set forth that strain P1L01T might represent a novel species of the genus Streptococcus. Additional phenotyping of strain P1L01T further supported this possibility. First, strain P1L01T was γ-hemolytic, whereas the other three strains investigated in this study showed α-hemolytic activity. Second, biochemical analysis of strain P1L01T demonstrated distinct characteristics compared to other strains in terms of the carbohydrate metabolism – with clear differences in acid production from melibiose, N-acetyl glucosamine, amygdaline, D-raffinose and β-gentiobiose. Additionally, strain P1L01T can be distinguished from other strains through α-galactosidase and alanyl-phenylalanyl-proline arylamidase activities. Based on phylogenetic, genotypic and phenotypic data, we therefore propose to classify this isolate as representative of a novel species of the genus Streptococcus, Streptococcus vaginalis sp. nov., in reference to its isolation from vaginal swabs. However, the question as to whether this novel species should be considered a pathogen or a cause of gynecological and/or obstetrics infections remains unanswered. Discussion While we found no clinical signs of infection in the patient from whom Streptococcus vaginalis sp. nov. was isolated, further research is needed to examine more rigorously its potential pathogenicity. Notably, bacteria of the S. anginosus group – which are closely related to the novel species of the genus Streptococcus identified in this study – have been shown to cause anogenital infections (mainly abscesses) and have been isolated in episiotomy site infections (Al Majid et al. 2020; Lampen and Bearman 2005; Rabe et al. 1988). The main virulence factors of the S. anginosus group include secreted toxins (exotoxins), hydrolytic enzymes, and capsular polysaccharides. To reveal the virulence potential of Streptococcus vaginalis sp. nov., further in vitro investigations would be required to evaluate its contribution to the cytotoxicity against human cell lines. Murine vaginal colonization models in preestrogenized mice may also be useful to investigate the mucosal biology of the novel species. In conclusion, we have confidently recognized a novel species of the genus Streptococcus, termed Streptococcus vaginalis sp. nov., using phylogenetic, genotypic and phenotypic identification methods. The novel species was isolated from vaginal swabs of a pregnant woman with diabetes and was most closely related to S. anginosus. The role of Streptococcus vaginalis sp. nov. in the pathogenesis of gynecological and/or obstetrics infections is currently questionable. More studies are necessary to clarify whether the novel species should be considered a pathogen or a cause of human infection. Acknowledgements We are grateful to Jung-Erh Yang and Hui-Jin Liao for their excellent technical assistance. Ethical statement All participants signed an informed consent to authorize the use of samples for research purposes. 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J Comput Biol 19:455-477. 2. ankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455-477. 3. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455-477. 3. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455-477. 4. Boers SA, Jansen R, Hays JP (2019) Understanding and overcoming the pitfalls and biases of next-generation sequencing (NGS) methods for use in the routine clinical microbiological diagnostic laboratory. Eur J Clin Microbiol Infect Dis 38: 1059-1070. 4. Boers SA, Jansen R, Hays JP (2019) Understanding and overcoming the pitfalls and biases of next-generation sequencing (NGS) methods for use in the routine clinical microbiological diagnostic laboratory. Eur J Clin Microbiol Infect Dis 38: 1059-1070. 5. Chor B, Hendy MD, Snir S (2006) Maximum likelihood Jukes-Cantor triplets: analytic solutions. Mol Biol Evol 23:626-632. 5. Chor B, Hendy MD, Snir S (2006) Maximum likelihood Jukes-Cantor triplets: analytic solutions. Mol Biol Evol 23:626-632. 6. References nov., isolated from the oral cavity of tufted capuchin. Int J Syst Evol Microbiol 66:1063-1067. 27. Saito M, Shinozaki-Kuwahara N, Hirasawa M, Takada K (2016) Streptococcus oricebi sp. nov., isolated from the oral cavity of tufted capuchin. Int J Syst Evol Microbiol 66:1063-1067. 28. Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. USFCC Newsl 20:16. 28. Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. USFCC Newsl 20:16. 28. Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. USFC 29. Scholl J, Nasioudis D, Boester A, Speleotes M, Grunebaum A, Witkin SS (2016) Group B streptococcus alters properties of vaginal epithelial cells in pregnant women. Am J Obstet Gynecol 214:383 e381-385. 29. Scholl J, Nasioudis D, Boester A, Speleotes M, Grunebaum A, Witkin SS (2016) Group B streptococcus alters properties of vaginal epithelial cells in pregnant women. Am J Obstet Gynecol 214:383 e381-385. 30. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512-526. 30. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512-526. 31. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876-4882. 31. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876-4882. 32. Yi L, Jin M, Li J, Grenier D, Wang Y (2020) Antibiotic resistance related to biofilm formation in Streptococcus suis. Appl Microbiol Biotechnol 104:8649-8660. 32. Yi L, Jin M, Li J, Grenier D, Wang Y (2020) Antibiotic resistance related to biofilm formation in Streptococcus suis. Appl Microbiol Biotechnol 104:8649-8660. 33. Yu HW, Lin HC, Yang PH, Hsu CH, Hsieh WS, Tsao LY, Chen CH, Lin HC, Tseng YC (2011) Group B streptococcal infection in Taiwan: maternal colonization and neonatal infection. Pediatr Neonatol 52:190-195. 33. Yu HW, Lin HC, Yang PH, Hsu CH, Hsieh WS, Tsao LY, Chen CH, Lin HC, Tseng YC (2011) Group B streptococcal infection in Taiwan: maternal colonization and neonatal infection. Pediatr Neonatol 52:190-195. 34. References Meier-Kolthoff JP, Auch AF, Klenk HP, Goker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14:60. 21. Niu L, Lu S, Lai XH, Hu S, Chen C, Zhang G, Yang J, Jin D, Wang Y, Lan R, Lu G, Xie Y, Ye C, Xu J (2017) Streptococcus himalayensis sp. nov., isolated from the respiratory tract of Marmota himalayana. Int J Syst Evol Microbiol 67, 256-261. 21. Niu L, Lu S, Lai XH, Hu S, Chen C, Zhang G, Yang J, Jin D, Wang Y, Lan R, Lu G, Xie Y, Ye C, Xu J (2017) Streptococcus himalayensis sp. nov., isolated from the respiratory tract of Marmota himalayana. Int J Syst Evol Microbiol 67, 256-261. 22. Opinion AC (2020) Prevention of Group B Streptococcal Early-Onset Disease in Newborns: ACOG Committee Opinion, Number 797. Obstet Gynecol 135, e51-e72. (doi: 10.1097/AOG.0000000000003668.) 22. Opinion AC (2020) Prevention of Group B Streptococcal Early-Onset Disease in Newborns: ACOG Committee Opinion, Number 797. Obstet Gynecol 135, e51-e72. (doi: 10.1097/AOG.0000000000003668.) Page 7/10 Page 7/10 23. Park JW, Wendt M, Heo GJ (2016) Antimicrobial activity of essential oil of Eucalyptus globulus against fish pathogenic bacteria. Lab Anim Res 32:87-90. 23. Park JW, Wendt M, Heo GJ (2016) Antimicrobial activity of essential oil of Eucalyptus globulus against fish pathogenic bacteria. Lab Anim Res 32:87-90. 24. Rabe LK, Winterscheid KK, Hillier SL (1988) Association of viridans group streptococci from pregnant women with bacterial vaginosis and upper genital tract infection. J Clin Microbiol 26:1156-1160. 24. Rabe LK, Winterscheid KK, Hillier SL (1988) Association of viridans group streptococci from pregnant women with bacterial vaginosis and upper genital tract infection. J Clin Microbiol 26:1156-1160. 25. Rzhetsky A, Nei M (1992) A simple method for estimating and testing minimum-evolution tree Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogene 26. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406-425. 26. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406-425. 26. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406-425. 27. Saito M, Shinozaki-Kuwahara N, Hirasawa M, Takada K (2016) Streptococcus oricebi sp. nov., isolated from the oral cavity of 27. Saito M, Shinozaki-Kuwahara N, Hirasawa M, Takada K (2016) Streptococcus oricebi sp. References Zbinden A, Mueller NJ, Tarr PE, Sproer C, Keller PM, Bloemberg GV (2012) Streptococcus tigurinus sp. nov., isolated from blood of patients with endocarditis, meningitis and spondylodiscitis. Int J Syst Evol Microbiol 62:2941-2945. 34. Zbinden A, Mueller NJ, Tarr PE, Sproer C, Keller PM, Bloemberg GV (2012) Streptococcus tigurinus sp. nov., isolated from blood of patients with endocarditis, meningitis and spondylodiscitis. Int J Syst Evol Microbiol 62:2941-2945. Figure 2 Phylogenetic tree analysis based on concatenated sequences of the two genes (rpoB and groEL) of S. vaginalis sp. nov. (strain P1L01T) and other species of the genus Streptococcus. The tree was reconstructed using the maximum-likelihood method with Kimura’s two-parameter model; the target region used for comparison was 1430 bp in length. Enterococcus faecalis CIP 103015T was used as an outgroup to root the trees. Bootstrap values (based on 1000 replications) are shown at branch points. The bar indicates 10% estimated sequence divergence. Phylogenetic tree analysis based on concatenated sequences of the two genes (rpoB and groEL) of S. vaginalis sp. nov. (strain P1L01T) and other species of the genus Streptococcus. The tree was reconstructed using the maximum-likelihood method with Kimura’s two-parameter model; the target region used for comparison was 1430 bp in length. Enterococcus faecalis CIP 103015T was used as an outgroup to root the trees. Bootstrap values (based on 1000 replications) are shown at branch points. The bar indicates 10% estimated sequence divergence. Figures Page 8/10 Figure 1 Phylogenetic tree analysis based on maximum-likelihood method with Kimura’s two-parameter model of S. vaginalis sp. nov. (strain P1L01T) and closely related species of the genus Streptococcus according to 16S rRNA sequences. GenBank accession numbers ar shown in parentheses. Enterococcus faecalis ATCC 19433T was used as an outgroup to root the trees. Numbers indicate bootstrap percentages (based on 1000 replications), and bootstrap values >70% are shown at branch points. The scale bar represents 0.002 substitutions per nucleotide position. Figure 1 Phylogenetic tree analysis based on maximum-likelihood method with Kimura’s two-parameter model of S. vaginalis sp. nov. (strain P1L01T) and closely related species of the genus Streptococcus according to 16S rRNA sequences. GenBank accession numbers are shown in parentheses. Enterococcus faecalis ATCC 19433T was used as an outgroup to root the trees. Numbers indicate bootstrap percentages (based on 1000 replications), and bootstrap values >70% are shown at branch points. The scale bar represents 0.002 substitutions per nucleotide position. Phylogenetic tree analysis based on maximum-likelihood method with Kimura’s two-parameter model of S. vaginalis sp. nov. (strain P1L01T) and closely related species of the genus Streptococcus according to 16S rRNA sequences. GenBank accession numbers are shown in parentheses. Enterococcus faecalis ATCC 19433T was used as an outgroup to root the trees. Numbers indicate bootstrap percentages (based on 1000 replications), and bootstrap values >70% are shown at branch points. The scale bar represents 0.002 substitutions per nucleotide position. Page 9/10 Figure 2 SupplementaryMaterials20210504.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryMaterials20210504.docx Page 10/10
W4311444953.txt	https://zenodo.org/record/7438242/files/11-16.pdf	tl		GERMANIYA MEHNAT QONUNCHILIGINING UMUMIY ASOSLARI	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,942	Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 1 \| ISSUE 7 \| 2022 GERMANIYA MEHNAT QONUNCHILIGINING UMUMIY ASOSLARI Samadov Rizamat Shuhrat o‘g‘li ANNOTATSIYA Ushbu ma’lumotnomada Germaniya mehnat qonunchiligining umumiy asoslari keltirib o‘tilgan. Mehnat shartnomasi, mehnat ta’tillari, ijtimoiy himoya va Germaniya mehnat qonunchiligiga aloqador boshqa ma;lumotlar normative hujjatlar orqali asoslat berilgan. ABSTRACT This reference cited the general foundations of German labor law. An employment contract, labor holidays, social protection and other information related to German labor law are given as justification through regulatory documents. АННОТАЦИЯ В этом справочнике приводятся общие основы трудового законодательства Германии. Трудовой договор, трудовой отпуск, социальная защита и другая информация, относящиеся к трудовому законодательству Германии, были обоснованы нормативными актами. Germaniya mehnat qonunchiligi ikki sohaga bo‘lingan: yakka tartibdagi mehnat huquqi va jamoaviy mehnat huquqi. Yakka tartibdagi mehnat qonunchiligi yakka(bir nafar) xodim va ish beruvchi o‘rtasidagi munosabatlarga taalluqlidir, jamoaviy mehnat qonunchiligi esa xodimlarning jamoaviy vakilligi va birlashmasini, shuningdek, xodimlar birlashmasining huquq va majburiyatlarini tartibga soladi. Germaniya mehnat qonunchiligi yagona mehnat kodeksiga birlashtirilmagan: asosiy manbalar federal qonunchilik, sud amaliyoti, jamoaviy bitimlar, mehnat kengashi bitimlari va individual mehnat shartnomalari. Qonuniy holatda, Germaniyada ishlashni istagan har bir xodimdan Germaniyaga kirishdan oldin resident maqomini hamda ishlash uchun ruxsatnoma talab qilinadi. Quyidagi shaxslar bundan mustasno: 1) Germaniya millatiga mansublar; 2) Yevropa Ittifoqi fuqarosi; 3) Yevropa iqtisodiy hududiga (EEA) aʼzo davlat fuqarosi (Islandiya, Lixtenshteyn, Norvegiya ); yoki 4) Shveytsariya fuqarosi. Germaniyada “Germaniya rezidenti to‘g‘risida”gi qonunga kiritilgan o‘zgartirishlar natijasida, agar mehnat shartnomasi taqdim etish imkoni bo‘lsa, endi “mehnat bozori testi” talab qilinmaydi. Bundan tashqari, uchinchi mamlakatlardan https://t.me/Erus_uz Multidisciplinary Scientific Journal December, 2022 11 Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 1 \| ISSUE 7 \| 2022 kelgan malakali ishchilar, agar ular zarur nemis tilini bilishini isbotlashlari va yashashlarini kafolatlashlari sharti bilan, ish qidirish uchun cheklangan muddatga Germaniyaga ko‘chib kelishlari mumkin. Chet ellik ish beruvchi xodimni yollash uchun mahalliy tashkilot tuzishi yoki u orqali ishlashi kerakmi? Yo‘q. Biroq, ish beruvchi qonuniy ijtimoiy ta’minot tizimiga ko‘ra, Germaniyada xodim bo‘lishi mumkin bo‘lgan shaxsni tayinlashi shart. Guruh ichidagi transferlar osonlashtirildi; Yevropa Ittifoqi fuqarosi bo‘lmagan shaxslar ma’lum sharoitlarda yangi "ICT-Card" yashash huquqiga ega bo‘lishlari mumkin, bu ularga Germaniyadagi tashkilotlar guruhi ichida uch yilgacha ishlash imkonini beradi. Bu bu holat qachonki mumkin bo‘ladi, qachonki ular Yevropa Ittifoqidan tashqaridagi boshqa tashkilotlar guruhi tomonidan yuborilgan bo‘lsa Bundan tashqari, boshqa Yevropa Ittifoqiga a’zo davlatda istiqomat qilayotgan(resident) va ishlayotgan uchinchi davlat fuqarolari, agar Germaniyada 90 kundan ortiq muddatga ishlash uchun jo‘natilishi kerak bo‘lsa, “mobil AKT-karta” uchun ariza topshirishlari mumkin. Qisqa muddatli topshiriq (ya’ni, 180 kunlik muddat ichida 90 kundan ortiq bo‘lmagan muddatga) bo‘lsa, yashash joyini tasdiqlovchi hujjat umuman talab qilinmaydi; faqat vakolatli organni (ya’ni, Migratsiya va qochqinlar bo‘yicha Federal idora) xabardor qilish kifoya. Demak, uchinchi mamlakat fuqarolari Yevropa Ittifoqiga a’zo turli davlatlarda bitta ruxsatnoma asosida ishlashlari mumkin. Ish beruvchi asosiy shartnoma shartlarini ish boshlaganidan keyin bir oydan kechiktirmay xodimga yozma ravishda taqdim etish bo‘yicha qonuniy majburiyatga ega. Ishga qabul qilishda minimal talablar. Ishga qabul qilish shartlari, asosan, nizomlar, jamoaviy bitimlar va mehnat kengashi shartnomalari bilan tartibga solinadi. Qoidaga ko‘ra, mehnat shartnomasi shartlari xodimning manfaatiga zarar yetkazadigan holatlarda ushbu qoidalardan chetga chiqish mumkin emas. Kelib chiqishi mumkin bo‘lgan nizolarni oldini olish uchun mehnat shartnomasi nemis tilida tuzilgani maqul.Biroq, bu qonuniy talab hisoblanmaydi. Mehnat shartnomasi. Umumiy qoida sifatida, mehnat shartnomasi cheklanmagan muddatga tuziladi. Agar shartnoma shartlari mehnat munosabatlariga kirishishdan oldin belgilab olingan bo‘lsa, muddatli mehnat shartnomalarini ham tuzish mumkin. Muddatli mehnat shartnomasi muddati tugagandan so‘ng yozma ravishda ogohlantirmasdan avtomatik ravishda tugaydi. Muddatli mehnat munosabatlari qonuniy asosda obyektiv tomonlari aniqlashtirilishi kerak. Obyektiv tomonlarning ba’zilari qonuniy jihatdan belgilab qo`yilgan bo‘ladi, masalan, ish hajmining ortganligi, ota-ona ta’tilida xodimni almashtirish. Agar obyektiv asoslar mavjud bo‘lmasa, muddatli mehnat munosabatlari eng ko‘pi bilan ikki yilgacha https://t.me/Erus_uz Multidisciplinary Scientific Journal December, 2022 12 Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 1 \| ISSUE 7 \| 2022 tuziladi. Agar mehnat munosabatlari muddat tugagandan keyin ham davom etayotgan bo‘lsa, shartnoma nomuayyan muddatga tuzilgan hisoblanadi. Ish beruvchi va xodim qonun bilan maksimal olti oylik muddat bilan cheklangan sinov muddati to‘g‘risida kelishib olishlari mumkin. Sinov muddati davrida ogohlantirish muddati ikki hafta hisoblanadi (yoki boshqacha tarzda kelishilgan bo‘lishi ham mumkin). Tomonlar sinov muddatiga rozi bo‘lishidan qat’i nazar, Ishdan bo‘shatishni himoya qilish to‘g‘risidagi qonun ishning dastlabki olti oyi davomida qo‘llanilmaydi. Ogohlantirish muddati. Ish beruvchi xodimni ogohlantirish muddati xodimning ushbu tashkilotdagi mehnat stajiga bog‘liq bo‘lib, xodim kamida 2 yillik stajga ega bo‘lsa 4 hafta oldin, 20 yildan uzoq bo‘lgan stajga ega bo‘lsa 7 oy oldin ogohlantirish lozim bo‘ladi. Agar mehnat shartnomasida boshqacha qoida nazarda tutilgan bo‘lmasa, qonuniy tarzda uzaytirilgan ogohlantirish muddatlari faqat ish beruvchi tomonidan ishdan bo‘shatish uchun qo‘llaniladi, xodim esa kalendar oyining oxirida yoki 15sanasida to‘rt haftalik ogohlantirish muddati bilan mehnat shartnomasini bekor qilishga haqli. Ko‘pchilik mehnat shartnomalari xodimlar ish beruvchi uchun qulay bo‘lgan ogohlantirish muddatini xodimga yuklaydi. Jamoa shartnomalarda uzoqroq yoki qisqaroq ogohlantirish muddatlari belgilanishi mumkin, individual mehnat shartnomalarida esa faqat uzoqroq ogohlantirish muddatlari belgilanishi mumkin. Ish tartibi va shartlari (masalan, eng ko‘p ish vaqti, eng kam haq to‘lanadigan ta’til va kasallik ta’tillari) qonunlar, jamoaviy shartnomalari va mehnat kengashi shartnomalari bilan tartibga solinadi. Individual mehnat shartnomasi ushbu qoidalardan xodimga zarar yetkazadigan tarzda chetga chiqa olmaydi. Germaniyaga vaqtincha ishlash uchun yuborilgan xodimlarning huquqlari odatda xorijiy mehnat qonunchiligi bilan belgilanadi. Ish beruvchi o‘z binolari ustidan nazoratga ega bo‘lganligi sababli va xodimlar ish joyidagi xavf-xatarlarga duchor bo‘lish xavfi bo‘lganligi sababli, ish beruvchi sog‘lom va xavfsiz ish joyini ta’minlashi shart. Shunday qilib, ish beruvchi barcha xonalarni, asboblarni va jihozlarni o‘rnatishi va ularga texnik xizmat ko‘rsatishi va ishni xodimlarni har qanday mumkin bo‘lgan zararlardan himoya qiladigan tarzda tashkil qilishi shart. Biroq, sog‘lom va xavfsiz ish joyi to‘g‘risidagi qoidalar sanoat sohasining turiga va muayyan ish joyida duch keladigan xavf darajasiga bog‘liq. Ish beruvchi xodimlar kompaniyaning Internet, telefon yoki elektron pochta tizimidan shaxsiy masalalar bo‘yicha, ish vaqtida yoki undan tashqarida foydalanishi mumkinmi yoki yo‘qmi va qay darajada foydalanishi to‘g‘risida qaror qabul qilishga haqlidir. Odatda, ruxsatisiz xodim shaxsiy masalalar uchun internetdan foydalanish huquqiga ega emas. Federal Mehnat sudining ta’kidlashicha, hatto aniq taqiq bo‘lmasa ham, xodimlar shuni https://t.me/Erus_uz Multidisciplinary Scientific Journal December, 2022 13 Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 1 \| ISSUE 7 \| 2022 e’tiborga olishlari lozimki, ish beruvchi xodimlarning tashkilot qurilmalaridan shaxsiy maqsadlarda foydalanishlariga toqat qiladi deb o‘ylamasligini. Agar xodim mehnat jihozlaridan shaxsiy foydalanish bo‘yicha taqiqni buzsa, ish beruvchi ogohlantirish berishga va hatto sharoitga qarab mehnat shartnomasini bekor qilishga haqlidir. Amalda ko‘pgina ish beruvchilar ma’lum darajada internetdan shaxsiy maqsadlarda foydalanishga ruxsat berishadi. Biroq, ruxsat berilgan taqdirda ham, shaxsiy maqsadlarda uchun internetdan foydalanishdagi kontentlarga va foydalanish vaqtiga nisbatan cheklov bo‘lishi kerak. Biz xodimning kompaniyadagi elektron pochtasidan shaxsiy maqsadlarda foydalanishni qatiy taqiqlashni tavsiya qilamiz, aks holda xodimning kompaniyadagi elektron pochtasini kuzatish yoki kirish og‘ir holat hatto jinoiy qilmish bo‘lishi ham mumkin, va hattoki bu xodimning shaxsiy manfaatlariga ham daxldor bo‘lishi mumkin (qachonki xodim kasallangan, ta’tildaligi yoki kompaniyadan ketgan holatlarda). Germaniyada xodimlar qonun bo‘yicha milliy ijtimoiy xavfsizlik tizimiga tegishlidir. Ijtimoiy ta’minot. Ijtimoiy sug‘urta kodeksi quyidagi asosiy prinsiplarni qamrab olgan qonuniy ijtimoiy sug‘urta tizimidan iborat: tibbiy sug‘urta, ishsizlik sug‘urtasi, hamshiralik parvarishi sug‘urtasi, pensiya va baxtsiz hodisalardan sug‘urta. Barcha ish haqi to‘lovlarida soliq va ijtimoiy sug‘urta badallari (pensiya, ishsizlik, sog‘liqni saqlash va hamshiralik parvarishi sug‘urtasi) bo‘ladi. Ular ish beruvchi tomonidan xodimning maoshidan ushlab qolinishi va tegishli muassasalarga to‘lanishi kerak. Umuman olganda, ish beruvchi va xodim har biri ijtimoiy sug‘urta badallarining yarmini to‘laydilar va ish beruvchilar ish haqiga qo‘shimcha ravishda o‘z ulushlarini, ma’lum maksimal miqdorlar qo‘llanilgan holda, xodimlarning yalpi ish haqi asosida to‘lashlari kerak. Xodimlarni baxtsiz hodisalardan sug‘urtalashga qo‘shgan ulushi faqat ish beruvchilar tomonidan amalga oshiriladi. Bayram va ta’tillar. Germaniya bo‘ylab davlat ta’tillari bir federal shtatniki boshqasinikidan farq qiladi. Maslan, Berlin, Quyi Saksoniyada 10 ta, Bavariya va Saarlandda 12 ta davlat ta’tillari bor. Har bir xodim 5 kunlik ish haftasi bilan hisoblanadigan 20 kunlik(shanba va yakshanba hisoblanmaydi) yillik mehnat ta’tili olish huquqiga egadir. Bu shuni anglatadiki, xodim kalendar yilida to‘rt haftalik yillik ta’tilni talab qilishi mumkin. Biroq, ko‘pchilik ish beruvchilar sanoat sohasidan kelib chiqib uzoqroq, ya’ni 25 kundan 30 kungacha bo‘lgan yillik mehnat ta’tilini beradi. Ayol xodimlar tug‘ruqdan 6 hafta oldin va tug‘ruqdan keyin 8 hafta pullik tug‘ruq ta’tiliga ega. Agar bir nafardan ortiq bola tug‘ilishi, muddatidan oldin tug‘ilish yoki nogiron bola tug‘ilishi sodir bo‘lsa, tug‘ruq ta’tili yana 12 haftaga cho‘zilishi mumkin. Ushbu davr mobaynida xodimga to‘lovlar qisman qonuniy tibbiy sug‘urtalovchi va qisman ish beruvchi tomonidan amalga oshiriladi. Bola tug‘ilgandan so‘ng, ham https://t.me/Erus_uz Multidisciplinary Scientific Journal December, 2022 14 Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 1 \| ISSUE 7 \| 2022 erkak va ham ayol xodimlar har bir bola uchun maksimal uch yillik ota-ona ta’tiliga chiqish huquqiga ega. Ushbu davrda ish beruvchi xodimga hech qanday to‘lovlarni amalga oshirishga majbur emas. Ota-onalik ta’tilining muddati tugagandan so‘ng, xodim avvalgi lavozimiga qaytadi. “Onalikni muhofaza qilish to‘g‘risida”gi qonunga muvofiq, homilador xodimlar, shuningdek, majburiy amaliyot o‘tayotgan shogirdlar, stajyorlar va talabalar homiladorlik davrida va bola tug‘ilgandan keyin to‘rt oy davomida ishdan bo‘shatishdan alohida himoyalanadi. Ish beruvchi nafaqat homilador xodimlar tomonidan bajarilgan ishlar uchun, balki kompaniyada olib boriladigan barcha ishlar uchun xavfni baholashni amalga oshirishi shart. Homilador xodimlarni himoya qilish bo‘yicha zarur chora-tadbirlar ish beruvchiga homiladorlik to‘g‘risida xabardor qilingandan so‘ng darhol amalga oshirilishi kerak va xodimga uning mehnat sharoitlariga (keyingi) tuzatishlarni muhokama qilish imkoniyati berilishi kerak. To‘rt haftalik ishdan so‘ng, xodim kasal bo‘lgan taqdirda, ish beruvchi tomonidan olti hafta davom etadigan to‘lovni olish huquqiga ega. Xodim kasallik ta’tilisiz ishlagan muntazam to‘lovni ish beruvchi to‘lashi kerak. 30 dan kam xodimga ega bo‘lgan kichik kompaniyalarda ish beruvchi kasallik to‘lovini qaytarishga imkon beradigan taqsimlash tartibida ishtirok etishi mumkin. Odatda, qonuniy kasallik nafaqalari 78 hafta davomida muntazam ish haqining 70% miqdorida to‘lanadi. Olti oylik ishdan so‘ng, og‘ir nogiron xodim 5 kunlik haftaga asoslanib, besh kunlik qo‘shimcha ta’tilni talab qilishi mumkin. Yuqorida aytib o‘tilgan qonuniy ta’tillardan tashqari har qanday ta’tillar (masalan, yaqin insoni vafot etgan yoki og‘ir bemor bo‘lgan holatdagi ta’tili, ko‘chish bilan bog‘liq ta’til) jamoa shartnomalari/ ishchi kengash shartnomalarida belgilanadi. Germaniya qonunchiligiga ko‘ra, mehnat munosabatlari o‘zaro rozilik bilan, muddatli shartnoma muddati tugashi yoki ikki tomonning birining ogohlantirishi bilan bekor qilinishi mumkin. Umumiy himoyaga kelsak, ish beruvchining xodimni ishdan bo‘shatish erkinligi Ishdan bo‘shatish to‘g‘risidagi qonun ("DPA") bilan sezilarli darajada cheklanadi, ishdan bo‘shatish quyidagi hollarda qo‘llaniladi: 1) biznes subyektida odatda o‘ndan ortiq xodim bo‘lsa; va 2) xodim bir korxonada yoki korxonada olti oy davomida uzluksiz ishlagan bo‘lsa. Korxona boshqa shaxsga o‘tkazilgan holatda xodimlarning huquqlari. O‘tkazuvchining barcha xodimlari mehnat shartnomalari shartlari va stajlari saqlanib qolgan holda avtomatik ravishda qabul qiluvchiga o‘tadilar. O‘tkazishdan oldin har bir manfaatdor xodim o‘tkazish, uning kelib chiqishi sabablari, ijtimoiy va huquqiy oqibatlari va qabul qiluvchi tomonidan rejalashtirilgan har qanday boshqa chorahttps://t.me/Erus_uz Multidisciplinary Scientific Journal December, 2022 15 Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 1 \| ISSUE 7 \| 2022 tadbirlar to‘g‘risida yozma ravishda xabardor qilinishi kerak. Xodim to‘g‘ri va to‘liq ma’lumot xatini olgan kundan boshlab bir oy ichida o‘z e’tirozining sabablarini ko‘rsatmasdan, ish joyini o‘tkazishga e’tiroz bildirishga haqli. Agar ma’lumot xati qonuniy talablarga mos kelmasa, e’tiroz bildirish huquqi faqat o‘tkazilish amalga oshirilgandan keyin bir necha yil o‘tgach tugatilishi mumkin. E’tiroz bo‘lsa, mehnat munosabatlari o‘tkazuvchi bilan davom etadi. Agar o‘tkazuvchi endi xodimga ish taklif qilish imkoniyatiga ega bo‘lmasa, operatsion sabablarga ko‘ra ishdan bo‘shatish ijtimoiy jihatdan qonuniy bo‘lishi mumkin. Sobiq mulkdor va huquqiy vorislar uchun qo‘yiladigan talablar. Qabul qiluvchiga o‘tkazish paytida mavjud bo‘lgan mehnat shartnomalari natijasida kelib chiqadigan barcha huquq va majburiyatlar yuklatiladi, shuningdek, qabul qiluvchi o‘tkazuvchi tomonidan zarar ko‘rgan xodimlar oldidagi pensiya majburiyatlari uchun javobgar bo‘ladi. Shu bilan birga, qabul qiluvchi qabul qilingan xodimlarga va uning boshqa xodimlariga teng munosabatda bo‘lishga majbur emas. Agar ishdan bo‘shatish transferga asoslangan bo‘lsa, ishdan bo‘shatish haqiqiy emas deb topiladi. FOYDALANILGAN ADABIYOTLAR RO‘YXATI: (REFERENCES) 1. German Residence Act 2. The dismissal Protection Act 3. Remuneration Transparency Act 4. German minimum Wage Act 5. German Protection against Infection Act 6. The Federal data Protection Act 7. Social Security Codes 8. Maternity Protection Act 9. German Constitution https://t.me/Erus_uz Multidisciplinary Scientific Journal December, 2022 16
https://openalex.org/W2146679517	https://europepmc.org/articles/pmc4443677?pdf=render	English	null	ENVIRONMENTS and EOL: identification of Environment Ontology terms in text and the annotation of the Encyclopedia of Life	Bioinformatics	2,015	cc-by	2,026	V C The Author 2015. Published by Oxford University Press. 1872 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Summary: The association of organisms to their environments is a key issue in exploring biodiver- sity patterns. This knowledge has traditionally been scattered, but textual descriptions of taxa and their habitats are now being consolidated in centralized resources. However, structured annotations are needed to facilitate large-scale analyses. Therefore, we developed ENVIRONMENTS, a fast dic- tionary-based tagger capable of identifying Environment Ontology (ENVO) terms in text. We evaluate the accuracy of the tagger on a new manually curated corpus of 600 Encyclopedia of Life (EOL) spe- cies pages. We use the tagger to associate taxa with environments by tagging EOL text content monthly, and integrate the results into the EOL to disseminate them to a broad audience of users. Availability and implementation: The software and the corpus are available under the open-source BSD and the CC-BY-NC-SA 3.0 licenses, respectively, at http://environments.hcmr.gr C t t ﬁli @h l j hl j @ k dk Supplementary information: Supplementary data are available at Bioinformatics online. Evangelos Pafilis1,, Sune P. Frankild2, Julia Schnetzer3,4, Lucia Fanini1, Sarah Faulwetter1, Christina Pavloudi1, Katerina Vasileiadou1, Patrick Leary5, Jennifer Hammock6, Katja Schulz6, Cynthia Sims Parr6,†, Christos Arvanitidis1 and Lars Juhl Jensen2, 1Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Heraklion, Crete, Greece, 2Disease Systems Biology Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 3Max Planck Institute for Marine Microbiology, Bremen, Germany, 4Jacobs University gGmbH, School of Engineering and Sciences, Bremen, Germany, 5Marine Biological Laboratory, Woods Hole, MA 02543, USA and 6National Museum of Natural History, Smithsonian Institution, Washington, DC 20013-7012, USA To whom correspondence should be addressed. †Present address: National Agricultural Library, USDA, Beltsville, MD 20705-2351, USA Associate Editor: Jonathan Wren To whom correspondence should be addressed. †Present address: National Agricultural Library, USDA, Beltsville, MD 20705-2351, USA Associate Editor: Jonathan Wren Received on November 13, 2014; revised on December 23, 2014; accepted on January 18, 2015 organisms with environmental descriptors (Buttigieg et al., 2013). The ontology comprises 1600 terms and is part of recommended (meta-)genomic metadata standards (Yilmaz et al., 2011). Having the environmental information contained in EOL annotated in the form of ENVO terms, rather than as free text, would enhance search capabilities and enable users to easily compile summary statistics on, for example, the ecological distribution of any taxa. However, manually annotating all EOL entries with ENVO terms would be ENVIRONMENTS and EOL: identification of Environment Ontology terms in text and the annotation of the Encyclopedia of Life Evangelos Pafilis1,, Sune P. Frankild2, Julia Schnetzer3,4, Lucia Fanini1, Sarah Faulwetter1, Christina Pavloudi1, Katerina Vasileiadou1, Patrick Leary5, Jennifer Hammock6, Katja Schulz6, Cynthia Sims Parr6,†, Christos Arvanitidis1 and Lars Juhl Jensen2, Bioinformatics, 31(11), 2015, 1872–1874 doi: 10.1093/bioinformatics/btv045 Advance Access Publication Date: 24 January 2015 Applications Note Bioinformatics, 31(11), 2015, 1872–1874 doi: 10.1093/bioinformatics/btv045 Advance Access Publication Date: 24 January 2015 Applications Note Data and text mining 6 Future work The ENVIRONMENTS tagger is applicable to other large sources of text than EOL. For example, it can be applied to text fields, such as isolation source, in Genbank (Hirschman et al., 2008). Combined with the SPECIES tagger (Pafilis et al., 2013), it can also be used to 1 Introduction organisms with environmental descriptors (Buttigieg et al., 2013). The ontology comprises 1600 terms and is part of recommended (meta-)genomic metadata standards (Yilmaz et al., 2011). Having the environmental information contained in EOL annotated in the form of ENVO terms, rather than as free text, would enhance search capabilities and enable users to easily compile summary statistics on, for example, the ecological distribution of any taxa. However, manually annotating all EOL entries with ENVO terms would be The Encyclopedia of Life (EOL; http://eol.org/) is a web resource offering biodiversity knowledge summaries of the world’s species to a vast audience (Parr et al., 2014). It currently aggregates content from more than 250 providers. These include textual descriptions about the biology, such as habitat, of more than 900 000 taxa. The Environment Ontology (ENVO) project aims to provide a controlled, structured vocabulary to support annotation of 1872 1873 ENVIRONMENTS and EOL clades Actinopterygii, Annelida, Arthropoda, Aves, Chlorophyta, Mammalia, Mollusca, Streptophyta. From these, we extracted the sections about behavior, biology, dispersal, distribution, ecology, habitat, legislation, migration, reproduction and trophic strategy. After discarding very short (<100 words) and very long (>1000 words) pages, we randomly selected 75 species from each of the eight clades. The resulting 600 species pages were randomly distrib- uted among six annotators; 20% of the pages were given to two an- notators to allow for assessment of inter-annotator agreement (IAA). Each person independently annotated ENVO terms in the text, with no knowledge of which pages had been assigned to a se- cond annotator. Based on the shared abstracts we find that the me- dian pairwise Cohen’s kappa is 0.65, implying that the overall IAA is acceptable despite the difficulty of the annotation task (a Cohen’s kappa value of 0 indicates random agreement, while a value of 1 total agreement). The corpus was partitioned into two sets of equal size that were used for development and final evaluation, respect- ively (see supplementary information). Fig. 1. Top: The “Overview” tab of the EOL taxon pages show a subset of the ENVO terms obtained through text mining; an extended list of such terms is available in the “Data” tab. Parts of the page have been resized to improve readability. Bottom: The latter list provides links to the EOL text sections where each term was found (highlighted in bold) 5 Annotation of EOL To realize the full potential of any text-mining system, it is import- ant that it is adopted by the broad community and that its results are disseminated to the intended end users. To this end, we have integrated the ENVIRONMENTS tagger with the EOL web re- source to provide users with ENVO terms for each taxon. Each month, we rerun the tagger on all English text in environment- related sections of EOL (see supplementary information). As of October 2014, this gave rise to 1 077 522 annotations of ENVO terms for 234 582 EOL taxa. We constructed a dictionary based on ENVO by extracting all names and synonyms from the OBO file (see supplementary informa- tion), excluding broad synonyms, obsolete terms, terms describing foods rather than environments and terms representing organisms and tissues, which are better captured by other ontologies. Names con- nected to multiple terms were assigned to the one that best captures its meaning, by ranking the terms based on whether the name was the primary name, an exact synonym, a narrow synonym or a related synonym of the term. Because ENVO usually lists only the singular noun forms, we automatically generated plural and adjective forms. We make these environment annotations available to end users in three different ways. First, we show them within the EOL taxon web pages, which provide links to the relevant paragraphs in the textual descriptions for each ENVO term (Fig. 1). Second, they can be queried through the web interface or the application program- ming interface of the new EOL/Traitbank semantic web data reposi- tory of organismal traits (http://eol.org/traitbank). Third, the full annotation dataset can be downloaded in tab-delimited format from http://download.jensenlab.org/EOL/. A small fraction of the names will result in many false positives due to homonymy. We created a block list of such names by inspecting text for all names that appeared more than 2000 times in Medline and EOL. To find important synonyms missing in ENVO, we tagged the habitat and ecology sections of 1342968 EOL pages that are not part of our evaluation set (see supplementary information), and inspected all words occurring more than 100 times in untagged text segments. Based on this analysis and false negatives found in the development part of the curated corpus, we added 142 synonyms to the dictionary. 4 Performance evaluation highly time demanding. An attractive alternative is to use text mining to automatically tag ENVO terms. So far, the few efforts to perform named entity recognition of environments have focused on tagging bacteria biotopes (Bossy et al., 2013) or made use of generic tools, not optimized for the task (Thessen and Parr, 2014). Since the ENVIRONMENTS tagger recognizes names within text and links them to ENVO terms, we benchmarked both aspects of its performance. To quantify to which extent the tagger recognizes the same text fragments as the annotators, we calculated precision and recall at the mention level, considering both exact and partially overlapping matches as true positives. On the evaluation part of the corpus, this resulted in 87.8% precision and 77.0% recall, corres- ponding to an F1 score of 82.0%. For the matches that were con- sidered true positive for the recognition task, we further evaluated if the tagger linked them to the same ENVO terms as the annotators did. In 87.1% of cases, the tagger and the annotator agreed on at least one ENVO term (see supplementary information). Here, we present ENVIRONMENTS, a tagger capable of iden- tifying ENVO terms with sufficient accuracy and speed to be useful for annotating large text corpora. To benchmark the method, we de- veloped a new gold standard corpus of manually annotated EOL taxon pages. Last but not least, we have extended the EOL web resource with ENVO terms for each taxon, which are automatically mined from their textual descriptions. 2 ENVO term tagger ENVIRONMENTS identifies ENVO terms in text using the same fast dictionary-based tagging engine as in Pafilis et al. (2013). The command- line tool requires only a single parameter, namely the path to a folder with the text files to be processed (see supplementary information). Funding Hirschman,L. et al. (2008) Habitat-lite: a GSC case study based on free text terms for environmental metadata. OMICS, 12, 129–136. The Encyclopedia Of Life Rubenstein Fellows Program [CRDF EOL-33066- 13/E33066], the LifeWatchGreece Research Infrastructure [384676-94/ GSRT/ NSRF(C&E)] and the Novo Nordisk Foundation Center for Protein Research [NNF14CC0001]. Paﬁlis,E. et al. (2013) The SPECIES and ORGANISMS resources for fast and accurate identiﬁcation of taxonomic names in text. PLoS One, 8, e65390. Parr,C.S. et al. (2014) The encyclopedia of life v2: providing global access to knowledge about life on earth. Biodivers. Data J., 2, e1079. Conflict of Interest: none declared. Thessen,A.E. and Parr,C.S. (2014) Knowledge extraction and se- mantic annotation of text from the encyclopedia of life. PLoS One, 9, e89550. 3 Manually curated corpus To construct an evaluation corpus that covers diverse environment types, we retrieved 313 269 EOL species pages representing the E.Pafilis et al. 1874 1874 Buttigieg,P.L. et al. (2013) The environment ontology: contextualising biolo- gical and biomedical entities. J. Biomed. Semantics, 4, 43. Buttigieg,P.L. et al. (2013) The environment ontology: contextualising biolo- extract species–environment pairs from the scientific literature, like legacy biodiversity literature (Gwinn and Rinaldo, 2009). Buttigieg,P.L. et al. (2013) The environment ontology: contextualising biolo- gical and biomedical entities. J. Biomed. Semantics, 4, 43. gical and biomedical entities. J. Biomed. Semantics, 4, 43. Gwinn,N.E. and Rinaldo,C. (2009) The biodiversity heritage library: sharing bi di i li i h h ld IFLA J 35 25 34 Gwinn,N.E. and Rinaldo,C. (2009) The biodiversity heritage library: sharing biodiversity literature with the world. IFLA J., 35, 25–34. Gw ,N. . a d a do,C. ( 009) e b od ve s ty e tage b a y: s a g biodiversity literature with the world. IFLA J., 35, 25–34. biodiversity literature with the world. IFLA J., 35, 25–34. References Yilmaz,P. et al. (2011) Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) spe- ciﬁcations. Nat. Biotechnol., 29, 415–420. Bossy,R. et al. (2013) BioNLP shared task 2013—an overview of the bacteria biotope task. ACL 2013. In: Proceedings of the BioNLP Shared Task 2013 Workshop, pp. 161–169. Bossy,R. et al. (2013) BioNLP shared task 2013—an overview of the bacteria biotope task. ACL 2013. In: Proceedings of the BioNLP Shared Task 2013 Workshop, pp. 161–169.
https://openalex.org/W2051728508	https://link.springer.com/content/pdf/10.1007/s11252-012-0268-x.pdf	English	null	Sustainable urban greening strategies for compact cities in developing and developed economies	Urban ecosystems	2,012	cc-by	14,128	Sustainable urban greening strategies for compact cities in developing and developed economies C. Y. Jim Published online: 24 October 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Published online: 24 October 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Published online: 24 October 2012 Published online: 24 October 2012 Published online: 24 October 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com # The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Urban greening contributes notably to quality of life and ecosystem services in cities. Compact cities in developing and developed countries are commonly beset by green- space deficit. Based on literature review supplemented by field studies in different cities, a sustainable urban greening strategy is proposed. Urban renewal and new developments without a greening vision could miss the opportunities to bring relief. The public and private sectors can join hands to insert plantable spaces into the urban fabric. Urban greenspaces (UGS) with good connectivity forming a green network to permeate the city constitute the hallmarks of a naturalistic design. Preservation and creation of natural areas with rich biodiversity offer a new dimension to UGS design. Greening benefits could be expressed in economic terms to complement conventional ecological-environmental emphasis. Out- standing trees could receive high-order conservation efforts, and trees in construction sites warrant enhanced protection. Tree transplanting demands an overhaul in concepts and skills. Improving roadside tree planting and maintenance offers a cost-effective way to upgrade the Abstract Urban greening contributes notably to quality of life and ecosystem services in cities. Compact cities in developing and developed countries are commonly beset by green- space deficit. Based on literature review supplemented by field studies in different cities, a sustainable urban greening strategy is proposed. Urban renewal and new developments without a greening vision could miss the opportunities to bring relief. The public and private sectors can join hands to insert plantable spaces into the urban fabric. Urban greenspaces (UGS) with good connectivity forming a green network to permeate the city constitute the hallmarks of a naturalistic design. Preservation and creation of natural areas with rich biodiversity offer a new dimension to UGS design. Greening benefits could be expressed in economic terms to complement conventional ecological-environmental emphasis. Out- standing trees could receive high-order conservation efforts, and trees in construction sites warrant enhanced protection. Tree transplanting demands an overhaul in concepts and skills. Urban Ecosyst (2013) 16:741–761 DOI 10.1007/s11252-012-0268-x Urban Ecosyst (2013) 16:741–761 DOI 10.1007/s11252-012-0268-x C. Y. Jim (*) Department of Geography, University of Hong Kong, Pokfulam Road, Pokfulam, Hong Kong e-mail: hragjcy@hku.hk Sustainable urban greening strategies for compact cities in developing and developed economies Improving roadside tree planting and maintenance offers a cost-effective way to upgrade the townscape. Ameliorating widespread soil limitations could remove a major hindrance to tree growth. Innovative ideas of development right transfer, street pedestrianization, river and canal revitalization, green roofs and green walls could mobilize hitherto underused plantable resources. Lacking appropriate institutional setup and scientific capability pose intractable bottlenecks. Innovative public policies and greening technologies are needed for sustained improvements. Amalgamating natural and social sciences in a multidisciplinary approach and reinforcing the link between science and public policies could overhaul greening. Improving roadside tree planting and maintenance offers a cost-effective way to upgrade the townscape. Ameliorating widespread soil limitations could remove a major hindrance to tree growth. Innovative ideas of development right transfer, street pedestrianization, river and canal revitalization, green roofs and green walls could mobilize hitherto underused plantable resources. Lacking appropriate institutional setup and scientific capability pose intractable bottlenecks. Innovative public policies and greening technologies are needed for sustained improvements. Amalgamating natural and social sciences in a multidisciplinary approach and reinforcing the link between science and public policies could overhaul greening. Keywords Compact city. Ecosystem service . Nature deficit . Precision planning . Sustainable development . Urban biodiversity. Urban ecology. Urban greenspace . Urban natural area Introduction The creation of cities since antiquity marked the triumph of humanity over changeable and harsh nature. One of the undesirable collaterals of urban living was detachment 742 Urban Ecosyst (2013) 16:741–761 from nature. When settlements were small, nature was situated nearby, allowing humans to maintain frequent contacts. As settlements grew bigger, nature was increas- ingly marginalized and it became scarce, distant and often neglected or degraded. Some urbanites began to miss, consciously and sublimally, the innate link with nature. They endeavored to find solace in created natural enclaves in cities (Conan 1986). Surrogates of nature were established in the form of domestic gardens. With a utilitarian bent, plants bearing edible fruits were initially domesticated and nurtured (Ferriolo 1989; Bowe 2010). The universal human preference for aesthetic objects soon triggered the adoption of ornamental plants. Trees with large and dense crowns were cultivated for shading from the hot sun and shielding from wind impact (Lawrence 1993). Initially, gardens were principally private domains for owners who could afford the means and the leisure to create and maintain them. The nobility and the aristocracy would establish them for personal gratification and as symbols of wealth and power. Some would maintain wild or semi-wild grounds for recreational hunting. Common people had no access to such private reserves. The continued urban expansion would occlude some private greenery in the sprawling process. The Industrial Revolution brought factories and workers into cities, together with abject poverty, low-quality housing, excessive building density, poor hygiene and health, and degraded environmental conditions. Responding to the deprivation, people flocked to the meager open and greenspaces accessible to the public, often associated with religious and public establishments and cemeteries. Governments were eventually urged to satisfy such demands by the novel institution of urban parks, which began in nineteenth century Britain (Lawrence 1988). Some private green areas were acquired by municipal governments and opened to the public. The bold innovation was soon adopted by other countries to become an obligatory and universal amenity. The year 2010 denotes a notable watershed in human and urbanization history, with half of the 7 billion population living in cities. The growth in population and large cities in recent decades occurred mainly in less-developed areas (United Nations Population Fund 2012). Most of the million- and mega-cities (over 10 million population) are now situated in less-developed countries (National Geographic 2012). Introduction A notable proportion of cities in less-developed countries, especially those with a long development history, tend to be compact (Jenks et al. 1996; Burton 2002) with tightly-packed urban form. With most lands usurped by buildings and roads, little interstices are left for greenery, resulting in grave deficiency in public open spaces (Jim 1989, 1998a). Urban redevelopments, new urban areas and new towns were sometimes built in haste with little regard to environmental quality (Olembo and de Rham 1987; Herz et al. 2003). The need to dilute the built-up harshness with green areas could be neglected (Marcotullio 2001). Opportunities to improve environmental quality and quality of life for millions of urban residents could be grasped in the spirit of sustainable development and smart growth (Hestmark 2000; Foo 2001). With increasing concern about climate change, cities are beset by the double scourge of global warming superimposed on urban heat island effect. Urban tree planting offers a viable way to climate proof cities, especially in high-density residential precincts (Hall et al. 2012) and a contribution to carbon offset and sequestration (Escobedo et al. 2010; Strohbach and Haase 2012). Some parts of cities in developed economies share similar problems encountered in compact cities in developing economies. The main objective of this study is to develop a comprehensive, operational and actionable strategy to overcome the physical, psychological and institutional barriers to greening compact cities mainly in developing economies and also in developed econ- omies (hereinafter referred to as “compact developing or developed cities” or “CDDC”). 743 Urban Ecosyst (2013) 16:741–761 The World Bank (2012) classifies countries in the world by income groups according to 2010 gross national income (GNI) per capita: low ≤US$1,005; lower middle US$1,006– 3,975; upper middle US$3,976–12,275; and high ≥US$12,276. Low-income and middle- income economies are often referred to as developing economies. The World Bank (2012) website lists the countries in each group. Hong Kong is put in the high income group, but its urban environment is one of the most compact in the world, sharing many greening limitations and environmental problems commonly found in cities in less developed countries. Introduction Related to the primary objective, this study aims at developing practical urban greening methods based on relevant concepts as well as exemplary practices: (a) the benefits of urban greening could be maximized by revamping entrenched thinking, practices and inertia; (b) decision makers could move from the conventional to the innovative mode, and adopt an integrated and long-term approach; (c) the entire cradle- to-grave spectrum of urban greening demands attention, encompassing plantable sites, planning, design, species assemblage, installation, maintenance, management, governance, and community expectations (Attorre et al. 2000; Jim and Chen 2006a); (d) notable gaps in knowledge have been identified for proposed improvement, such as the contribution to regional biodiversity and carbon sequestration (Takahashi et al. 2008), and landscape and conservation planning (Jim and Chen 2009a, b); and (e) the framework could provide hints on future research priorities to strengthen the locally-relevant knowledge base and appropriate technology, and to inform management. Methods This study is mainly based on literature review, supplemented by field studies in over 100 cities in 25 countries, and research and practice experience in urban forestry in Hong Kong and other south China cities. The exemplars could serve to verify or validate the applicability of the chosen urban greening approach. Where appropriate, literature is cited (175 items in total) to lend support. Learning from field studies, however, often cannot be supported by literature, but they refer mainly to good practices rather than elucidation of concepts. As the bulk of urban ecology and urban greening researches are conducted in developed cities, their examples and good practi- ces would be selected to inform the strategy for CDDC. The more limited studies in developing cities, if appropriate, would be included in the study. Besides expounding underlying principles, the practical and actionable applications were emphasized. A multidisciplinary and transdisciplinary approach (James et al. 2009) has been adopted to encompass relevant concepts from urban biogeography, urban ecology, urban forestry, urban horticulture, urban landscape ecology, urban environmental planning, and urban soil science. The results and discussion of the study, given in the ensuing part of the paper, are structured according to a natural progression based on three main stages in the planning- management stream of urban greening: (1) Applying urban ecological principles to urban greening design, greenspace geometry, urban biodiversity enrichment, and holistic assess- ment of urban greening benefits; (2) Protecting nature-in-city assets, involving spontaneous natural remnants, champion-caliber tree stock, tree preservation in construction sites, sal- vaging outstanding trees by transplanting, and timely tree care; and (3) Augmenting greening opportunities, such as planting at narrow roadsides, ameliorating soil constraints, and introducing greenery into compact areas. 744 Urban Ecosyst (2013) 16:741–761 Adopt ecological design Cities have most soils sealed by buildings, roads or paving materials (Scalenghe and Marsan 2009). The spatial variations in permeable or evaporating surfaces determine the capacity of a city to admit greenery (Arnold and Gibbons 1996; James and Bound 2009). The hetero- geneous urban ecosystem demands diverse conservation treatments (Band et al. 2005). Both public and private lands could contribute to the greening endeavor (Troy et al. 2007). Urban ecological principles have seldom been enlisted to optimize the configuration and ingre- dients of urban greening. The conventional approach is dominated by ornamental or aesthetic considerations of landscape plants. Most urban greenspaces (UGS) were designed to achieve neat geometric patterns and well-manicured parkland landscape with limited inherited or created nature. Such outdated mentality has a strong inertia to linger in both developing and developed countries. Modern UGS could avoid excessive regimentation of ingrained mainstream landscaping by emulating nature’s diversity, variability, changeability and flexibility (Henke and Sukopp 1986; Gordon 1990; Cook and Lier 1994). The naturalistic or ecological design could create UGS with more natural species composition, community assemblage, biomass structure, and spatial pattern (Cole 1986; Cook 2002). The muffled senses of people living in cities could be revitalized by diverse visual, audio, olfactory and tactile stimuli offered by natural ingredients. Instead of making all UGS wild, which may not be practicable, a compromise could be reached between popular expectations and nature provision. A spectrum of sites with different degree of naturalness could match the local land-use and biotope pattern (Breuste and Wohlleber 1998; Löfvenhaft et al. 2002). A large site could accommodate a variety of habitats, whereas a small site could focus on one. The pre-urbanization ecosystems around a city, including the common and rare types, could be inherited or emulated. Local ecological knowledge and has to be reinforced by research to design naturalistic UGS. Optimize greenspace geometry and connectivity Applying urban ecological principles Adopt ecological design Optimize greenspace geometry and connectivity Where continuity could not be achieved in developed areas, a series of small sites with short inter-site distance could provide stepping stones for humans and wildlife. (3) Long corridors or linear green patches They provide extended edges with the surrounding built-up matrix, so that more residents can access conveniently, preferably by walking less than 400 m or 10 min (Müller-Perband 1979; Burgess et al. 1988). For two UGS with a similar area, the linear one with a larger catchment of potential users is preferred. Well-connected UGS permit movement of wildlife (including pollinators and dispersal agents) and propa- gules. Intra-urban corridors connected to peri- and extra-urban natural areas could augment such biotic movements. g (4) Patches or corridors parallel to natural linear features Patches or corridors oriented parallel to streams or coastlines could enhance urban ecological networks (Ignatieva et al. 2010). Water bodies provide an important land- scape element for joint development of greenways and blueways. People’s affinity to water edges can increase the UGS appeal. It also helps to restore or conserve natural waterways or canals that run through cities. (5) (5) High quality green site edges The edges between green and non-green areas could enhance ecological and landscape qualities. They should preferably be soft (natural), wide, gradual and curvilinear. Thus fences or walls that create abrupt and unfriendly barriers should be avoided. (5) High quality green site edges The edges between green and non-green areas could enhance ecological and landscape qualities. They should preferably be soft (natural), wide, gradual and curvilinear. Thus fences or walls that create abrupt and unfriendly barriers should be avoided. It is desirable to mingle green patches or corridors with the built-up matrix, such that UGS are situated close to people to generate a nature-in-city ambience. At the urban edge where develop- ments interface with natural areas, tongues or wedges of peri-urban natural areas could extend into built-up areas in an interfingering pattern (Frey 2000; Caspersen and Olafsson 2010). Small remnant nature pockets embedded in built-up areas could be kept in the wild state and designated as urban natural areas (UNA) by statutory zoning. With peninsulas of nature extending into the city and islands of nature punctuating the city, the landscape, amenity, ecological and air-quality benefits could spill into the urban matrix. In landuse planning, opportunities for nature to intermix with the city should be assiduously realized (Carr and Lane 1993). Optimize greenspace geometry and connectivity The pertinent benefits of creating spatially connected and contiguous UGS have escaped the attention of some planners in CDDC. Natural ecosystems tend to be well connected to each other, with unimpeded flow of energy, materials, organisms, propagules and genes across contiguous boundaries. UGS, however, are usually demarcated as isolated entities sur- rounded by rather inhospitable built-up areas (Kong et al. 2010; Tian et al. 2011). The main green sites, urban parks, are often square or rectangular plots surrounded by roads. Some large parks are penetrated, traversed or severed by roads. The harsh and alien conditions of roads and buildings form barriers to the movement of wildlife and people. They also generate spillover impingement on UGS environmental quality to reduce their capacity to support and nurture nature. Applying the concepts of landscape ecology (Hersperger 1994; Dramstad et al. 1996; Breuste et al. 2008) and landscape ecological urbanism (Steiner 2011) could improve spatial design of UGS. They could be analyzed as three principal entities, namely patch (broad green areas), corridor (linear green areas), and matrix (surrounding built-up areas), in conjunction with the ancillary issue of edge (interface between patches or corridors with matrix). The key geometrical properties of UGS, such as location, size, shape, orientation, and distance from and connectivity with other green patches, could be molded to enhance their ecosystem services (Davey 1998; Young 2010): 745 Urban Ecosyst (2013) 16:741–761 (1) Large patches or wide corridors They provide adequate spaces to accommodate a variety of habitats and species, especially those demanding a relatively large minimum site. They also raise the ratio between site area and edge to reduce edge impacts where the UGS interfaces with incompatible or nuisance-generating land uses. (1) Large patches or wide corridors They provide adequate spaces to accommodate a variety of habitats and species, especially those demanding a relatively large minimum site. They also raise the ratio between site area and edge to reduce edge impacts where the UGS interfaces with incompatible or nuisance-generating land uses. (2) Connectivity between patches or corridors Green sites that are close to each other or contiguous can form a green network to permeate the built-up matrix. Green patches linked by green corridors or greenways (Flink and Searns 1993) form an integrated UGS system to enmesh built up areas (Flores et al. 1998; Jim and Chen 2003). Enrich greenspace biodiversity Enrich greenspace biodiversity Habitat diversity could be enhanced to raise biodiversity in UGS design (Alvey 2006). For new green sites, the manicured horticultural design could be complemented by the naturalistic- ecological approach (Henke and Sukopp 1986). Based on the hemeroby concept (Jalas 1955; Sukopp 1972), urban habitats range from natural, to emulated or created natural, semi-natural or ruderal, and entirely artificial. Some UGS in CDDC cluster at the artificial end of the spectrum, reflecting the inertia of traditional landscape design (Qureshi and Breuste 2010). To redress the bias, new or revamped UGS could admit more natural elements. Nature-oriented designs are often less expensive to build, and largely self-sustaining with minimum maintenance needs to reduce recurrent upkeeping costs. They meet the demand for natural areas within and near cities (Johnston 1990; Tzoulas and James 2010) with features that over-designed and expensive urban parks fail to deliver (Thompson 2002). Biodiversity in UGS could be enhanced especially with native species representative of the regional flora (Smale and Gardner 1999). For relatively large sites (e.g. >5,000 m2), not less than 25 % of the area could be earmarked for naturalistic greening. An ecological survey could identify existing natural habitats for incorporation into the naturalistic design. Species enrichment could be incorporated into the refurbishment scheme. Differ- ent sites could serve different ecological functions to depart from the common visual- ornamental bias. For large sites, the ecological-park design could include a mosaic of representative habitats. Using natives in urban greening is often hampered by a knowledge gap. The landscape profession and industry often have limited understanding or experience with indigenous species. They tend to adhere to a standard palette of common horticultural species for a given climatic zone, resulting in stereotype landscape designs and species assemblages with a disproportionate amount of exotic species. Local botanical-ecological research could be developed to identify, test and extend the use of indigenous plants in UGS. Botanic gardens with research facilities and orientation could play a key role in filling the knowledge gap (Maunder et al. 2001a, b; Dosmann 2006). The research findings could be applied to nurture a modern horticultural-nursery industry to produce native planting materials, and a land- scape profession to promote their use. Advocating natives should not exclude exotics. Cities are characterized by a diverse range of site conditions, far more than its countryside, which can accommodate a surpris- ingly varied urban flora. Optimize greenspace geometry and connectivity Existing green sites at the urban fringe could be ecologically upgraded to enhance ecosystem services. Nature can best be preserved; if not, emulated nature (defined as a planted area with species composition and biomass structure that are similar to a comparable natural vegetation type) could be created as surrogates (Baines and Smart 1991). A comprehensive assessment of urban ecological endowments could optimize a city-wide UGS master plan. Such plans could embrace ecological considerations and avoid domina- tion by locational and economic factors. To usher high-quality nature into the city, green plans could make good use of ecological realities and potentials. Sites of high ecological 746 Urban Ecosyst (2013) 16:741–761 value regardless of size could be protected. In particular, small pockets of biodiversity hotspots, unique habitats, remnant native vegetation, or heritage trees, could be captured in an embracing conservation package. Pristine or minimally disturbed ecosystems offer outstanding candidates, and disturbed sites could be evaluated for recuperative potential with or without human assistance. In neighborhoods suffering from nature deficit, suitable localities could be identified for creation of natural pockets. Evaluate holistic benefits of greenspace In planning and designing UGS, there is a tendency to focus on site area or green-area per capita at the expense of high-level benefits such as ecosystem services. The narrow outlook could discourage adoption of the ecological design. The appraisal of achievement in urban greening and benefits to the community could encompass the multiple and less tangible ecosystem services, such as improvement in air quality, enhancement of stormwater quality, and suppression of the urban heat island effect. A holistic assessment could gage the wide spectrum of ecological functions. Key attributes such as species richness, species diversity index, native-exotic ratio, remnant natural component, and fidelity to local or regional natural habitats, could be rated (Jim and Chen 2009c). Other factors that contribute to the ecological-environmental well-being of the city could enter the equation, such as biomass structure, vegetation coverage, planting density, vegetation height, leaf area index, open soil and evaporative surface, and soil connected to natural ground. Cities with different natural history, development mode, disturbance regime, nature conservation performance, and UGS provision demand different assessment methods. Local urban-ecological research could equip the assessment scheme and judge the quality of UGS plans. A small site with high scores may bring more ecosystem services than a large site with low scores. The importance of natural pocket parks in compact city areas could be stressed (Nordh et al. 2009). Selected criteria could be emphasized to guide and fulfill certain ecological-planning objectives. Individual sites could set specific targets, such as the minimum species richness and leaf area index. Such a precision UGS planning methodology could bring a renaissance to landscape design with sustainable benefits to urban ecology and residents. Cost-benefit analysis could enhance understanding of urban greening benefits (Dochinger 1980; Nowak and Dwyer 2000). As people are more amenable to monetary than ecological values, the benefits and amenities could be expressed in dollars. As nature-in-city is not a marketable commodity, indirect methods permit objective-scientific valuation (Chen and Jim 2008). Two strands of methods have been applied to the economic assessment of urban greening. The contingent valuation method explores citizens’ willingness-to-pay to use UGS, to prevent UGS loss (Jim and Chen 2006b; Lo and Jim 2012), or to preserve urban biodiversity (Chen and Jim 2010). Enrich greenspace biodiversity The capacity of UGS to support a wide assortment of species could be filled by a complement of natives and exotics. Natural sites could mainly hold natives, whereas semi-natural and artificial sites could accommodate both. A plant introduction office could develop a strategy and an action plan to screen systematically worthwhile exotic species from other places, and promote suitable ones to the landscape industry. This orderly importation could replace the random introduction, legally or illegally, of exotic plants by companies or individuals. Unregulated activities operating outside the quarantine regime should be controlled to avoid phytosanitation problems and inadvertent transfer of pest organisms and instant pathogens. 747 Urban Ecosyst (2013) 16:741–761 Evaluate holistic benefits of greenspace The hedonic pricing method analyzes the home buying behavior by isolating the proportion of the property transaction price attributed to urban greening and other natural areas (Garrod and Willis 1994; Jim and Chen 2006c, 2007). The results could estimate the monetary worth of urban green areas, which could be compared with establishment and maintenance costs (Jim and Chen 2009d). The dollar values assigned to UGS could justify the allocation of public funds for urban greening, and help to compete for resources in times of budgetary constraints. In applying the monetary valuation methods, the problems and challenges in methodology and hence the results should be fully acknowl- edged (Carson et al. 2001; Bateman et al. 2002; Malpezzi 2003). Preserve and nurture spontaneous nature Such persistent attitude has unneces- sarily eliminated or degraded high-caliber natural sites. Paradoxically, in their place we create parodies of nature that incur high capital and recurrent expenditures. Landuse zoning could be revamped to rescue nature in cities (Goldsmith 1988). Natural areas with high conservation value or are critically endangered can be proactively identified and protected by zoning and related legal instruments (Mills et al. 2009; Freeman and Bell 2011). Habitats with significant indigenous vegetation and wildlife should receive special protection (Breuste 2004). Instead of treating development land as a blank sheet, green fields could be designated as urban natural areas (UNA) with protected-area status analogous to countryside conservation areas. Natural patches and corridors could be preserved as far as possible in their present state (Henke and Sukopp 1986), for incorporation in a green network preferably connected to the city’s countryside (cf. Section on Adopt Ecological Design in Urban Green- ing). Remnant woodlands with high biodiversity, ecological value and ecosystem services should receive special attention (Croci et al. 2008; Jim 2011a, b). Core habitats and migration corridors could be preserved in a smart-growth and spatial-planning package. Spokes, fingers or tongues of linear UNA lands extending into built-up areas create a desirable spatial pattern to maximize nature’s benefits. Such greenways (Flink and Searns 1993) permit fresh air, clean water and wildlife to enter the city via natural expressways. The riparian corridor with existing woodlands that traverse a city is particularly valuable for habitat and biodiversity conservation (Pennington et al. 2010). Residential areas could have convenient access to nature and enjoyment of ecosystem services (Freeman and Bell 2011). Where natural areas are deficient, they could be nurtured with the careful site and soil preparation and native plant species to emulate regional wooded ecosystems (Johnston 1990; Lee and Thompson 2005). Disturbed natural areas could be repaired or restored by enrich- ment planting and elimination of undesirable elements to foster reconstitution of natural ecosystem (Borgmann and Rodewald 2005). Parts of existing green sites could be enriched with natural ingredients in terms of species composition and biomass structure. Where the climatic zone permits, woodland as the most complex ecosystem could be established by an afforestation program (Tartaglia-Kershaw 1982; Harmer 1999). The overarching spatial planning principle is to create adjacency and interpenetration between natural and urbanized areas. The health and vigor of UNA could indicate urban livability and environmental sustainability. Preserve and nurture spontaneous nature Preserve and nurture spontaneous nature Existing natural areas of high ecological worth in CDDC are often neglected or destroyed. Especially in the course of recent rapid growth, natural areas with spontaneous flora and fauna could be annihilated. Green fields in the urban fringe or the countryside envelope are 748 Urban Ecosyst (2013) 16:741–761 intruded by urban sprawl. Some islands of remnant nature in the urban matrix, especially the interstitial wilderness (Jorgensen and Tylecote 2007) and semi-natural sites with significant spontaneous components (Towne 1998; Millard 2008), could be ruined by development. Occasionally, the loss of nature is partly compensated by emulated natural areas in UGS. Preservation of natural sites within developed areas too often escapes the attention of city planners (Qureshi et al. 2010). Paradoxically, the land for new UGS have to be prepared at a high cost, often involving elimination of natural features and replacement by stereotype and simple parkland landscape with limited ecosystem benefits (Fernández-Juricic 2000; Hess and King 2002). intruded by urban sprawl. Some islands of remnant nature in the urban matrix, especially the interstitial wilderness (Jorgensen and Tylecote 2007) and semi-natural sites with significant spontaneous components (Towne 1998; Millard 2008), could be ruined by development. Occasionally, the loss of nature is partly compensated by emulated natural areas in UGS. Preservation of natural sites within developed areas too often escapes the attention of city planners (Qureshi et al. 2010). Paradoxically, the land for new UGS have to be prepared at a high cost, often involving elimination of natural features and replacement by stereotype and simple parkland landscape with limited ecosystem benefits (Fernández-Juricic 2000; Hess and King 2002). High-quality nature can as far as practicable be kept in development areas instead of establishing new UGS from scratch. Valuable natural remnants are often replaced by humanized greenery. Occasionally, isolated well-wooded religious sites or sacred groves are retained in built-up areas (Jim 2003a; Hong et al. 2007; Ormsby 2011). However, the natural landform, soil, water and vegetation of the preserved sites tend to be modified or partly replaced (Williams et al. 2005). Urban living has probably muffled if not distorted our value judgment of nature. Wild areas are commonly and erroneously construed as inferior or unsuitable UGS (Mazzotti and Morgenstern 1997). Nature has to exist in urbanized areas on human terms, to be simplified, sanitized, contained, tamed, regimented and manicured. Conserve champion quality trees Champion quality trees, as the most outstanding remnants of nature in cities, are often advertently and inadvertently damaged. In the course of urban development, a tiny subset of trees could perform exceptionally well in tree form, stature, vigor and life span. Local residents often develop sentimental attachment to them, sometimes elevated to veneration or worship. The cream of the tree stock could be identified by six criteria: species, dimensions, structure, performance, location, and special considerations such as species rarity, special ecological value, unique habitat, unusual tree form or dimensions, connection with notable personalities or events, landmark specimen, and historical significance (Jim 1994a, b). Champion trees are comparable to cultural heritage (antiquities and monuments) due to intimate association with the local community (Jim 2004b, 2005b, c). They denote out- standing representatives and ambassadors of nature in cities, and could be treated as the collective natural-cum-cultural heritage of the community. In many cities, they are inade- quately protected against damages by natural and human causes (Jim 2003b, 2005a). The alarming losses echo the lack of understanding and appreciation of an irreplaceable com- munity heritage. The major predisposing causes for losses are related to construction and trenching-roadwork activities, with some succumbing to age and health problems. Most cities have inherited only a small number of champion trees as ecological gems. The fast development and renewal of some CDDC have continued to damage this precious endow- ment. They deserve intensive monitoring, care and statutory protection. A dedicated urban tree ordinance (Jim and Liu 2000; Jim 2002) could be enacted, with a section covering special measures to guard them against harm. A systematic champion tree survey can establish a scientific database (Council of Tree and Landscape Appraisers 2000) to deepen appreciation of tree heritage value and reinforce tree management. They also constitute a benchmark to determine the level of compensation and penalty in case of damage (Jim 2004a). The data can allow development of a monetary valuation of the prized trees (Jim 2006). A champion tree register should cover developed lands and sites earmarked for development, and as an integral part of the tree ordinance (Randall and Clepper 1977). Professional tree-care guidelines and tree workers at the highest international standard could ensure high-grade maintenance. Cities could learn from the best practices to improve the protection regime. The out- standing trees could be conserved collaterally with their sites and environs. Preserve and nurture spontaneous nature Cities that permit nature to thrive are likely to permit humans to thrive. 749 Urban Ecosyst (2013) 16:741–761 Conserve champion quality trees Heritage trees would have witnessed the vicissitudes of their surroundings for decades if not centuries. Nearby urban fabrics of historical significance could have lingered with their natural partners, to deserve co-conservation and co-management with the elite trees. The overall townscape, including the housing styles and the high-quality vegetation cover in private lots, qualify as a holistic conservation entity. The conservation area concept that protects countryside and natural habitats could cover urban sites with high-caliber trees. A sizeable area with a notable number of champion trees could be designated as tree conservation areas. Roads lined with champion trees could be designated as tree conservation roads. Smaller sites including a special habitat such as a remnant woodland enclave, an old stone wall with hanging mature trees (Jim 1998a; Jim and Chen 2010), or old buildings with interesting spontaneous tree growth (Jim and Chen 2011), could be designated as tree conservation sites. The tree conservation strategy should take into account the past, present and future considerations in the spirit of transgenerational urban forestry and arboriculture (Jim 2005c). Tree conservation should pay special attention to protecting present and future growth spaces. Besides the above-ground expansion space, the often neglected subterranean rooting 750 Urban Ecosyst (2013) 16:741–761 room should be guarded against intrusion and degradation. If a particularly valuable tree is encountered and the site conditions permit, a three-dimensional tree protection zone (TPZ) could be delineated (Jim 1988). For champion trees that have not attained their potential dimensions, the TPZ should allow for future growth. In the below-ground environment, a sufficient soil mass not less than 1 m deep and as wide as the final crown spread should be provided to permit future root expansion. No excavation should be permitted in the TPZ. For trees cloistered by adjacent developments and do not have sufficient rooms to demarcate the TPZ, any future changes should prevent deterioration of tree growth conditions. Ameliorative measures could be applied to improve conditions for tree growth. Adjoining redevelopments should include setback (a town planning term meaning the required distance separating a building or structure from a property line) to enlarge the TPZ. Champion trees should be recorded on statutory zoning plans. Instead of denoting the tree location only, the boundary of the TPZ should be shown on the map like cultural relicts. Conserve champion quality trees The explanatory notes of the zoning plan could provide details on tree species, dimensions, protection justifications, and precautions to prevent damages. Developers and professionals involved in the development process could interpret the zoning plans and participate actively in their protection. Protecting trees in construction sites Protecting trees in construction sites Many CDDC are redeveloped and expanded at a fast rate, incurring many incongruous encounters between trees and development (Bowers 1999), which often conflict with existing trees (Watson and Neely 1995). The lack of effective statutory measures could fail to protect outstanding trees in construction sites. Trees prescribed for in situ preservation could be badly mistreated, resulting in degradation, injuries or death. The special skills required to protect trees in construction sites are often lacking. Preserved trees could be felled or killed due to improper treatment and poor protective measures. Effective measures could salvage more high-quality trees associated with construction. Tree preservation in construction sites requires detailed planning with regard to building footprint and foundation spread, access roads and ancillary paved areas (Jim 1988). The development process could match modern environmental protection practices. Instead of treating the development site as a blank sheet, an ecological site survey should identify trees or ecological habitats deserving preservation. A precision planning takes into account real- world site conditions, and treats outstanding natural features as assets rather than liabilities. Instead of development plans that are apathetic and antagonistic to nature, they should be sympathetic and synergistic. The size, shape, orientation and location of buildings and roads could be adjusted to match conservation needs. The inputs of landscape professionals should be solicited at the early rather than the late stage of the development stream. y g p Tree-protection practices in construction sites in CDDC have room for improvement (Morell 1992). Besides low standard, tree protection is beset by inadequate supervision of site workers. The half-hearted efforts and cavalier attitudes suggest lack of commitment. Site crews often take calculated risks to expedite construction work at the expense of trees. Clearly written guidelines should be prepared and enforced (Matheny and Clark 1998), to be supplemented by training in relevant concepts, skills and regulations. The knowledge transfer should permeate all levels from the management to the professionals, technicians and workers. Developers and construc- tion companies should learn tree-survey and timely preventive and precautionary measures at the earliest opportunity (Ames and Dewald 2003). The engineers and architects at the frontline of the construction project should take care of the quality control of tree-related tasks. As an incentive to tree preservation, that high quality trees could augment property value (Anderson and Cordell 1988) should be emphatically brought home. Transplant trees as the last resort High-quality trees affected by development are often indiscriminately recommended for transplanting rather than in situ preservation. Developers are tempted to eliminate obstacles to construction activities and the development. Application for transplanting is often attempted to transfer the trees to locations in or outside the subject site. The option of transplanting outstanding trees, especially large specimens, should be taken as the last resort. Moving a large tree often weakens and disfigures it, substantially reducing its landscape value and useful life span, and negating the very purpose of keeping it (Jim 1995; Watson and Himelick 1997; Harris 2004). Compensatory planting in lieu of preserving trees should be scrutinized with caution, for new planting could hardly make up for magnificent old trees. To transplant a large tree, a tree specialist and a structural engineer should team up to execute the complex task. It should be prepared in advance to reduce the transplant shock and to increase the chance of success. The indispensable phased root pruning stage is too commonly omitted. Ideally, a tree should be transplanted with all its roots in the undisturbed soil envelope. During the move, the soil in the rootball must not be disturbed or deformed to avoid injuring the roots. In practice, moving all roots is not possible, because the rootball will be too large and heavy. Recent tree root research suggests moving a manageable amount of roots, so that the rootball could be lifted and transported. The recommended minimum rootball, with width ten times the trunk diameter at breast height (DBH) (Watson and Himelick 2005) and depth of 1–2 m, is dependent on tree size. Adhering to the prescribed rootball size entails losing over 80 % of a tree’s roots, hence roots have to be cut in preparing the rootball. The drastic root loss imposes extreme stresses on tree functions and health (Jim 1995). Some trees that cannot tolerate may perish; others may struggle to survive in a feeble state. It takes years for an injured tree to heal, and full recovery may not be feasible. To reduce massive root amputation in rootball preparation, roots should be pruned in phases with intervening recuperative periods. For a large tree, root pruning demands four phases each separated by at least 6 months, requiring transplant work to start 2 years in advance. Due to ignorance of transplanting concepts and practices, this advanced planning horizon is seldom realized. Protecting trees in construction sites 751 Urban Ecosyst (2013) 16:741–761 A common weak link is the poor and inconsistent quality of tree survey reports (TSR), upon which decisions on trees are made. A substandard or inaccurate TSR could mislead and muddle. A guideline could explain specific TSR requirements with the help of a sample report (American Society of Consulting Arborists 2004; British Standards Institution 2012). A professional TSR should cover information on individual trees, including location, species, dimensions (tree height, trunk diameter and crown spread), structural integrity, health, performance, habitat condition, tree defects and disorders, special features, future growth prognosis, and color photographs of the whole tree as well as its notable parts and environs. It should provide specific recommendations and justifications for retention, transplanting and felling. Every effort should be taken to minimize transplanting and felling. The key task of TSR preparation should only be entrusted to a tree specialist. Transplant trees as the last resort The receiving site should be carefully chosen and prepared to permit healthy growth of the new migrant. It should provide adequate room for future expansion. The soil, drainage and microclimatic (wind and solar exposure, tempera- ture and humidity) conditions should match the source site. The precision arboriculture practice (Powell 1992; Starbuck 2007; ArborMaster 2012) should be based firmly on careful site and tree assessment. 752 Urban Ecosyst (2013) 16:741–761 Upgrade tree-care quality and timeliness The low-quality and delayed tree care cannot nurture the next generation of healthy and robust trees. Arboriculture and urban forestry in some cities are constrained by weak links and gaps in knowledge and applications. Trees commonly suffer from inadequate and unprofessional maintenance (Watson and Himelick 1997; Harris 2004). Newly planted trees may receive more attention mainly due to the post-planting maintenance and warranty contract of 1 to 2 years. Long-term and preventive tree care is often inadequate. Mature trees including champion specimens, could be neglected. Most urban trees encounter more growth problems than those in the countryside, requiring more care which is often not provided. The urban tree management package could focus more on tree care to treat it as a long-term cradle-to-grave enterprise. Trees in CDDC are often inflicted by chronic problems originated from poor-quality planting materials. They relate to poor nursery practice and inability to maintain a high and consistent standard. The common weaknesses include lack of vigor, crossed branches, v-crotch, unbalanced crown, crooked or curved trunk, multiple stems, wounds, decays, sparse foliage, kinked or girdling roots, injured roots, pest and disease infestation, and tiny rootball (Jim 1997b). Such problems existing at the seedling and sapling stages would in time develop into long-term liabilities and potential hazards of mature trees. The problems begin in the nurseries, associated with the lack of selection of seeds and seedlings to eliminate weaklings, and little corrective pruning and branch training. Nursery production methods demand an overhaul to meet vigorous planting-material specifications. Substandard planting materials should be resolutely rejected. Poor tree management in some CDDC is related to inadequate and infrequent tree inspection and the lack of a comprehensive assessment method. The visual tree assessment (VTA) based on the body language of trees (Mattheck and Breloer 1994) should be supplemented by instrumental tests, and conducted by qualified specialists in good time. The VTA results should inform preventive or pre-emptive maintenance to reduce the future need for care. Transplant trees as the last resort The problems could be classified by the degree of exigency and given timely treatments without procrastination. The tree-care staff should receive training commensurate with staff grade and job responsibilities. Technical skills in horticulture and arboriculture demand formal training coupled with on-the-job practices. The tree-care team should be led by an urban forester who should be a tree specialist educated to the degree level or above. Continuing professional development should be mandatory to acquire the latest concepts and practices. Establish- ment an urban forester post with an attractive compensation package could strengthen leadership and quality in the tree team. Overseas secondment and professional visits could encourage exchange of ideas and best practices based on first-hand experience. Some CDDC lack urban forestry knowledge especially on native species, which hinders urban greening work. Research conducted in developed countries may not be directly transferable to the less-developed world. The shortage of relevant basic and applied research calls for strengthening of local knowledge repertoire and capability. Networking of scientists and practitioners in an urban forestry league, grouped by geographical regions, could facilitate sharing of research resources and findings. Augmenting greening opportunities Hone planting techniques at narrow roadsides Hone planting techniques at narrow roadsides Roadside trees are the most cost-effective and conspicuous way to enhance the cityscape. Conventional planting techniques can hardly raise the quantity and quality of street trees in 753 Urban Ecosyst (2013) 16:741–761 CDDC. The roadside space above and below the ground is commonly inadequate or unsuitable for trees (de la Chevallerie 1986; Jim 1997a, b). In old districts, buildings usually have 100 % site coverage with little setback from the lot frontage for trees. Sidewalks are usually too narrow or too heavily used to afford tree planting (Kuhns et al. 1985; Evans et al. 1990). In this context, the old districts recommended for enhanced greening are those with little or no cultural, historical, heritage or conservation value, and have been designated or approved for urban redevelopment. In high-density areas, the footpaths are usually covered by awnings or arcades. In medium- and low-density areas, the limited setbacks are often cordoned off by walls and paved with concrete for parking or other non-green uses. City planning departments often prescribe the width of roadside planting strips to be the greatest width feasible given the required minimum paved width of the sidewalk established under transport regulations (e.g. New York City 2012). Some cities maintain lists of recommended street tree species of different final dimensions based on the width of the planting strip (e.g. Portland City 2012). A proposed minimum setback of 3 m along road- sides could create a planting strip to accommodate small to medium trees in densely-packed areas (Jim 1997a, 1999). Wider setback, where site conditions permit, can provide space for larger trees. The required land could be derived from redevelopment sites or conversion of vehicular carriageway. Building awning and advertisement signs should not intrude into the tree strip. The underground space should be reserved for tree roots, and buried utilities and other subsurface installation should not infringe. The unimpeded soil in the strip should be 1 m deep to accommodate tree-root requirements (Perry 1982; Day et al. 2010). Where space is limited, a narrower roadside planting strip of preferably 1 m wide could allow the growth of shrubs and herbs. To encourage setback, incentives such as the transfer of development right of the setback strip to the remainder of the plot or bonus plot ratio could be offered. Wider setback should be targeted to accommodate large trees for notable landscape improvement (Jim 1999). Embrace innovative greening ideas and sites Urban-greening planners and managers could think out-of-the-box to find innovative op- portunities and solutions. In old urban areas, redevelopment offer chances to increase plantable spaces within and at the frontage of building sites. In densely-packed neighbor- hoods with inadequate UGS provision, brownfield sites could become UGS to bring relief (Doick et al. 2009). Brownfield sites colonized spontaneously by vegetation could be conserved (Schadek et al. 2009). The development right of the enlisted land parcel could be transferred to new development areas where UGS provision is well-planned and suffi- cient. Such shifting of development right, applied systematically, could gradually improve the landscape and environmental qualities of old districts. Enabling incentives and planning laws could encourage developers to participate in landuse restructuring. Some side streets in CDDC are hardly used by vehicles, which could be diverted to nearby roads to permit pedestrianization and greening. Simple planting with beautiful flowering trees could be preferred by residents than expensive hard landscape. However, emergency vehicle access often restricts planting opportunities in narrow pedestrianized streets. Thus planting is limited on the edge and on one side only; where the streets are too narrow, planting is not permitted. Planting small trees that can be run over by emergency vehicles offer a possible solution. As emergency vehicle accesses are seldom used, most of the sites could be covered by greenery instead of hard paving, with the proviso that the vegetation could be sacrificed if it is necessary. It is relatively easy and fast to replace the lost small trees. The amenity value of old river courses or canals is often neglected in old districts. Some may become contaminated or clogged, and others may be covered by a deck. They could be restored and revitalized by cleaning the water course in conjunction with greening of the banks to create blueways bordered by greenways (Bae 2011). As people have strong affinity for water, particularly the land-water interface, such transformed amenity strips could serve as linear urban parks (Van Der Windt and Swart 2008). The water surfaces together with vegetation could lower air temperature and ameliorate the urban heat island effect. The waterfront area facing the harbor in some cities could similarly be upgraded to create a green promenade with attendant benefits. Many city areas have on-street car parking at the curbside. Hone planting techniques at narrow roadsides For setback >3 m, a road-median planting strip could be provided in addition to the lot-frontage one. Hard landscape could accompany the new green strips improve significantly the roadside pedestrian environment. A comprehensive and integrated approach to introduce greenery into old city areas could be instituted. Ameliorate pervasive urban soil constraints Urban soils especially at roadsides are often not conducive to healthy tree growth, yet they are seldom improved in greening programs (Bartens et al. 2010). The main physical problems are shallow soil layer, obstruction by rocks, building foundations and utility control boxes, excessive amount of stones and sand, poor soil structure, soil compaction, and sealing of the soil surface by concrete or asphalt (Jim 1993, 1998a, b; Perry 1994). The soil environment has poor aeration, limited moisture holding capacity and impeded drainage (Morgenroth and Buchan 2009). The poor chemical soils properties impose additional constraints, notably contamination by construction rubbles with calcareous concrete and cement fragments that raise the soil reaction to the harmful alkaline range. Urban soils are the sink for pollutants brought by run-on water, rainfall and gravity settlement from the atmosphere (Craul 1980; Jim 1998c). The shortage of available nutrients especially the essential nitrogen and phosphorus commonly dampens tree growth (Jim 1998d, e). The poor urban soil quality is often neglected in tree programs (Bullock and Gregory 1991; Craul 1992). The common misconception is that any soil can grow trees, and soil deficiencies can be easily rectified. Thus trees are often planted in poor site soil without improvement. Inferior soil trapped in the urban landscape after construction is difficult to 754 Urban Ecosyst (2013) 16:741–761 ameliorate or replace. Such poor soil cannot nurture healthy trees, resulting in heavy management liabilities. As soil materials are relatively inexpensive, poor site soils should be replaced by a good soil mix down to about 1 m deep before trees are planted (Lindsey and Bassuk 1991). Localized improvement in a tree pit cannot help. At roadsides, a continuous soil corridor could be installed, with a reinforced concrete slab cover to return walkable surface to pedestrians. Roots along the corridor can share the soil and spread out to enhance growth and anchorage. Embrace innovative greening ideas and sites The area between two contiguous parking spaces could accommodate an at-grade (at the same level as the road surface) tree pit with its soil protected from vehicular compaction by a metal grille. Alternatively, a raised planter could be installed. Streets that are otherwise treeless could be greened. Some cities have tramways or railways laid along roads often at the median position. The rather extensive strips are usually paved with concrete, asphalt or stones. They could be replaced with soil and herbaceous vegetation. Besides turfgrass, native flowering herbs could significantly improve the streetscape. Where the tracks are shared with vehicular traffic, a porous paving system could carry the traffic and permit some herb growth. Many European cities have extensive green tracks, adding a new dimension to urban greening. CDDC could modify the method to suit local conditions. Conclusion Urban greening has a long tradition in many cities, but in some CDDC it is beset by multiple constraints and inertia. Fast urban expansion has strained the quality of the environment and urban life. Urban greening could be degraded and neglected in the hasty rush towards economic growth. Development and greening could join hand to contribute to sustainable development and smart growth (Gatrell and Jensen 2002). An overhauled urban planning regime could treat greening as an indispensable infrastructure, rather than dispensable options to fill residual niches. The naturalistic or ecological approach could transform UGS design. Aligning the spatial pattern of UGS with nature in a well-connected network could significantly improve their benefits and functions. They bring bonuses such as reduction in the capital and recurrent costs of greening programs, and higher level of user satisfaction. The technical solutions are not difficult to apply, and they could be adjusted and refined to suit the local environment. Relevant urban forestry and arboricultural concepts and skills could be acquired by education and training. The quality of tree professionals and workers, and associated equipment, are amenable to improvement. The more difficult hurdles lie in the institutional bottlenecks and psychological barriers. Modifying the administrative and statutory regimes could facilitate development of enabling government policies in landscape and conservation planning (Gordon et al. 2009; Hill et al. 2010). Enacting a comprehensive urban greening ordinance can provide impetus and a structured framework to greening work. Encouraging public participation and engagement could make urban greening relevant and welcome by citizens. The persistent adherence to the urbanized strand of environmental determinism, that compact cities cannot become meritorious green cities, has stifled initiatives and enthusiasm. The general neglect of tree care could be rectified by the long-term vision of transgenera- tional urban forestry and arboriculture. The inertia of sticking to the old-fashioned, anach- ronistic and often erroneous techniques presents another hindrance. Cities could be compact as well as green, with meticulous attention to every aspect of the urban greening complex. The overriding concern of quality has to permeate all greening plans and tasks. The knowledge gap in urban greening, with particular reference to the use and care of native species in urban horticultural applications, needs to be filled by research in conjunction with knowledge exchange and transfer. It highlights inadequacies in research infrastructure, scien- tists, funding, and an enabling research environment. 755 Urban Ecosyst (2013) 16:741–761 In the pervasive compact developments, plantable space at the ground level is limited. The largely bare flat roof tops could be enlisted for green roof installation. Where the loading capacity is limited, the light-weight extensive green roof could be adopted. Where the roof slab is strong, the intensive green roof with small trees and shrubs provide high-quality sky gardens (Jim 2008; Tian and Jim 2011). Green roofs offer recreational areas to supplement the ground-level UGS stock. Moreover, they provide environmental and ecological benefits such as temperature cooling, air pollutantremoval,noisereduction,stormwaterdischargereductionandwaterqualityimprovement, and wildlife habitats (Dunnett and Kingsbury 2004; Jim and Tsang 2011; Jim and Peng 2012). Numerous building facades and walls offer vertical greening opportunities that have hardly been realized in CDDC (Alexandri and Jones 2008). Appropriate technology and materials could be evaluated to suit local conditions (Jim and He 2011). Native species, preferably flowering climbers, could be tested by field experiments. Green walls bring benefits similar to green roofs, plus the more prominent visual amenity to invigorate the cityscape. Conclusion The principal determinant of a successful urban greening program is the successful synergy of researchers, practitioners, and decision makers. If they could excel in unison, urban greening could be facilitated and upgraded. 756 Urban Ecosyst (2013) 16:741–761 A major difference between a developing and developed economy is that the former often finds a balance between development and nature, whereas the latter tends to tilt towards development. A developed economy is more likely to stress research and integration of findings into policies and practices (Mazzotti and Morgenstern 1997). A hallmark of a sustainable city is the embodiment of sufficient natural ingredients in a permeating configuration into the built-up matrix. The basic precept is to optimize the factors to include healthy trees into the town plan (Petit et al. 1995), which is highly feasible. The major obstacles lie in the administrative, political and policy realms (Duvernoy 1995; Bowers 1999). We need an innovative spirit and vision to take urban greening ideas and ideals to fruition in CDDC. Acknowledgments The research grant support kindly provided by the Dr Stanley Ho Alumni Challenge Fund and the Government Matching Fund is gratefully appreciated. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. References Alexandri E, Jones P (2008) Temperature decreases in an urban canyon due to green walls and green roofs in diverse climates. Build Environ 43:480–493 Alvey AA (2006) Promoting and preserving biodiversity in the urban forest. Urban For Urban Green 5:195– 201 American Society of Consulting Arborists (2004) A consultant’s guide to writing effective reports. Rockville, MD Ames B, Dewald S (2003) Working proactively with developers to preserve urban trees. 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